Guo Asu 0010E 16735

Nuclear Magnetic Resonance (NMR) Spectroscopic Characterization of Nanomaterials
and Biopolymers
by
Chengchen Guo
A Dissertation Presented in Partial Fulfillment

of the Requirements for the Degree
Doctor of Philosophy
Approved February 2017 by the

Graduate Supervisory Committee:
Jeffery L. Yarger, Co-Chair

Gregory P. Holland, Co-Chair
Daniel A. Buttry
Xu Wang
ARIZONA STATE UNIVERSITY
May 2017
ABSTRACT
Nanomaterials have attracted considerable attention in recent research due to their
wide applications in various fields such as material science, physical science, electrical
engineering, and biomedical engineering. Researchers have developed many methods for
synthesizing different types of nanostructures and have further applied them in various
applications. However, in many cases, a molecular level understanding of nanoparticles
and their associated surface chemistry is lacking investigation. Understanding the surface
chemistry of nanomaterials is of great significance for obtaining a better understanding of
the properties and functions of the nanomaterials. Nuclear magnetic resonance (NMR)
spectroscopy can provide a familiar means of looking at the molecular structure of
molecules bound to surfaces of nanomaterials as well as a method to determine the size
of nanoparticles in solution. Here, a combination of NMR spectroscopic techniques
including one- and two-dimensional NMR spectroscopies was used to investigate the
surface chemistry and physical properties of some common nanomaterials, including for
example, thiol-protected gold nanostructures and biomolecule-capped silica
nanoparticles.
Silk is a natural protein fiber that features unique properties such as excellent
mechanical properties, biocompatibility, and non-linear optical properties. These
appealing physical properties originate from the silk structure, and therefore, the
structural analysis of silk is of great importance for revealing the mystery of these
impressive properties and developing novel silk-based biomaterials as well. Here, solid-
state NMR spectroscopy was used to elucidate the secondary structure of silk proteins in
N. clavipes spider dragline silk and B. mori silkworm silk. It is found that the Gly-Gly-X
i
(X=Leu, Tyr, Gln) motif in spider dragline silk is not in a β-sheet or α-helix structure and
is very likely to be present in a disordered structure with evidence for 31-helix
confirmation. In addition, the conformations of the Ala, Ser, and Tyr residues in silk
fibroin of B. mori were investigated and it indicates that the Ala, Ser, and Tyr residues
are all present in disordered structures in silk I (before spinning), while show different
conformations in silk II (after spinning). Specifically, in silk II, the Ala and Tyr residues
are present in both disordered structures and β-sheet structures, and the Ser residues are
present primarily in β-sheet structures.
ii
DEDICATION
To my Family
iii
ACKNOWLEDGMENTS
First and foremost I would like to express my sincere gratitude to my Ph.D. advisor Prof.
Jeffery Yarger and Prof. Gregory Holland for their constant support and great guidance.
They have been outstanding mentors to me, and I thank them for giving me the
opportunity to work with them and a great deal of freedom to conduct research in the
group as well. I also appreciate all their contributions of time, ideas, patience, motivation,
and funding to make my Ph.D. career productive. I could not have imagined having a
better advisor and mentor for my Ph.D. study.
I would like to thank all the members in Jeff’s group including current and former group
members. I thank all of the former graduate students and postdoctoral researchers in the
group who helped me in different ways at different times: Dr. Xiangyan Shi, Dr. Stephen
Davidowski, Dr. John Addison, Dr. Dian Xu, and Dr. Daniel Finkelstein Shapiro. I also
extend my gratitude to Dr. Brian Cherry and Dr. Samrat Amin in the MRRC facility.
Their encyclopedic knowledge of NMR spectroscopy and instrumentation is awe-
inspiring and they have helped me on many NMR experiments.
I would also like to thank my undergraduate advisor, Prof. Gi Xue for his kind and
inspiring guidance at the early stage of my research career. He has led me to the right
track of being a good, motivated and independent scientist.
Lastly, I would like to express my deepest gratitude to my family for all their love and
encouragement. I thank my parents for raising me up and supporting me in all my
pursuits. And most of all, I give my special thank to my beautiful and lovely wife, Jie
Ding for her constant love, patience, support, and encouragement in my life.
iv
TABLE OF CONTENTS
Page
LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES .............................................................................................................x
CHAPTER
1 GENERAL INTRODUCTION .................................................................................1
1.1 Nuclear Magnetic Resonance (NMR) Spectroscopy........................................1
1.2 Gold Nanoparticles ...........................................................................................8
1.3 Silica Nanoparticles ........................................................................................11
1.4 Silk Materials..................................................................................................13
1.5 References ......................................................................................................21
2 CHARACTERIZING GOLD NANOPARTICLES BY NMR SPECTROSCOPY ....
..............................................................................................................................34
2.1 Introduction ....................................................................................................34
2.2 Materials and Methods ...................................................................................36
2.3 Results and Discussion ...................................................................................39
2.4 Conclusions ....................................................................................................48
2.5 References ......................................................................................................49
2.6 Supplementary Materials ................................................................................54
3 ALANINE ADSORPTION AND THERMAL CONDENSATION AT THE
INTERFACE OF FUMED SILICA NANOPARTICLES ......................................56
3.1 Introduction ....................................................................................................56
3.2 Materials and Methods ...................................................................................59
v
CHAPTER Page
3.3 Results and Discussion ...................................................................................64
3.4 Conclusions ....................................................................................................91
3.5 References ......................................................................................................92
3.6 Supplementary Materials ................................................................................99
4 INVESTIGATING LYSINE ADSORPTION ON FUMED SILICA
NANOPARTICLES ..............................................................................................103
4.1 Introduction ..................................................................................................103
4.2 Materials and Methods .................................................................................106
4.3 Results and Discussion .................................................................................110
4.4 Conclusions ..................................................................................................124
4.5 References ....................................................................................................124
4.6 Supplementary Materials ..............................................................................131
5 SECONDARY STRUCTURE OF GLY-GLY-X (X=LEU, TYR, GLN) MOTIF IN
SPIDER DRAGLINE SILK INVESTIGATED BY SOLID-STATE NMR
SPECTROSCOPY.................................................................................................136
5.1 Introduction ..................................................................................................136
5.4 Conclusions ..................................................................................................148
5.5 References ....................................................................................................148
6 PROTEIN SECONDARY STRUCTURE IN BOMBYX MORI SILK
INVESTIGATED BY SOLID-STATE NMR SPECTROSCOPY .......................157
vi
CHAPTER Page
6.1 Introduction ..................................................................................................157
6.4 Conclusions ..................................................................................................173
6.5 References ....................................................................................................173
6.6 Supplementary Materials ..............................................................................179
REFERENCES ................................................................................................................185
vii
LIST OF TABLES
Table Page
1.1. Comparison of Mechanical Properties of Silk Materials and Other Materials ......15
2.1. Summary of 1H and 13C Resonances of Thiol-capped AuNPs ...............................44
2.2. Summary of DOSY Experiments on Thiol-capped AuNPs ...................................47
3.1. Summary of TGA Results for Alanine Adsorption on Fumed Silica Nanoparticles
................................................................................................................................67
3.2. Summary of 13C and 15N Chemical Shifts of U-[13C/15N]-L-alanine and 13C/15N-Ala/SiO2
Samples .............................................................................................................................. 70
3.3. Summary of Alanine Adsorption and Thermal Condensation on Fumed Silica
Nanoparticles from Thermal Analysis ............................................................................... 80
3.4. Assignments of FTIR band ................................................................................................ 83
4.1. Effect of Initial Concentration of Solution on Lysine Adsorption on Fumed Silica
Nanoparticles from TGA ......................................................................................114
4.2. 13C and 15N Chemical Shifts of Lysine and Lysine/SiO2 Samples ......................117
4.3. Calculated 1H, 13C and 15N Chemical Shielding Values and Extrapolated Chemical
Shifts for Lysine and Lysine/silanol Complex from DFT Calculations...............123
4.4. Summary of Lysine/silanol Complex from DFT Calculations ............................135
5.1. 13C Chemical Shifts for N. clavipes Dragline Silk, Random Coil, and Polypeptides
with Defined Secondary Structures ......................................................................145
6.1. 13C Chemical Shifts of Amino Acid Residues in Degummed B. mori Silk and
Lyophilized B. mori Silk, Solid Polypeptides with Known Secondary Structures,
and Random Coil Conformations.........................................................................167
viii
Table Page
6.2. 13C Chemical Shifts of Amino Acid Residues in Liquid Silk in B. mori Gland,
Regenerated B. mori Silk Solution, Degummed B. mori Silk and Lyophilized B.
mori Silk ..............................................................................................................183
ix
LIST OF FIGURES
Figure Page
1.1. Energy Level Diagram for Spin with I=1/2 in a Magnetic Filed 𝑩𝟎 ....................3
1.2. Schematic Representation of Sample Position in MAS NMR..............................5
1.3. NMR Pulse Sequences Involving CP Transfer .....................................................6
1.4. Illustration of the Structural and Dynamic Investigation of Proteins using NMR
Spectroscopy .........................................................................................................7
1.5. TEM Image of AuNPs Synthesized using the Method Developed by Turkevich
and Coworkers ......................................................................................................9
1.6. Partial MaSp1 and MaSp2 Primary Sequences for N. clavipes Dragline Silk....15
1.7. Illustration of Microstructural Model for Spider Silk .........................................17
1.8. Total Production of Silk over the World within the Most Recent Six Years .....18
1.9. Primary Sequences of One Domain in B. mori Silk Fibroin...............................20
2.1. UV-Vis Spectra of Purified C8H17S-AuNPs and GS-AuNPs, Luminescence
Spectrum of GS-AuNPs with An Excitation Wavelength of 500 nm .................40
2.2. TEM Images of C8H17S-AuNPs and GS-AuNPs ................................................40

1
2.3. H NMR Spectra of C8H17SH, C8H17S-AuNPs, GSH, and GS-AuNPs ..............41
1
2.4. H-1H COSY NMR Spectra of C8H17S-AuNP and GS-AuNPs, and 1H-13C
HSQC NMR Spectra of C8H17S-AuNPs and GS-AuNPs ...................................45

1
2.5. H NMR Spectrum of Purified C8H17S-AuNPs with DMSO at 298 K ..............46
2.6. 2D DOSY NMR Spectra of C8H17S-AuNPs and GS-AuNPs with δ = 2 ms, Δ =
50 ms at 298 K ....................................................................................................47
x
Figure Page
1
2.7. H-1H COSY NMR Spectra of C8H17SH and GSH, and 1H-13C HSQC NMR
Spectra of C8H17SH and GSH .............................................................................54
2.8. TGA of the Purified C8H17S-AuNPs and GS-AuNPs .........................................55
2.9. 2D DOSY NMR Spectra of C8H17S-AuNPs and GS-AuNPs with δ = 2 ms, Δ =
50 ms at 298 K (Full Scale) ................................................................................55
3.1. TEM Images and Raman Spectra of Fumed Silica Nanoparticles and Colloidal
Silica Nanoparticles ............................................................................................66
3.2. The Amount of Alanine Adsorbed on Fumed Silica Nanoparticles as a Function
of Initial Aqueous Alanine Concentration at Room Temperature and Near
Neutral pH (pH=6.7) ...........................................................................................67

1
3.3. Hà13C CP-MAS NMR Spectra and 1Hà15N CP-MAS NMR Spectra of U-
[13C/15N]-L-alanine, 13
C/15N-Ala/SiO2-0.10M (“hydrated” and ”dry”), and
13
C/15N-Ala/SiO2-0.03M (“hydrated” and ”dry”) ...............................................69
1
3.4. Hà13C 2D HETCOR MAS NMR Spectra of “hydrated” 13
C/15N-Ala/SiO2-
0.10M and “dry” 13C/15N-Ala/SiO2-0.10M.........................................................71

1
3.5. Hà15N 2D HETCOR MAS NMR spectra of (A) “hydrated” 13C/15N-Ala/SiO2-
13
0.10M and (B) “dry” C/15N-Ala/SiO2-0.10M. Experiments were conducted
with a 1.0 ms contact time at 400 MHz and a MAS frequency of 35 kHz .........72
1
3.6. Hà13C 2D HETCOR MAS NMR Spectra of “hydrated” 13
C/15N-Ala/SiO2-
0.03M and “dry” 13C/15N-Ala/ SiO2-0.03M........................................................72

1
3.7. Hà15N 2D HETCOR MAS NMR Spectra of “hydrated” 13
C/15N-Ala/SiO2-
0.03M and “dry” 13C/15N-Ala/SiO2-0.03M.........................................................73
xi
Figure Page
1
3.8. Hà13C and 1Hà15N 2D HETCOR MAS NMR Spectra of “dry” 13
C/15N-
Ala/SiO2-0.03M with Contact Times of 2.0 ms and 0.25 ms .............................76
3.9. Schematic Representation of Alanine Adsorption on Fumed Silica
Nanoparticles Surfaces in “hydrated” and “dry” State .......................................77
3.10. DTG Curves of Bulk Alanine and Ala/SiO2 (solid, black) Samples as a Function
of Initial Concentration of Amino Acids in the Adsorption Solutions ...............78
3.11. DTG Curves for Ala/SiO2-0.03M, Alanine Anhydride, and Alanine
Anhydride/SiO2-0.03M .......................................................................................80
3.12. 1Hà13C and 1Hà15N CP MAS Spectra of 13C, 15N-Alanine, Alanine Anhydride,
13
C, 15N-Ala/SiO2-0.03M, and 13C, 15N-Ala/SiO2-0.03M Incubated at 170 °C for
Three Hours ........................................................................................................82
3.13. FTIR Spectra of Fumed Silica Nanoparticles, Alanine, Ala/SiO2-0.03M,
Ala/SiO2-0.03M Incubated at 170 °C for Three Hours, Product Washed off
Nanoparticles after Thermal Incubation, and Alanine Anhydride ......................83
3.14. 1H NMR Spectra of Alanine, Alanine Anhydride, the Peptide Formed on Fumed
Silica Nanoparticles, and Peptide Formed at Colloidal Silica Nanoparticles .....84
3.15. Schematic Representation of Thermal Condensation of Alanine at 170 °C
Forming Alanine Anhydride on Fumed Silica Nanoparticles.............................85
xii
Figure Page
3.16. Raman Spectra of Alanine Adsorbed Fumed Silica Nanoparticles (Ala/SiO2-
0.03M), Ala/SiO2-0.03M after Thermal Incubation at 170 °C for Three Hours,
Alanine Adsorbed Colloidal Silica Nanoparticles, and Alanine Adsorbed
Colloidal Silica Nanoparticles after Thermal Incubation at 170 °C for Three
Hours; Raman spectra of Fumed Silica and Alanine Adsorbed Fumed Silica
Nanoparticles after Thermal Incubation at 170 °C for Three Hours ..................89
3.17. 1H MAS NMR Spectra of Dried Fumed Silica Nanoparticles, 13

C/15N-alanine,
13
“hydrated” C/15N-Ala/ SiO2-0.03M, “hydrated” 13
C/15N-Ala/ SiO2-0.10M,
“dry” 13C/15N-Ala/ SiO2-0.03M, and “dry” 13C/15N-Ala/ SiO2-0.10M ...............99
3.18. 1Hà13C and 1Hà15N 2D HETCOR NMR Spectra of 13C, 15N-Ala/ SiO2-0.03M,
13
C, 15N-Ala/ SiO2-0.03M Incubated at 170 °C for Three Hours .....................100
3.19. 1H-13C HSQC NMR Spectra of Alanine Anhydride and Peptide Formed at
Fumed Silica Nanoparticles Surfaces by Incubating Ala/SiO2-0.03M at 170 °C
For Three Hours ................................................................................................100
3.20. 1H NMR Spectra of Alanine and Peptide Formed at Fumed Silica Nanoparticles
Surfaces by Incubating Ala/SiO2-0.03M at 170 °C for Three Hours ...............101
3.21. TGA and DTG Curves for Alanine Adsorbed Colloidal Silica Nanoparticles
with a Heating Rate of 5 °C/min .......................................................................101
3.22. FTIR Spectra of Colloidal Silica Nanoparticles, Alanine, Alanine Adsorbed
Colloidal Silica Nanoparticles, Alanine Adsorbed Colloidal Silica Nanoparticles
Incubated at 170 °C for Three Hours, and Alanine Anhydride ........................102
3.23. Raman Spectra of Alanine and Alanine Anhydride in Crystalline Form .........102
xiii
Figure Page
4.1. Summary of Different L-lysine Forms and Charged States of Silica Surface as a
Function of pH Values ......................................................................................104
4.2. Bulk Lysine and Lysine/silanol Complex Models Used in DFT Calculations:
Lysine and Lys-H-OSiH3 ..................................................................................110

1
4.3. H NMR Spectra of Natural Abundance Bulk Lysine at MAS Speeds of 30 kHz
and 67 kHz at a 1H Larmor Frequency of 800 MHz and 850 MHz, respectively;
1
H-1H BABA NMR Spectrum of Natural Abundance Bulk Lysine at Spinning
Speed of 67 kHz at a 1H Larmor Frequency of 850 MHz ................................112
4.4. TGA Curves of Lysine/silica Samples as a Function of Initial Concentration of
Lysine in the Adsorption Solution; The Amount of Lysine Adsorbed on Silica
as a Function of Initial Concentration of Lysine at Room Temperature and
pH=10.0 ............................................................................................................113
1
4.5. Hà13C CP-MAS NMR Spectra of Bulk lysine, 13
C, 15
N-Lysine·2HCl, 13
C,
15 13 15
N-Lysine·2NaCl, Lys/SiO2-0.01M, and C, N-Lysine/SiO2 (0.01 M);
1
Hà15N CP-MAS NMR Spectra of Bulk Lysine, 13C, 15N-lysine·2HCl, and 13C,
15
N-Lysine/SiO2 (0.01 M). ................................................................................116
1
4.6. H NMR Spectra of Natural Abundance Bulk Lysine and 13C, 15N-Lysine/SiO2
at a MAS Speed of 67 kHz ...............................................................................117

1
4.7. H-13C 2D HETCOR NMR Spectrum of 13
C, 15
N-Lysine/SiO2 with Different
Mixing Times (0.25 ms, 2.0 ms) .......................................................................118

1
4.8. Hà13C CP-MAS NMR Spectra of Lys/SiO2-0.01M, Lys/SiO2-0.10M, and
Lys/SiO2-0.10M/NaCl ......................................................................................120
xiv
Figure Page
4.9. Schematic Representation of the Favorable Model for Lysine Adsorption on
Fume Silica Nanoparticles Surfaces .................................................................123
4.10. 1H-13C HETCOR NMR Spectrum of Bulk Lysine ...........................................131

13
4.11. 2D C-13C Through-bond Double-quantum (DQ)/Single-quantum (SQ)
Refocused INADEQUATE NMR Spectrum of 13C, 15N-Lysine·2HCl ............132

15
4.12. N-13C 2D HETCOR NMR Spectrum of 13C, 15N-lysine/SiO2 .......................133
4.13. 2D 13C-13C Through-space Correlation NMR Spectrum of 13C, 15N-Lysine·2HCl
...........................................................................................................................134
13
4.14. Extrapolated Curve of C Chemical Shift Derived from the Experimental and
Calculated 13C Chemical Shift Values of Bulk Lysine .....................................135
5.1. Typical Repetitive Amino Acid Sequences of Silk Proteins for N. clavipes .......137
5.2. Illustration of a Microstructural Model for Spider Silk .......................................137
5.3. SEM Image of N. Clavipes Dragline Silk ............................................................141
5.4. Consensus Primary Amino Acid Sequence for N. clavipes MaSp1 and 1Hà13C
CP-MAS NMR Spectrum of 13C-labeled N. clavipes Dragline Silk ....................143

13
5.5. 2D C-13C Correlation Spectra of 13
C labeled N. clavipes Spider Dragline Silk
Collected with a Long Dipolar Assisted Rotational Resonance (DARR) Mixing
Period of 1 s .........................................................................................................146
6.1. 1Hà13C CP-MAS Spectra of Degummed Silkworm Silk Collected from B. mori
Silkworms that were Fed with [U-2H/13C/15N]-Serine, [U-2H/13C/15N]-
Phenylalanine, [U-13C]-Glucose Enriched Food, and Regular Food that was not
Isotopically Enriched............................................................................................162
xv
Figure Page
13
6.2. 2D Through-bond C-13C DQ/SQ Refocused INADEQUATE NMR Correlation
Spectrum of Degummed Silk Collected from B. mori Silkworms that were Fed
with [U-2H/13C/15N]-Serine ..................................................................................165

13
Spectrum of Lyophilized Silk Collected from B. mori Silkworms that were Fed
with [U-2H/13C/15N]-Serine ..................................................................................166

13
Spectrum of Degummed Silk Collected from B. mori Silkworms that were Fed
with [U-2H/13C/15N]-Phenylalanine......................................................................169
6.5. 13C Direct Spectrum of the Degummed Silkworm Silk Collected from B. mori
Silkworms that were Fed with [U-2H/13C/15N]-Phenylalanine; Deconvolution
Results of the Tyr Cβ Regions in 13C NMR Spectrum ........................................170

13
Spectrum of Lyophilized Silkworm Silk Made from B. mori Silkworms that were
Fed with [U-2H/13C/15N]-Phenylalanine...............................................................171
6.7. 1Hà13C CP-MAS Spectra of Degummed Silk Collected from B. mori Silkworms
that were Fed with [U-2H/13C/15N]-Serine, Lyophilized Silk Made from [U-
2
H/13C/15N]-Serine Labeled Degummed B. mori Silk, Degummed Silk Collected
from B. mori Silkworms that were Fed with [U-2H/13C/15N]-Phenylalanine, and
Lyophilized Silk Made from [U-2H/13C/15N]-Phenylalanine Labeled Degummed
B. mori Silk ..........................................................................................................182
xvi
CHAPTER 1
General Introduction
1.1 Nuclear Magnetic Resonance (NMR) Spectroscopy
1.1.1 Introduction to Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy is a non-destructive molecular
characterization tool that requires little perturbation of the analyzed system, while
providing exceptional details about the structure and dynamics of molecules, and the
chemical environments of constituent atomic nuclei. It relies on the phenomenon of
nuclear magnetic resonance which was first observed experimentally in 1946 by Bloch et
al at Stanford [1, 2] and Purcell et al at Harvard [3]. Over the past 50 years, NMR
spectroscopy has bloomed into a major characterization tool in organic chemistry,
analytical chemistry and structural biology with several revolutionary milestones such as
Fourier transform (FT) NMR spectroscopy [4], two-dimensional (2D) NMR method [5],
and nuclear overhauser effect (NOE) spectroscopy [6]. The Nobel Prize in Chemistry
1991 was awarded to Richard R. Ernst for his contribution in developing FT-NMR and
its derivatives, and the Nobel Prize in Chemistry 2002 was awarded to Kurt Wüthrich for
demonstrating that the NOE could be exploited using two-dimensional NMR
spectroscopy to determine the three-dimensional structures of biological macromolecules
in solution. In recent years, with the continuous development of NMR methodology
along with the instrument upgrade, the time efficiency of NMR experiments, particular
multi-dimensional NMR experiments, and the signal sensitivity have been improved
significantly, which makes it possible to investigate complicated biological systems
within a reasonable time period. Due to this, NMR spectroscopy has become a premier
1
technique that rivals X-ray crystallography in structural biology for studying the structure
and dynamics of proteins [7, 8].
1.1.2 Basic Principles of NMR Spectroscopy
When a sample is placed in a magnet, the nuclei in the molecules generate a bulk
macroscopic magnetization originating from the interaction of the nuclear magnetic
moment with the applied external magnetic field. For nuclei with a non-zero spin angular
momentum (NMR active), this interaction results in splitting energy levels according to
the rules of quantum mechanics that is known as Zeeman splitting. For proton with spin
I=1/2, in a magnetic field, two energy levels are defined (Figure 1.1) with an energy
difference between two levels (∆𝐸) described by
∆𝐸 = ℎ𝛾𝐵! /2𝜋 Equation 1.1
where 𝛾 is the magnetogyric ratio and 𝐵! is the external magnetic field. As shown in
Figure 1.1, the lower state (labeled as α), in which the nuclear magnetic moment is
!
parallel to the external magnetic field corresponds to 𝑚! = + !, while the upper state
(labeled as β), in which the nuclear magnetic moment is antiparallel to the external
!
magnetic field corresponds to 𝑚! = − !. The observed frequency in NMR spectroscopy
can be expressed in terms of the magnetogyric ratio and the external magnetic field:
𝜈 = ∆𝐸/ℎ = 𝛾𝐵! /2𝜋 Equation 1.2
where 𝜈 is the resonant frequency in hertz (Hz).
If nuclei have the same magnetogyric ratio and are placed in exactly identical
external magnetic fields, the observed frequencies are equal. However, this is barely the
case in practice because the nuclei in the molecules have characteristic chemical
2
environments where the nuclei sense slightly different magnetic fields from each other.
The ability for NMR spectroscopy to detect these small changes in the local magnetic
field makes it a powerful technique in differentiating nuclei in the molecules with rich
chemical environmental information encoded.
∆E
E
α
B0
Figure 1.1. Energy level diagram for spin with I=1/2 in a magnetic filed 𝑩𝟎 .
From an experimental standpoint, the observation of frequencies of nuclei can be
achieved with two types of NMR spectrometers, continuous-wave (CW) NMR
spectrometers and pulsed NMR spectrometers. CW NMR spectrometers were used
frequently in the very early decades of NMR spectroscopy development, but soon have
been largely replaced with pulsed NMR spectrometers due to the better sensitivity and
signal-to-noise ratio with pulsed NMR spectrometers [8]. In pulsed NMR, a pulse is
applied to disturb the system from equilibrium and then the response of the system to the
disturbance was monitored. The signals from nuclei are extracted and analyzed using
Fourier transform (FT), so pulsed NMR is also called pulsed FT-NMR. In modern NMR
spectroscopy, numerous pulse sequences have been developed and applied for obtaining
the structural and dynamic information of nuclei in various systems.

3
1.1.3 Solid-state NMR Spectroscopy
Due to the presence of anisotropic interactions in solids, the resonance in solid-
state NMR spectrum is typically broad, which limits the application of solid-state NMR
spectroscopy in the structural analysis of molecules at the early stage of development.
However, with some great improvement in solid-state NMR methodology such as magic-
angle-spinning (MAS) [9], cross-polarization (CP) [10, 11], advanced multiple-pulse
sequences, and enhanced probe designs, solid-state NMR spectroscopy has been
recognized as an invaluable tool in the structural and dynamic analysis of
biomacromolecules like amyloids [12, 13], membrane proteins [14, 15], and silks [16, 17]
that are typically not soluble in solutions. In addition, it has also been widely used for
other applications in a variety of fields including chemistry, geology, and materials
science [18-23]. The following two paragraphs introduce the basic principles of magic-
angle-spinning (MAS) and cross-polarization (CP).
Magic-angle-spinning (MAS) was first developed by Andrew et al. in 1958 and
has been used to enhance the sensitivity in solid-state NMR spectroscopy [9]. In
comparison to static NMR spectroscopy, MAS NMR spectroscopy results in narrower
resonances in NMR spectra because the anisotropic interactions in solids including
chemical shift anisotropic interaction, dipolar coupling and quadruple coupling are
extensively averaged under MAS. In principle, all these anisotropic interactions have an
orientation term (3cos2θ-1)/2 (θ is the angle between the interaction vector and the
external magnetic field) [24]. To average out the anisotropic interactions in samples, the
axis of the sample holder (rotor) is placed at the magic angle (θ=54.74°) with respect to
the external magnetic field in MAS NMR spectroscopy (Figure 1.2). When the rotor is
4
spun at a speed over the anisotropic interactions in Hz, the orientation-dependent
anisotropic interactions are averaged to an integrated tensor whose orientation is along
the rotor axis. Since the rotor is fixed at the magic angle (θ) with respect to the external
magnetic filed, the tensor is averaged out with (3cos2θ-1)/2 equals zero. As a
consequence, the resonance in the spectra becomes narrower [25]. However, in practice,
the MAS speed is difficult to reach the level that averages out all the anisotropic
interactions, the resonance obtained in MAS NMR is still broader than that in classical
liquid NMR. Overall, with the MAS technique, the sensitivity and signal-to-noise ratio
are significantly enhanced, which leads to a rapid development and wide application of
solid-state NMR spectroscopy.
B0
54.74o
Figure 1.2. Schematic representation of sample position in MAS NMR.
Cross-polarization (CP) is one of the most common methods in solid-state NMR
spectroscopy which was first discovered by Pine et al. in 1973 with the demonstration of
13
CP C NMR spectra of solid adamantane and benzene [10]. CP is usually used for
observing dilute spins such as 13C and 15N, where the magnetization of an abundant spin
(I) is transferred to a dilute spin (S) via the heteronuclear dipolar coupling. In order to
5
achieve the best transferring efficiency, in practice, the field strengths of the radio
frequency (rf) applied to spins I and S must fulfill the Hartmann-Hahn condition [11]. A
simple NMR pulse sequence involving CP transfer is shown in Figure 1.3A, where an
excitation rf pulse (π/2 pulse) is applied to spin I and the generated magnetization is
transferred to spin S through CP, which is also called spin-lock process [26]. Typically, a
rf field with ramped-amplitude is applied to one spin to circumvent the imperfect CP
condition induced by MAS and hardware instability. By using CP method, the signal-to-
noise ratio for the dilute spin (S) is significantly enhanced with a theoretical factor of
𝛾! /𝛾! . Additionally, CP can be used to obtain spatial information as well since it relies on
heteronuclear dipolar coupling which is an interaction depending on the spatial distances
of nuclei. Figure 1.3B shows a pulse sequence for obtaining spatial information using CP
method, where the spatial information is encoded in the delay time (t1) between the
excitation rf pulse and CP transfer.
A B
π/2 π/2
I CP Decoupling I t1 CP Decoupling
S CP S CP
Figure 1.3. NMR pulse sequences involving CP transfer. (A) is the simplest version, while (B) is
a modified version used for obtaining spatial information of nuclei in the sample.
1.1.4 Structural Analysis of Proteins using NMR Spectroscopy
Upon its discovery, NMR spectroscopy is well known for its strong ability in
identifying atoms/groups involved in different chemical environments. This benefits
organic chemistry considerably at the early stage of NMR spectroscopy since it provides
6
a convenient way to resolve structures for thousands of natural and synthetic organic
molecules. With the discovery of multi-dimensional and dipole-dipole interaction based
NMR spectroscopy such as correlation spectroscopy (COSY), nuclear Overhauser effect
spectroscopy (NOESY), and heteronuclear single quantum correlation spectroscopy
(HSQC), NMR spectroscopy has soon become a powerful and indispensable technique
for solving structures of large molecules like proteins, which leads to a booming research
area called protein NMR [27].
Figure 1.4. Illustration of the structural and dynamic investigation of proteins using NMR
spectroscopy.
In principle, NMR spectroscopy makes three main contributions to the success of
determining protein structures and they are summarized in Figure 1.4. First, by
combining single and multi-dimensional NMR spectroscopies, a full list of chemical
shifts including 1H, 13

C, and 15
N for every amino acid residues can be obtained. These
7
chemical shifts have strong correlations to protein secondary structures, so it makes the
prediction of whole/local protein structure possible by assigning amino acid residues.
Second, dipole-dipole interaction based NMR spectroscopy provides an excellent way to
obtain distances between nearby atoms/groups that can be further used to predict or refine
2
the protein structures. Third, relaxation time (T1/T2) measurements or H NMR
spectroscopy can be used as a good tool for investigating the dynamics of proteins since
the relaxation times or the 2H patterns of specific groups correlates strongly with the
dynamics of the groups [23, 28].
1.2 Gold Nanoparticles
Gold nanoparticles (AuNPs) have a rich history in chemistry, dating back to
ancient Roman times over 2000 years ago where they were used as pigments for staining
glasses. The modern era of AuNPs research began over 150 years ago with the work of
Michael Faraday, who was possibly the first to illustrate the colloidal gold solution have
different properties from bulk gold [29]. Since then, the AuNPs have fascinated scientists
for over a century and are now widely utilized in chemistry, biology, engineering, and
medicine [29-39]. Over the last few decades, high-yielding and reliable methods for the
synthesis of AuNPs with different morphologies and properties have been developed [36,
40, 41]. The synthesized AuNPs have a variety of unique properties, including size- and
shape-dependent optical and electronic properties, a high surface area-to-volume ratio,
surface plasmon resonance, and the abilities to be functionalized with ligands containing
functional groups such as thiols, amines, and phosphine, which exhibit affinity to gold
surfaces [29, 35, 36]. Due to this, AuNPs are considered as an excellent scaffold for
8
developing sensors by anchoring a variety of ligands such as oligonucleotides, proteins,
and antibodies via functional groups [38, 42].
The scientific synthesis of colloidal gold nanoparticles can be traced back to
Michael Faraday’s work in 1857, in which a gold hydrosol solution was prepared by
reduction of an aqueous solution of chloroaurate with phosphorus dissolved in carbon
disulfide. Later in 1951, Turkevich and coworkers developed one of the most popular
approaches for the synthesis of AuNPs, in which citrate was used to reduce chloroauric
acid (HAuCl4) in water [39]. In this method, citric acid acts as both reducing and
stabilizing agent, providing Au NPs with a diameter of about 20 nm (Figure 1.5). This
method has then been investigated and optimized by several researchers since its first
discovery to enable high-yielding and high-quality AuNPs synthesis, and precise control
over AuNPs sizes [43].
Figure 1.5. TEM image of AuNPs synthesized using the method developed by Turkevich and
coworkers.
During the last two decades with nanotechnology came into being, a considerable
number of synthetic methods for AuNPs have been discovered and reported. One of the
9
most famous methods was developed by Brust and Schiffrin in 1994 [41], in which a
two-phase synthetic strategy was created, utilizing strong thiol−gold interactions to
protect AuNPs with thiol ligands. Briefly, AuCl4- is transferred from aqueous phase to
organic phase (toluene) using the surfactant tetraoctylammonium bromide (TOAB). Then
dodecanethiol is added to the organic phase to initiate the first reduction of AuCl4-,
followed by a quick addition of strong reducing agent, sodium borohydride (NaBH4). The
AuNPs are formed in toluene with sizes in the range of 1.5-5 nm. Due to the strong thiol-
gold interaction, these thiol-protected AuNPs feature super stability in both dry and wet
states. This impressive property soon has attracted considerable attention in nanoscience
community. As a result, extensive studies have been carried out on the synthesis of
different types of thiol-protect AuNPs using ligands such as benzenethiolates [44],
glutathione [45, 46], and DNA [42].
Besides developing synthetic methods for AuNPs, physical characterization of
AuNPs including the size, morphology, optical properties and surface chemistry is
another focus in AuNPs research. Transmission electron microscopy (TEM) and scanning
electron microscopy (SEM) are used as primary tools to determine the sizes of AuNPs
while UV-vis/fluorescent spectroscopy and IR/Raman spectroscopy are used for
understanding the optical properties and surface chemistry of AuNPs, respectively.
Recently, mass spectroscopy and single crystal X-ray crystallography have been
demonstrated to be excellent tools for characterizing gold nanoclusters. Mass
spectroscopy has been used for investigating the ligand density of nanostructures,
molecular formula for molecular-like nanoclusters and charge-state of nanocluster [47-
50]. Single crystal X-ray crystallography not only can determine the size of the
10
nanostructures and but also the exact stacking patterns of atoms [44, 51-55]. However,
this technique requires sufficient very good quality single crystals to give well-defined
diffractions, which limits the technique to be utilized extensively. Although these
characterization techniques can give abundant information on the sizes, surface coverage,
optical properties, and even the crystal structures for AuNPs. The surface chemistry of
the inorganic (Au core)-organic (ligand) interface still remains poorly understood. In
Chapter 2, the author focuses on investigating the surface chemistry and size of AuNPs
with advanced NMR spectroscopy techniques. The NMR spectroscopy is chosen as the
primary technique because it is a well-suited technique for in situ analysis of chemical
structure, surface chemistry, and dynamics.
1.3 Silica Nanoparticles
Silica (SiO2) is one of the most common inorganic materials on Earth and has
been extensively investigated for decades due to its diverse properties such as
polymorphism and biocompatibility [56, 57]. It has widespread applications ranging from
structural materials to catalysis and as a medium for drug delivery [58-66]. Amorphous
silica nanomaterials including fumed silica, mesoporous silica and colloidal silica
nanoparticles are one class of synthetic silica materials, featuring small particle sizes and
high surface areas [56, 59, 67, 68].
Over 40 years ago, Stöber et al. first synthesized colloidal silica nanoparticles with
a high homogeneity in solution by means of controlled hydrolysis and condensation
reactions [67]. The synthesis is carried out in alcoholic solutions using ammonia as a
morphological catalyst and alkyl silicates such as tetraethyl orthosilicate (TEOS) as the
11
silicon source. By varying the ratio of water, well-defined spherical silica nanoparticles
with a wide range of sizes can be synthesized.
Mesoporous silica nanoparticles is a type of nanoscale mesoporous silica developed
in recent years that has a solid framework with porous structures and large surface area
[59, 69]. They are typically synthesized based on the well-established Stöber method but
with a few modifications, in which surfactants were used to generate the porosity and
control the pore morphologies. Due to the unique properties, such as high surface area,
tunable pore size with a narrow distribution, and good chemical and thermal stability,
mesoporous silica nanoparticles have been widely used in various controlled release
applications such as drug delivery [57, 60, 64].
Fumed silica (Cab-O-Sil) nanoparticles are another class of synthetic nanomaterials
composed of amorphous structures with extensively high surface area and nanoscale size
[68]. They are prepared at high temperature by hydrolyzing silicon tetrachloride vapor in
a flame followed by rapid quenching to room temperature. Many studies have been
carried out on the surface chemistry at fumed silica nanoparticle interfaces because of the
considerable utility of high surface area amorphous silicates [70-73]. These studies have
shown that the surface chemistry of fumed silica nanoparticles is very different from
mesoporous and colloidal silica nanoparticles due to the presence of metastable strained
ring structures in fumed silica that result from the fast quenching during the synthesis of
the material in a flame. This unique property has been found to closely relate with the
high surface reactivity of the fumed silica nanoparticles [74].
During the past decades, silica nanomaterials have been studied extensively from
experimental synthesis to structural characterization to biomedical applications [57].
12
Additionally, the adsorption and thermal condensation of amino acids on silica
nanomaterials have attracted considerable attention in recent years, which is thought to be
the very first step towards understanding the interaction between biomolecules and silica
nanomaterials including the varying toxicity of different silica nanoparticle types [74].
According to the literature review, considerable experimental studies have been carried
on mesoporous and colloidal silica nanomaterials, but very limited effort has been put on
understanding the behaviors of amino acid on fumed silica nanoparticles. In Chapter 3
and Chapter 4, the author focuses on the investigations of alanine and lysine on fumed
silica nanoparticles by NMR spectroscopy, respectively, including the adsorption and
thermal condensation of the amino acids at the interfaces.
1.4 Silk Materials
1.4.1 General Introduction to Silk Materials
Silks are generally defined as protein polymers that are produced naturally by some
Lepidoptera larvae such as silkworms, spiders, scorpions, and flies. Silkworm silk and
spider silk are the most studied and widely used, and are excellent materials for
biomedical applications such as drug delivery and tissue engineering because of their
biocompatibility, biodegradability, and extraordinary mechanical properties [58]. Spider
silks are known as the strongest and toughest natural fibers, possessing unrivalled
extensibility and high tensile strength. Silkworm silk has been used as suture material for
centuries and recently has gained considerable attention as a biomaterial with various
medical applications due to its slow rate of degradation in vivo and its ability to be
fabricated into multiple types of materials such as fibers, films, gels, and foams [59].
13
1.4.2 Spider silk
Spider silk is produced by spiders, ancient arachnids that have evolved for over
400 millions years [75]. Orb-weaving spiders like Nephila clavipes can produce up to
seven different types of silks that originate from different abdominal glands [76, 77].
These silks vary widely in morphologies and mechanical properties and have been
optimized for performing specific functional roles [78-81]. Major ampullate (MAA) silk,
also called dragline silk, and minor ampullate (MIA) silk are used to construct the frame
and radii of the web, respectively [82]. Flagelliform silk is used for making the core
filaments of the orb web’s capture thread [78]. The capture thread filaments are coated
with another type of ‘silk’ originating from the aggregate gland, which is not a hard
filament but an aqueous solution of small and highly hygroscopic peptides as well as
sticky glycoproteins. The web threads are anchored to the vegetation and affixed to one
another by a type of silk cement from the pyriform glands. Additionally, the egg sack of
the spiders is constructed by very fine silk filaments originating from the tubuliform or
cylindriform gland and one type of aciniform gland, while another type of aciniform
filament is used for a multitude of other purposes such as strengthening the cement
matrix [77].
Among all types of spider silks, dragline silk, is the toughest one with extraordinary
strength that exceeds most of the man-made materials and even stronger than steel by
weight, together with the impressive extensibility (Table 1.1). It is used not only to
construct the outer frame and radii of the orb-shaped web but also as a hanging lifeline
that allows the spider to evade and/or escape from predators. Due to the impressive
14
mechanical properties, dragline silk has been extensively studied in recent decades [16,
76, 83-88].
Table 1.1. Comparison of mechanical properties of silk materials and other materials
Young’s modulus Ultimate strength Breaking strain Toughness
Materials Ref.
(GPs) (MPa) (%) (MJ/m3)
Spider dragline silk
10.9 875 16.7 - [81]
(Nephila clavipes)
Spider dragline silk
10 1100 27 160 [82]
(Araneus)
Silkworm silk
10-17 300-740 4-26 70-78 [89, 90]
(Bombyx mori)
Collagen 0.0018-0.046 0.9-7.4 24-68 - [91]
Wool 0.5 200 50 60 [82]
Nylon 1.8-5 430-950 18 80 [82]
Kevlar 49TM 130 3600 2.7 50 [82]
The core constituents of dragline silk are two fibrous proteins produced in the
major ampullate gland that are called major ampullate spidroin 1 (MaSp1) and major
ampullate spidroin 2 (MaSp2), and each has a molecular weight of 250-350 kDa [79, 88].
Figure 1.6 shows the known partial sequences of MaSp1 and MaSp2 proteins in N.
clavipes dragline silk, which is the major focus of spider silk research in recent years, as
well as the focus in this thesis.
MaSp1
QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQG
GYGGLGSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAA
GGAGQGGYGGGNQGAGRGGQGAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAA
AAAGGAGQGGYGGLGGQGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGGAGQGGL
GGQGAGQGAGASAAAAGGAGQGGYGGLGSQGAGRGGEGAGAAAAAAGGAGQGGYGGLG
GQGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAGGAGQGGLGGQGAGQGAGAAAAAAGG
AGQGGYGGLGSQGAGRGGLGGQGAGAVAAAAAGGAGQGGYGGLGSQGAGRGGQGAGAA
AAAAGGAGQRGYGGLGNQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGNQGAGRGG
15
QGAAAAAGGAGQGGYGGLGSQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGL
GSQGSGRGGLGGQGAGAAAAAAGGAGQGGLGGQGAGQGAGAAAAAAGGVRQGGYGGLG
SQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGY
GGVGSGASAASAAASRLSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGASILVFLDV
MSSFKLFSRLFLLLSRS
MaSp2
PGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAAAGPGGYGPGQQGPGGYGPGQQGP
GRYGPGQQGPSGPGSAAAAAAGSGQQGPGGYGPRQQGPGGYGQGQQGPSGPGSAAAAS
AAASAESGQQGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGP
GQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAASGPGQQGPGGYGPGQQGPGG
YGPGQQGLGPGSAAAAAAAGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAAGPGGYGPGQ
QGPGGYGPGQQGPSGAGSAAAAAAAGPGQQGLGGYGPGQQGPGGYGPGQQGPGGYGPG
SASAAAAAAGPGQQGPGGYGPGQQGPSGPGSASAAAAAAAAGPGGYGPGQQGPGGYAPG
QQGPSGPGSASAAAAAAAAGPGGYGPGQQGPGGYAPGQQGPSGPGSAAAAAAAAAGPGG
YGPAQQGPSGPGIAASAASAGPGGYGPAQQGPAGYGPGSAVAASAGAGSAGYGPGSQASA
AASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEIVSA
CVTILSSSSIGQVNYGAASQFAQVVGQSVLSAF
Figure 1.6. Partial MaSp1 and MaSp2 primary sequences for N. clavipes dragline silk. The
poly(Ala) and poly(Gly-Ala) motif are highlighted in red, indicating the sequence forming –sheet
structures in silk. Gly-Gly-X or Gly-X-Gly (X=Gln, Tyr, Leu) motifs in MaSp1 and Gly-Pro-Gly
motif in MaSp2 are highlighted in blue and green, respectively.
Both proteins contain a large, central, repetitive domain that consists of
approximately 100 tandem copies of a 30 to 40 amino acid repeat sequence. For MaSp1,
the consensus repeat includes poly(Ala), poly(Gly-Ala), Gly-Gly-X or Gly-X-Gly
(X=Gln, Leu, Tyr) motifs and very low proline content. In contrast, the MaSp2 consensus
repeat has significant proline content and characteristic motifs such as Gly-Pro-Gly-X-X
(X-X = Gln-Gln, Gly-Tyr). A combination of NMR spectroscopy and X-ray diffraction
(XRD) has illustrated that each type of repetitive motifs form distinct secondary structure
and the basic unit of the fiber assemblies [16, 76, 83-88, 92]. It is these secondary
structures formed by different types of repetitive sequences provide the elasticity and
toughness for spider silk. Specifically, ploy(Ala) and ploy(Gly-Ala) form β-sheet
(dominated by antiparallel) structures and these β-sheet structure are organized into
16
nanocrystallites with a approximate size of 2×5×7 nm [86, 93]. Simulation study has
indicated that the hydrogen bonds in the noncrystalline domains together with the highly
ordered organization of the nanocrystallites are responsible for silk strength [94]. In
addition, Gly-Gly-Ala motif in MaSp1 and Gly-Pro-Gly-X-X motif in MaSp2 have been
characterized to be present in 31-helical and type II β-turn structures, respectively, and
they are thought to construct the amorphous domains in silk fibers [84, 92]. A general
microstructural picture of spider silk is shown in Figure 1.7.
With a goal of understanding the reasons of silks’ incredible mechanical property,
considerable efforts have been made to understand the molecular structure of spider silk
using different characterization techniques including NMR spectroscopy, X-ray
diffraction, and vibrational spectroscopy. However, the clear structures of Gly-Gly-X
(X=Leu, Tyr, Gln) motifs in MaSp1 other than Gly-Gly-Ala have not been well
understood yet. In Chapters 5, the author focuses on elucidating the secondary structure
of the Gly-Gly-X (X=Leu, Tyr, Gln) motifs in MaSp1 by using advanced solid-state
NMR spectroscopy.

Crystalline Domain Amorphous Domain
Figure 1.7. Illustration of microstructural model for spider silk.

17
1.4.3 Silkworm silk
Silks have been used commercially in textile production for centuries and it is
recognized as one of the most studied protein-based materials in history. Among all
different kinds of silks, the silk spun from the domesticated Bombyx mori (B. mori)
silkworm is the most extensively produced silk that has been used in textile industry
nowadays with an estimated worldwide annual production of more than 200000 tons
according to report by International Sericultural Commission (Figure 1.8). Although the
main economic impact is concentrated in China, India, and Uzbekistan recently, it is
expected to see a worldwide impact in the near future because of the ongoing rapid
development of silk-based products and materials.
Figure 1.8. Total production of silk over the world within the most recent six years.
Over the past 70 years, researchers from a wide range of fields including chemistry,
physics, biology, and engineering have been attracted by silkworm silk and considerable
effort has been put into understanding the properties, structures, and applications of
silkworm silk as evidenced by a booming of research publications on silk related topics

18
[16, 17, 95-101]. Among all studies, a majority of work focuses on B. mori silk since it is
a domesticated silkworm with a large annual production and a popular use in textile
industry for centuries.
The Bombyx mori cocoon is composed of a raw silk fiber with 700-1500 m in
length and 10-20 µm in diameter. The raw silk fiber consists of two parallel fibroin fibers
held together with a layer of sericin on their surfaces [89]. Upon degumming the cocoon
and removing the sericin, soft and shiny fibroin fibers can be obtained and this is the
primary material used in the textile industry. Moreover, it is worthy to note here that most
research has been carried on fibroin fibers rather than raw cocoons. According to
literature, fibroin fibers have a combination of excellent mechanical properties, unique
optical properties, attractive biocompatibility, biodegradability, and thermal stability. Due
to this, they has been recognized as one of the most ideal natural materials for developing
man-made biomaterials and offer unlimited opportunities for functionalization and
biological integration. B. mori silk fibers show impressive mechanical properties when
controllably spun, where the mechanical properties of fibers (strength: 1 GPa, failure
strain: 20%) have been shown to be nearly as impressive spider dragline silk [89].
Recently, researchers have shown that silk fibroin can be utilized and processed into a
variety of material formats including films, hydrogels, sponges, nano- and micro-scale
fibers, and microspheres [99-101].
Silk fibroin consists of a heavy (H) chain of 390 kDa and a light (L) chain of 26
kDa connected by a disulfide bond at the C-terminus of H-chain, forming a H-L complex.
The H-L complex also binds a glycoprotein named P25 (30 kDa) in a ratio of 6:1 via
hydrophobic interactions to form an elementary micellar unit [102-104]. In silk fibroin,
19
the H-chain serves as the main structural component responsible for the excellent
mechanical properties due to its ability to form different kinds of secondary structures,
which could further organized into nano/micro hierarchy structures via molecular
assembly [105]. L-chain plays little mechanical role overall as its size is much smaller
than H-chain, and additionally, no significant secondary structures were identified for L-
chain. The amino acid composition of the heavy chain (in mol%) is dominated by four
amino acids: Gly (46%), Ala (30%), Ser (12%), and Tyr (5.3 %) [106]. The protein
sequence of the heavy chain contains a large number of repetitive sequences that are
organized into 12 domains and the amino acid sequence of one domain is shown in
Figure 1.9. According to the literature, the repetitive sequences can be roughly classified
into three primary motifs: (1) a highly repetitive GAGXGA (X=S, Y, V) sequence that
makes up the bulk of the crystalline/semi-crystalline regions [107, 108]; (2) a relatively
less repetitive GAAS sequence, which may serve as a “sheet-breaking” motif [109]; (3) a
sequence used to separate the domains and form the amorphous regions in fibroin fibers,
e.g., TGSSGFGPYVANGGYSGYEYAWSSESDFGT. It is presumed that the excellent
mechanical properties of silk fibers are closely related to the secondary structures of these
characteristic sequences in silk fibroin, which are further organized into hierarchical
structures. So obtaining a clear molecular picture of the secondary structures in silk
fibroin is very critical for interpreting the superior nature of silk fibroin properly.
GAGAGSGAGAGAGAGAGSGAGAGYGAGVGAGYGAGYGAGAGAGYGAGAGSGTGSG
AGAGSGAGAGYGAGVGAGYGAGAGSGAAFGAGAGAGAGSGAGAGSGAGAGSGAGA
GSGAGAGSGAGAGYGAGYGAGVGAGYGAGAGSGAASGAGAGSGAGAGSGAGAGSG
AGAGSGAGAGSGAGAGYGAGVGAGYGAGYGAGAGAGYGAGAGSGAASGAGAGSGA
GAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSGAGAGYGAGAGSGAAS
GAGAGAGAGAGTGSSGFGPYVANGGYSGYEYAWSSESDFGTGS (Linker)
Figure 1.9. Primary sequences of one domain in B. mori silk fibroin.
20
The secondary structure of silk fibroin has been studied extensively with a variety
of techniques including X-ray diffraction [105, 110-117], solid-state NMR spectroscopy
[16, 17, 97, 107-109, 118-138], and vibrational spectroscopy [110, 139-146] over the past
decades. The combination of these structural characterization techniques has illustrated
that in silk fibers, GAGAGS motif and GAGAGY motif flanking the GAGAGS
repetitive sequences form β-sheet structures and are further organized to form β-sheet
nanocrystallites, while in liquid silk before spinning GAGAGX (X=S, Y, V) was
hypothesized to be in type II β-turn structures [147]. According to the review on
previously reported studies, it is found that very limited effort has been put on elucidating
the structural features of the Tyr residues in comparison to the extensive studies on the
Gly, Ala, and Ser residues which are frequently found in the (GA)n and GAGAGS
repetitive sequences. A thorough study is necessary to get a clear structural understanding
on the Tyr residues since tyrosine is very likely to play important roles in the structure
and dynamics of silk fibroin because of its unique side chain structures. In Chapter 6, the
author focuses on understanding the structural role of the Tyr residues in silk fibroin,
together with a brief structural investigation on the Ala and Ser residues with advanced
solid-state NMR spectroscopy.
1.5 References
[1] F. Bloch, Nuclear Induction, Phys. Rev., 70 (1946) 460-474.
[2] F. Bloch, W.W. Hansen, M. Packard, Nuclear Induction, Phys. Rev., 69 (1946) 127.
[3] E.M. Purcell, H.C. Torrey, R.V. Pound, Resonance absorption by nuclear magnetic
moments in a solid, Phys. Rev., 69 (1946) 37-38.
[4] R.R. Ernst, W.A. Anderson, Application of fourier transform spectroscopy to

magnetic resonance, Rev. Sci. Instrum., 37 (1966) 93-102.
21
[5] W.P. Aue, E. Bartholdi, R.R. Ernst, Two-dimensional spectroscopy. Application to
nuclear magnetic resonance, J. Chem. Phys., 64 (1976) 2229-2246.
[6] A.W. Overhauser, Polarization of nuclei in metals, Phys. Rev., 92 (1953) 411-415.
[7] K. Wuthrich, The way to NMR structures of proteins, Nature Struct. Biol., 8 (2001)
923-925.
[8] E.D. Becker, A brief history of nuclear magnetic resonance., Anal. Chem., 65
(1993) 295A-302A.
[9] E.R. Andrew, A. Bradbury, R.G. Eades, Nuclear magnetic resonance spectra from a
crystal rotated at high speed, Nature, 182 (1958) 1659-1659.
[10] A. Pines, M.G. Gibby, J.S. Waugh, Proton-enhanced NMR of dilute spins in solids,
J. Chem. Phys., 59 (1973) 569-590.
[11] S.R. Hartmann, E.L. Hahn, Nuclear double resonance in the rotating frame, Phys.
Rev., 128 (1962) 2042-2053.
[12] O.N. Antzutkin, J.J. Balbach, R.D. Leapman, N.W. Rizzo, J. Reed, R. Tycko,
Multiple quantum solid-state NMR indicates a parallel, not antiparallel,
organization of beta-sheets in Alzheimer's β-amyloid fibrils, Proc. Natl. Acad. Sci.,
97 (2000) 13045-13050.
[13] R. Tycko, Solid state NMR studies of amyloid fibril structure, Annu. Rev. Phys.
Chem., 62 (2011) 279-299.
[14] F. Creuzet, A. McDermott, R. Gebhard, K. Van Der Hoef, M.B. Spijker-Assink, J.

Herzfeld, J. Lugtenburg, M.H. Levitt, R.G. Griffin, Determination of membrane
protein structure by rotational resonance NMR: bacteriorhodopsin., Science, 251
(1991) 783-786.
[15] O.C. Andronesi, S. Becker, K. Seidel, H. Heise, a. Howard S Young, M. Baldus,

Determination of membrane protein structure and dynamics by magic-angle-
spinning solid-state NMR spectroscopy, J. Am. Chem. Soc., 127 (2005) 12965-
12974.
[16] T. Asakura, Y. Suzuki, Y. Nakazawa, K. Yazawa, G.P. Holland, J.L. Yarger, Silk
structure studied with nuclear magnetic resonance, Prog. Nucl. Magn. Reson.
Spectrosc., 69 (2013) 23-68.
[17] T. Asakura, Y. Suzuki, Y. Nakazawa, G.P. Holland, J.L. Yarger, Elucidating silk
structure using solid-state NMR, Soft Matter, 9 (2013) 11440-11411.
[18] D.A. Torchia, Solid state NMR studies of protein internal dynamics, Ann. Rev.
Biophys. Bioeng., 13 (1984) 125-144.
22
[19] D.D. Laws, H.-M.L. Bitter, A. Jerschow, Solid-state NMR spectroscopic methods
in chemistry Angew. Chem. Int. Ed., 41 (2002) 3096-3129.
[20] C. Dybowski, S. Bai, Solid-state NMR spectroscopy, Anal. Chem., 80 (2008) 4295-
4300.
[21] S. Paasch, E. Brunner, Trends in solid-state NMR spectroscopy and their relevance
for bioanalytics., Anal. Bioanal. Chem., 398 (2010) 2351-2362.
[22] A. Roehrich, G. Drobny, Solid-state NMR studies of biomineralization peptides and

proteins., Acc. Chem. Res., 46 (2013) 2136-2144.
[23] P. Schanda, M. Ernst, Studying dynamics by magic-angle spinning solid-state NMR

spectroscopy: Principles and applications to biomolecules, Prog. Nucl. Magn.
Reson. Spectrosc., 96 (2016) 1-46.
[24] M.J. Duer, Solid state NMR spectroscopy: Principles and applications, John Wiley
& Sons, 2008.
[25] E.R. Andrew, The narrowing of NMR spectra of solids by high-speed specimen
rotation and the resolution of chemical shift and spin multiplet structures for solids,
Prog. Nucl. Magn. Reson. Spectrosc., 8 (1971) 1-39.
[26] B.H. Meier, Cross polarization under fast magic angle spinning: Thermodynamical
considerations, Chem. Phys. Lett., 18 (1992) 201-207.
[27] J. Cavanagh, W.J. Fairbrother, A.G. Palmer, N.J. Skelton, Protein NMR
spectroscopy: Principles and practice, Elsevier Academic Press, 2007.
[28] R.W. Schurko, Ultra-wideline solid-state NMR spectroscopy, Acc. Chem. Res., 46
(2013) 1985-1995.
[29] D.A. Giljohann, D.S. Seferos, W.L. Daniel, M.D. Massich, P.C. Patel, C.A. Mirkin,
Gold nanoparticles for biology and medicine, Angew. Chem. Int. Ed., 49 (2010)
3280-3294.
[30] O.S. Wolfbeis, An overview of nanoparticles commonly used in fluorescent

bioimaging., Chem Soc Rev, 44 (2015) 4743-4768.
[31] T. Vo-Dinh, Y. Liu, A.M. Fales, H. Ngo, H.-N. Wang, J.K. Register, H. Yuan, S.J.
Norton, G.D. Griffin, SERS nanosensors and nanoreporters: golden opportunities in
biomedical applications., WIREs Nanomed. Nanobiotechnol., 7 (2015) 17-33.
[32] K. Saha, S.S. Agasti, C. Kim, X. Li, V.M. Rotello, Gold nanoparticles in chemical
and biological sensing, Chem. Rev., 112 (2012) 2739-2779.
[33] M.A. Hayat, Colloidal gold: Principles, methods, and applications, Elsevier, 2012.
23
[34] R. Wilson, The use of gold nanoparticles in diagnostics and detection, Chem Soc
Rev, 37 (2008) 2028-2019.
[35] R.A. Sperling, P.R. Gil, F. Zhang, M. Zanella, W.J. Parak, Biological applications
of gold nanoparticles, Chem Soc Rev, 37 (2008) 1896-1908.
[36] M. Grzelczak, J. Pérez-Juste, P. Mulvaney, L.M. Liz-Marzán, Shape control in gold

nanoparticle synthesis, Chem Soc Rev, 37 (2008) 1783-1710.
[37] S. Eustis, M.A. El-Sayed, Why gold nanoparticles are more precious than pretty
gold: Noble metal surface plasmon resonance and its enhancement of the radiative
and nonradiative properties of nanocrystals of different shapes, Chem Soc Rev, 35
(2006) 209-217.
[38] M.-C. Daniel, D. Astruc, Gold nanoparticles: Assembly, supramolecular chemistry,

quantum-size-related properties, and applications toward biology, catalysis, and
nanotechnology, Chem. Rev., 104 (2004) 293-346.
[39] J. Turkevich, P.C. Stevenson, J. Hillier, A study of the nucleation and growth
processes in the synthesis of colloidal gold, Discuss. Faraday Soc., 11 (1951) 55-75.
[40] Y. Sun, Y. Xia, Shape-controlled synthesis of gold and silver nanoparticles,

Science, 298 (2002) 2176-2179.
[41] M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R. Whyman, Synthesis of thiol-

derivatised gold nanoparticles in a two-phase liquid–liquid system, J. Chem. Soc.,
Chem. Commun., (1994) 801-802.
[42] C.A. Mirkin, R.L. Letsinger, R.C. Mucic, J.J. Storhoff, A DNA-based method for
rationally assembling nanoparticles into macroscopic materials., Nature, 382 (1996)
607-609.
[43] X. Ji, X. Song, J. Li, Y. Bai, W. Yang, X. Peng, Size control of gold nanocrystals in
citrate reduction: The third role of citrate, J. Am. Chem. Soc., 129 (2007) 13939-
13948.
[44] M.W. Heaven, A. Dass, P.S. White, K.M. Holt, R.W. Murray, Crystal structure of
the gold nanoparticle [N(C8H17)4][Au25(SCH2CH2Ph)18], J. Am. Chem. Soc., 130
(2008) 3754-3755.
[45] Y. Negishi, K. Nobusada, T. Tsukuda, Glutathione-protected gold clusters

revisited: bridging the gap between gold(I)−thiolate complexes and thiolate-
protected gold nanocrystals, J. Am. Chem. Soc., 127 (2005) 5261-5270.
[46] Y. Shichibu, Y. Negishi, H. Tsunoyama, M. Kanehara, T. Teranishi, T. Tsukuda,

Extremely high stability of glutathionate-protected Au25 clusters against core
etching, Small, 3 (2007) 835-839.
24
[47] H. Hinterwirth, S. Kappel, T. Waitz, T. Prohaska, W. Lindner, M. Lämmerhofer,
Quantifying thiol ligand density of self-assembled monolayers on gold
nanoparticles by inductively coupled plasma-mass spectrometry, ACS Nano, 7
(2013) 1129-1136.
[48] N.K. Chaki, Y. Negishi, H. Tsunoyama, Y. Shichibu, T. Tsukuda, Ubiquitous 8 and

29 kda gold:alkanethiolate cluster compounds: Mass-spectrometric determination of
molecular formulas and structural implications, J. Am. Chem. Soc., 130 (2008)
8608-8610.
[49] Z. Wu, C. Gayathri, R.R. Gil, R. Jin, Probing the structure and charge state of
glutathione-capped Au25(SG)18 clusters by NMR and mass spectrometry, J. Am.
Chem. Soc., 131 (2009) 6535-6542.
[50] H. Qian, Y. Zhu, R. Jin, Atomically precise gold nanocrystal molecules with
surface plasmon resonance, Proc. Natl. Acad. Sci. U.S.A., 109 (2012) 696-700.
[51] R. Jin, C. Zeng, M. Zhou, Y. Chen, Atomically precise colloidal metal nanoclusters
and nanoparticles: Fundamentals and opportunities, Chem. Rev., 116 (2016) 10346-
10413.
[52] A. Das, T. Li, K. Nobusada, C. Zeng, N.L. Rosi, R. Jin, Nonsuperatomic

[Au23(SC6H11)16]− nanocluster featuring bipyramidal Au15 kernel and trimeric
Au3(SR)4 motif, J. Am. Chem. Soc., 135 (2013) 18264-18267.
[53] T. Dainese, S. Antonello, J.A. Gascón, F. Pan, N.V. Perera, M. Ruzzi, A. Venzo, A.
Zoleo, K. Rissanen, F. Maran, Au25(SEt)18, a nearly naked thiolate-protected Au25
cluster: Structural analysis by single crystal X-ray crystallography and electron
nuclear double resonance, ACS Nano, 8 (2014) 3904-3912.
[54] C. Zeng, T. Li, A. Das, N.L. Rosi, R. Jin, Chiral structure of thiolate-protected 28-
gold-atom nanocluster determined by X-ray crystallography, J. Am. Chem. Soc.,
135 (2013) 10011-10013.
[55] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Structure of a thiol
monolayer-protected gold nanoparticle at 1.1Å resolution, Science, 318 (2007) 426-
430.
[56] D.W. Schaefer, K.D. Keefer, Structure of random porous materials: Silica aerogel,
Phys. Rev. Lett., 56 (1986) 2199-2202.
[57] D. Tarn, C.E. Ashley, M. Xue, E.C. Carnes, J.I. Zink, C.J. Brinker, Mesoporous
silica nanoparticle nanocarriers: biofunctionality and biocompatibility, Acc. Chem.
Res., 46 (2013) 792-801.
25
[58] H. Maleki, L. Durães, C.A. García-González, P. del Gaudio, A. Portugal, M.
Mahmoudi, Synthesis and biomedical applications of aerogels: Possibilities and
challenges, Adv. Colloid Interface Sci., 236 (2016) 1-27.
[59] F. Tang, L. Li, D. Chen, Mesoporous silica nanoparticles: Synthesis,

biocompatibility and drug delivery, Adv. Mater., 24 (2012) 1504-1534.
[60] J.A. Hubbell, A. Chilkoti, Nanomaterials for drug delivery, Science, 337 (2012)
303-305.
[61] C.E. Ashley, E.C. Carnes, G.K. Phillips, D. Padilla, P.N. Durfee, P.A. Brown, T.N.
Hanna, J. Liu, B. Phillips, M.B. Carter, N.J. Carroll, X. Jiang, D.R. Dunphy, C.L.
Willman, D.N. Petsev, D.G. Evans, A.N. Parikh, B. Chackerian, W. Wharton, D.S.
Peabody, C.J. Brinker, The targeted delivery of multicomponent cargos to cancer
cells by nanoporous particle-supported lipid bilayers, Nat. Mater., 10 (2011) 389-
397.
[62] C.-H. Lee, T.-S. Lin, C.-Y. Mou, Mesoporous materials for encapsulating enzymes,
Nano Today, 4 (2009) 165-179.
[63] S. Hudson, J. Cooney, E. Magner, Proteins in mesoporous silicates, Angew. Chem.

Int. Ed., 47 (2008) 8582-8594.
[64] C. Bharti, U. Nagaich, A.K. Pal, N. Gulati, Mesoporous silica nanoparticles in

target drug delivery system: A review., Int J Pharm Investig, 5 (2015) 124-133.
[65] S. Kwon, R.K. Singh, R.A. Perez, E.A. Abou Neel, H.-W. Kim, W. Chrzanowski,
Silica-based mesoporous nanoparticles for controlled drug delivery, J Tissue Eng, 4
(2013) 2041731413503357.
[66] I.I. Slowing, J.L. Vivero-Escoto, C.-W. Wu, V.S.-Y. Lin, Mesoporous silica
nanoparticles as controlled release drug delivery and gene transfection carriers.,
Adv. Drug Deliv. Rev., 60 (2008) 1278-1288.
[67] W. Stöber, A. Fink, E. Bohn, Controlled growth of monodisperse silica spheres in

the micron size range, J. Colloid Interface Sci., 26 (1968) 62-69.
[68] D.W. Schaefer, A.J. Hurd, Growth and structure of combustion aerosols: Fumed
silica, Aerosol Sci. Technol., 12 (1990) 876-890.
[69] R.I. Nooney, D. Thirunavukkarasu, Y. Chen, a. Robert Josephs, A.E. Ostafin,

Synthesis of nanoscale mesoporous silica spheres with controlled particle size,
Chem. Mater., 14 (2002) 4721-4728.
[70] C.C. Liu, G.E. Maciel, The fumed silica surface: A study by NMR, J. Am. Chem.
Soc., 118 (1996) 5103-5119.
26
[71] M. Zaborski, A. Vidal, G. Ligner, H. Balard, E. Papirer, A. Burneau, Comparative
study of the surface hydroxyl groups of fumed and precipitated silicas. I. Grafting
and chemical characterization, Langmuir, 5 (1990) 447-451.
[72] J.P. Gallas, J.C. Lavalley, A. Burneau, O. Barres, Comparative study of the surface
hydroxyl groups of fumed and precipitated silicas. 4. Infrared study of
dehydroxylation by thermal treatments, Langmuir, 7 (1990) 1235-1240.
[73] A. Burneau, O. Barres, J.P. Gallas, J.C. Lavalley, Comparative study of the surface
hydroxyl groups of fumed and precipitated silicas. 2. Characterization by infrared
spectroscopy of the interactions with water, Langmuir, 6 (1990) 1364-1372.
[74] H. Zhang, D.R. Dunphy, X. Jiang, H. Meng, B. Sun, D. Tarn, M. Xue, X. Wang, S.
Lin, Z. Ji, R. Li, F.L. Garcia, J. Yang, M.L. Kirk, T. Xia, J.I. Zink, A. Nel, C.J.
Brinker, Processing pathway dependence of amorphous silica nanoparticle toxicity:
colloidal vs pyrolytic, J. Am. Chem. Soc., 134 (2012) 15790-15804.
[75] W.A. Shear, J.M. Palmer, J.A. Coddington, P.M. Bonamo, A devonian spinneret:
Early evidence of spiders and silk use, Science, 246 (1989) 479-481.
[76] R.V. Lewis, Spider silk: Ancient ideas for new biomaterials, Chem. Rev., 106
(2006) 3762-3774.
[77] F. Vollrath, D. Porter, Spider silk as archetypal protein elastomer, Soft Matter, 2
(2006) 377-385.
[78] C.Y. Hayashi, R.V. Lewis, Evidence from flagelliform silk cDNA for the structural
basis of elasticity and modular nature of spider silks., J. Mol. Biol., 275 (1998) 773-
784.
[79] M. Xu, R.V. Lewis, Structure of a protein super fiber: Spider dragline silk, Proc.
Natl. Acad. Sci., 87 (1990) 7120-7124.
[80] O. Emile, A.L. Floch, F. Vollrath, Biopolymers: Shape memory in spider draglines,
Nature, 440 (2006) 621-621.
[81] P.M. Cunniff, S.A. Fossey, M.A. Auerbach, J.W. Song, D.L. Kaplan, W.W. Adams,
R.K. Eby, D. Mahoney, D.L. Vezie, Mechanical and thermal properties of dragline
silk from the spider Nephila clavipes, Polymer. Adv. Tech., 5 (1994) 401-410.
[82] J.M. Gosline, P.A. Guerette, C.S. Ortlepp, K.N. Savage, The mechanical design of
spider silks: From fibroin sequence to mechanical function., J. Exp. Biol., 202
(1999) 3295-3303.
[83] C.J. Benmore, T. Izdebski, J.L. Yarger, Total X-ray scattering of spider dragline
silk, Phys. Rev. Lett., 108 (2012) 178102-178104.
27
[84] G.P. Holland, M.S. Creager, J.E. Jenkins, R.V. Lewis, J.L. Yarger, Determining
secondary structure in spider dragline silk by carbon−carbon correlation solid-state
NMR spectroscopy, J. Am. Chem. Soc., 130 (2008) 9871-9877.
[85] C. Riekel, M. Müller, F. Vollrath, In situ X-ray diffraction during forced silking of
spider silk, Macromolecules, 32 (1999) 4464-4466.
[86] C. Riekel, C. Branden, C. C, C. Ferrero, F. Heidelbach, M. Muller, Aspects of X-

ray diffraction on single spider fibers, Int. J. Biol. Macromol., 24 (1999) 179-186.
[87] B.L. Thiel, C. Viney, L.W. Jelinski, β-sheets and spider silk, Science, 273 (1996)
1480-1481.
[88] R.V. Lewis, Spider silk: the unraveling of a mystery, Acc. Chem. Res., 25 (1992)
392-398.
[89] Z. Shao, F. Vollrath, Surprising strength of silkworm silk, Nature, 418 (2002) 741-
741.
[90] C. Vepari, D.L. Kaplan, Silk as a biomaterial, Prog. Polym. Sci., 32 (2007) 991-
1007.
[91] L. Yang, K.O. van der Werf, C.F.C. Fitié, M.L. Bennink, P.J. Dijkstra, J. Feijen,
Mechanical properties of native and cross-linked type I collagen fibrils, Biophys. J.,
94 (2008) 2204-2211.
[92] J.E. Jenkins, M.S. Creager, E.B. Butler, R.V. Lewis, J.L. Yarger, G.P. Holland,
Solid-state NMR evidence for elastin-like β-turn structure in spider dragline silk,
Chem. Commun., 46 (2010) 6714-6713.
[93] D.T. Grubb, L.W. Jelinski, Fiber morphology of spider silk: the effects of tensile
deformation, Macromolecules, 30 (1997) 2860-2867.
[94] S. Keten, Z. Xu, B. Ihle, M.J. Buehler, Nanoconfinement controls stiffness, strength
and mechanical toughness of beta-sheet crystals in silk, Nat. Mater., 9 (2010) 359-
367.
[95] J.G. Hardy, L.M. Römer, T.R. Scheibel, Polymeric materials based on silk proteins,
Polymer, 49 (2008) 4309-4327.
[96] L.-D. Koh, Y. Cheng, C.-P. Teng, Y.-W. Khin, X.-J. Loh, S.-Y. Tee, M. Low, E.
Ye, H.-D. Yu, Y.-W. Zhang, M.-Y. Han, Structures, mechanical properties and
applications of silk fibroin materials, Prog. Polym. Sci., 46 (2015) 86-110.
[97] T. Asakura, K. Okushita, M.P. Williamson, Analysis of the structure of Bombyx

mori silk fibroin by NMR, Macromolecules, 48 (2015) 2345-2357.
28
[98] R.F.P. Pereira, M.M. Silva, V. de Zea Bermudez, Bombyx mori silk fibers: an
outstanding family of materials, Macromol. Mater. Eng., 300 (2014) 1171-1198.
[99] B. Kundu, R. Rajkhowa, S.C. Kundu, X. Wang, Silk fibroin biomaterials for tissue
regenerations, Adv. Drug Deliv. Rev., 65 (2013) 457-470.
[100] H. Tao, D.L. Kaplan, F.G. Omenetto, Silk materials - a road to sustainable high
technology, Adv. Mater., 24 (2012) 2824-2837.
[101] D.N. Rockwood, R.C. Preda, T. Yücel, X. Wang, M.L. Lovett, D.L. Kaplan,
Materials fabrication from Bombyx mori silk fibroin, Nature Protocols, 6 (2011)
1612-1631.
[102] C. Zhou, F. Confalonieri, M. Jacquet, R. Perasso, Z. Li, J. Janin, Silk fibroin:

structural implications of a remarkable amino acid sequence, Proteins, 44 (2001)
119-122.
[103] K. Yamaguchi, Y. Kikuchi, T. Takagi, A. Kikuchi, F. Oyama, K. Shimura, S.

Mizuno, Primary structure of the silk fibroin light chain determined by cDNA
sequencing and peptide analysis, J. Mol. Biol., 210 (1989) 127-139.
[104] P. Nony, J.C. Prudhomme, P. Couble, Regulation of the P25 gene transcription in
the silk gland of Bombyx, Biol. Cell, 84 (1995) 43-52.
[105] R.E. Marsh, R.B. Corey, L. Pauling, An invesitgation of the structure of silk
fibroin, Biochim. Biophys. Acta, 16 (1955) 1-34.
[106] W.A. Schroeder, L.M. Kay, B. Lewis, N. Munger, The amino acid composition of
Bombyx mori silk fibroin and of tussah silk fibroin, J. Am. Chem. Soc., 77 (1955)
3908-3913.
[107] T. Asakura, J. Yao, T. Yamane, K. Umemura, A.S. Ulrich, Heterogeneous structure

of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy, J.
Am. Chem. Soc., 124 (2002) 8794-8795.
[108] T. Asakura, J. Yao, 13C CP/MAS NMR study on structural heterogeneity in

Bombyx mori silk fiber and their generation by stretching, Protein Sci., 11 (2002)
2706-2713.
[109] T. Asakura, R. Sugino, T. Okumura, Y. Nakazawa, The role of irregular unit,

GAAS, on the secondary structure of Bombyx mori silk fibroin studied with 13C
CP/MAS NMR and wide-angle X-ray scattering, Protein Sci., 11 (2002) 1873-1877.
[110] A. Martel, M. Burghammer, R.J. Davies, E. Di Cola, C. Vendrely, C. Riekel, Silk

fiber assembly studied by synchrotron radiation SAXS/WAXS and Raman
spectroscopy, J. Am. Chem. Soc., 130 (2008) 17070-17074.
29
[111] L.F. Drummy, B.L. Farmer, R.R. Naik, Correlation of the β-sheet crystal size in
silk fibers with the protein amino acid sequence, Soft Matter, 3 (2007) 877-882.
[112] T. Asakura, T. Yamane, Y. Nakazawa, T. Kameda, K. Ando, Structure of Bombyx

mori silk fibroin before spinning in solid state studied with wide angle X-ray
scattering and 13C cross-polarization/magic angle spinning NMR, Biopolymers, 58
(2001) 521-525.
[113] Y. Takahashi, M. Gehoh, K. Yuzuriha, Structure refinement and diffuse streak

scattering of silk (Bombyx mori), Int. J. Biol. Macromol., 24 (1999) 127-138.
[114] M. Demura, M. Minami, a. Tetsuo Asakura, T.A. Cross, Structure of Bombyx mori
silk fibroin based on solid-state NMR orientational constraints and fiber diffraction
unit cell parameters, J. Am. Chem. Soc., 120 (1998) 1300-1308.
[115] B. Lotz, H.D. Keith, Crystal structure of poly(L-Ala-Gly)II: a model for silk I, J.
Mol. Biol., 61 (1971) 201-215.
[116] M.G. Dobb, R.D.B. Fraser, T.P. Macrae, The fine structure of silk fibroin, J. Cell
Biol., 32 (1967) 289-295.
[117] R.D. Fraser, T.P. Macrae, F.H. Stewart, Poly-L-alanylglycyl-L-alanylglycyl-L-

serylglycine: a model for the crystalline regions of silk fibroin., J. Mol. Biol., 19
(1966) 580-582.
[118] E. Callone, S. Dirè, X. Hu, A. Motta, Processing influence on molecular

assembling and structural conformations in silk fibroin: elucidation by solid-state
NMR, ACS Biomater. Sci. Eng., 2 (2016) 758-767.
[119] S.-W. Ha, H.S. Gracz, A.E. Tonelli, S.M. Hudson, Structural study of irregular
amino acid sequences in the heavy chain of Bombyx mori silk fibroin,
Biomacromolecules, 6 (2005) 2563-2569.
[120] T. Asakura, K. Ohgo, K. Komatsu, M. Kanenari, K. Okuyama, Refinement of

repeated β-turn structure for silk I conformation of Bombyx mori silk fibroin using
13
C solid-state NMR and X-ray diffraction methods, Macromolecules, 38 (2005)
7397-7403.
[121] T. Asakura, K. Ohgo, T. Ishida, P. Taddei, P. Monti, R. Kishore, Possible

implications of serine and tyrosine residues and intermolecular interactions on the
appearance of silk I structure of Bombyx mori silk fibroin-derived synthetic
peptides: high-resolution 13C cross-polarization/magic-angle spinning NMR study,
[122] T. Asakura, Y. Nakazawa, E. Ohnishi, F. Moro, Evidence from 13C solid-state

NMR spectroscopy for a lamella structure in an alanine-glycine copolypeptide: A
30
model for the crystalline domain of Bombyx mori silk fiber, Protein Sci., 14 (2005)
2654-2657.
[123] J. Yao, K. Ohgo, R. Sugino, R. Kishore, T. Asakura, Structural analysis of Bombyx

mori silk fibroin peptides with formic acid treatment using high-resolution solid-
state 13C NMR spectroscopy, Biomacromolecules, 5 (2004) 1763-1769.
[124] J. Yao, Y. Nakazawa, T. Asakura, Structures of Bombyx mori and Samia cynthia
ricini silk fibroins studied with solid-state NMR, Biomacromolecules, 5 (2004)
680-688.
[125] T. Asakura, K. Suita, T. Kameda, S. Afonin, A.S. Ulrich, Structural role of tyrosine
in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics
on a model peptide prepared as silk I and II., Magn. Reson. Chem., 42 (2004) 258-
266.
[126] T. Kameda, Y. Nakazawa, J. Kazuhara, T. Yamane, T. Asakura, Determination of

intermolecular distance for a model peptide of Bombyx mori silk fibroin, GAGAG,
with rotational echo double resonance, Biopolymers, 64 (2002) 80-85.
[127] T. Asakura, R. Sugino, J. Yao, H. Takashima, R. Kishore, Comparative structure

analysis of tyrosine and valine Residues in unprocessed silk fibroin (Silk I) and in
the processed silk fiber (Silk II) from Bombyx mori using solid-state 13C, 15N, and
2
H NMR, Biochemistry, 41 (2002) 4415-4424.
[128] T. Asakura, J. Ashida, T. Yamane, T. Kameda, Y. Nakazawa, K. Ohgo, K.

Komatsu, A repeated β-turn structure in poly(Ala-Gly) as a model for silk I of
Bombyx mori silk fibroin studied with two-dimensional spin-diffusion NMR under
off magic angle spinning and rotational echo double resonance, J. Mol. Biol., 306
(2001) 291-305.
[129] H. Kimura, S. Kishi, A. Shoji, H. Sugisawa, K. Deguchi, Characteristic 1H

chemical shifts of silk fibroins determined by 1H CRAMPS NMR, Macromolecules,
33 (2000) 9682-9687.
[130] T. Kameda, Y. Ohkawa, K. Yoshizawa, E. Nakano, T. Hiraoki, A.S. Ulrich, T.

Asakura, Dynamics of the tyrosine side chain in Bombyx mori and Samia cynthia
ricini silk fibroin studied by solid state 2H NMR, Macromolecules, 32 (1999) 8491-
8495.
[131] T. Kameda, Y. Ohkawa, K. Yoshizawa, J. Naito, A.S. Ulrich, T. Asakura,

Hydrogen-bonding structure of serine side chains in Bombyx mori and Samia
cynthia ricini silk fibroin determined by solid-state 2H NMR, Macromolecules, 32
(1999) 7166-7171.
[132] S.J. He, R. Valluzzi, S.P. Gido, Silk I structure in Bombyx mori silk foams, Int. J.
Biol. Macromol., 24 (1999) 187-195.
31
[133] T. Asakura, M. Minami, R. Shimada, M. Demura, M. Osanai, T. Fujito, M.
Imanari, A.S. Ulrich, 2H-labeling of silk fibroin fibers and their structural
characterization by solid-state 2H NMR, Macromolecules, 30 (1997) 2429-2435.
[134] T. Asakura, M. Demura, T. Date, N. Miyashita, K. Ogawa, M.P. Williamson, NMR

study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR
chemical shift contour plots, Biopolymers, 41 (1997) 193-203.
[135] M. Ishida, T. Asakura, M. Yokoi, H. Saito, Solvent- and mechanical-treatment-

induced conformational transition of silk fibroins studied by high-resolution solid-
state 13C NMR spectroscopy, Macromolecules, 23 (1990) 88-94.
[136] H. Saito, Conformation-dependent 13C chemical shifts: A new means of

conformational characterization as obtained by high-resolution solid-state 13C
NMR, Magn. Reson. Chem., 24 (1986) 835-852.
[137] T. Asakura, A. Kuzuhara, T. Ryoko, S. Hazime, Conformational characterization of

Bombyx mori silk fibroin in the solid state by high-frequency 13C cross polarization-
magic angle spinning NMR, X-ray diffraction, and infrared spectroscopy,
Macromolecules, 18 (1985) 1841-1845.
[138] H. Saito, R. Tabeta, T. Asakura, Y. Iwanaga, A. Shoji, T. Ozaki, I. Ando, High-

resolution 13C NMR study of silk fibroin in the solid state by the cross-
polarization-magic angle spinning method. Conformational characterization of silk I
and silk II type forms of Bombyx mori fibroin by the conformation-dependent 13C
chemical shifts, Macromolecules, 17 (1984) 1405-1412.
[139] F. Paquet-Mercier, T. Lefèvre, M.l. Auger, M. Pézolet, Evidence by infrared

spectroscopy of the presence of two types of β-sheets in major ampullate spider silk
and silkworm silk, Soft Matter, 9 (2013) 208-215.
[140] S. Ling, Z. Qi, D.P. Knight, Z. Shao, X. Chen, Synchrotron FTIR

microspectroscopy of single natural silk fibers, Biomacromolecules, 12 (2011)
3344-3349.
[141] T. Lefèvre, F. Paquet-Mercier, S. Lesage, M.-E. Rousseau, S. Bédard, M. Pézolet,

Study by Raman spectromicroscopy of the effect of tensile deformation on the
molecular structure of Bombyx mori silk, Vib. Spectrosc, 51 (2009) 136-141.
[142] E. Van Nimmen, K. De Clerck, J. Verschuren, K. Gellynck, T. Gheysens, J.

Mertens, L. Van Langenhove, FT-IR spectroscopy of spider and silkworm silks,
Vib. Spectrosc, 46 (2008) 63-68.
[143] T. Lefèvre, M.-E. Rousseau, M. Pézolet, Protein secondary structure and

orientation in silk as revealed by Raman spectromicroscopy, Biophys. J., 92 (2007)
2885-2895.
32
[144] P. Taddei, P. Monti, Vibrational infrared conformational studies of model peptides
representing the semicrystalline domains of Bombyx mori silk fibroin, Biopolymers,
78 (2005) 249-258.
[145] P. Taddei, T. Asakura, J. Yao, P. Monti, Raman study of poly(alanine-glycine)-

based peptides containing tyrosine, valine, and serine as model for the
semicrystalline domains of Bombyx mori silk fibroin, Biopolymers, 75 (2004) 314-
324.
[146] M.-E. Rousseau, T. Lefèvre, L. Beaulieu, T. Asakura, M. Pézolet, Study of protein

conformation and orientation in silkworm and spider silk fibers using Raman
microspectroscopy, Biomacromolecules, 5 (2004) 2247-2257.
[147] Y. Suzuki, T. Yamazaki, A. Aoki, H. Shindo, T. Asakura, NMR study of the

structures of repeated sequences, GAGXGA (X = S, Y, V), in Bombyx mori liquid
silk, Biomacromolecules, 15 (2014) 104-112.
33
CHAPTER 2
Characterizing Gold Nanoparticles by NMR Spectroscopy
2.1 Introduction
Noble metal nanomaterials, such as gold/silver nanomaterials have been studied
extensively in recent years because of their wide applications in catalysis, surface-
enhanced Raman scattering, biological imaging, drug delivery, and cancer therapy [1-11].
Gold nanoparticles (AuNPs) can be synthesized and functionalized with a variety of
ligands through Au-S chemical bonds or electrostatic interaction [2, 12-22]. These
ligands control the nucleation and growth of nanoparticles and provide chemical and
colloidal stability as well [2, 22-24]. Besides developing synthetic methods for AuNPs,
physical characterization of AuNPs including the size, morphology, optical property and
surface chemistry is another focus in AuNPs research. Many techniques have been
developed in the past decades and utilized to investigate the morphologies, structures,
optical properties and surface chemistry of nanoparticles [2, 25, 26]. Transmission
electron microscopy (TEM) and scanning electron microscopy (SEM) are able to
determine the size and to investigate morphology while optical spectroscopies such as
UV-vis spectroscopy, fluorescent spectroscopy, Raman spectroscopy, and IR
spectroscopy are used for understanding the optical properties and surface chemistry of
gold nanomaterials. Recently, mass spectroscopy and single crystal X-ray crystallography
have been demonstrated to be excellent tools for characterizing gold nanoclusters. Mass
spectroscopy has been used for investigating the ligand density of nanostructures,
molecular formula for molecular-like nanoclusters and charge state of nanoclusters [27-
30]. Single crystal X-ray crystallography not only can determine the size of
34
nanostructures but also the exact stacking patterns of atoms [5]. However, this technique
requires sufficient very good quality single crystals to give well-defined diffractions,
which limits the technique to be utilized extensively. Until recently, only the structures of
Au23(SC6H11)16 [31], Au25(PET)18 [32], Au25(SEt)18 [33], Au28(TBBT)20 [34], Au36(SPh-
tBu)24 [35], Au38(SR)24 [36], and Au102(p-MBA)44 [37] has been successfully determined
by X-ray crystallography. Comparing with all these techniques, nuclear magnetic
resonance (NMR) spectroscopy has been demonstrated to be a universal and versatile
technique for the characterization of nanomaterials [38, 39]. One- and multi-dimensional
NMR spectroscopy combined with homo- and hetero-nuclear NMR spectroscopy
provides considerable methods for characterizing nanostructures including purity, ligand
density and surface chemistry [29, 40-47]. Furthermore, diffusion-ordered NMR
spectroscopy (DOSY) [48] is able to determine the size of nanoparticles in solution state
[15, 49-51].
In this work, we demonstrate that NMR spectroscopy can be applied as a
universal tool for characterizing different types of gold nanoparticles, determining the
purity, size, structure of ligands, and Au/ligand ratio. Briefly, 1H direct, two-dimensional
(2D) correlation spectroscopy (COSY), and 2D heteronuclear single quantum correlation
(HSQC) spectroscopy were performed to investigate the ligand structures on the surfaces
and to determine the purity and Au/ligand ratio. DOSY was applied to measure the
diffusion coefficient of ligands binding to the nanostructures, which is further used to
estimate the diameter of nanoparticles. By combining different kinds of NMR
spectroscopy techniques, the purity, size and surface chemistry of gold nanoparticles can
be characterized very efficiently in time and cost.
35
2.2 Materials and Methods
2.2.1 Chemicals and Materials
Gold(III) chloride hydrate (HAuCl43H2O, 99.999 %), octanethiol (C8H17SH,
98.5%), tetraoctylammonium bromide (TOAB, 98%), sodium borohydride (NaBH4, ≥
99%), L-glutathione, reduced (≥ 99%, Aldrich), toluene (ACS reagent, ≥ 99.5 %),
methanol (ACS reagent, ≥ 99.8 %), ethanol (ACS reagent, ≥ 99.5 %), acetonitrile (ACS
reagent, ≥ 99.5 %), and isopropyl alcohol (ACS reagent, ≥ 99.5 %) were purchased from
Sigma Aldrich and all chemicals were used as received. Deuterated chloroform (CDCl3,
99.9%) and deuterated water (D2O, 99.9%) were purchased from Cambridge Isotope Inc.
and was used as received.
2.2.2 Synthesis of Octanethiol-capped Gold Nanoparticles (C8H17S-AuNPs)
C8H17S-AuNPs were prepared as follows. Briefly, an aqueous solution of
HAuCl43H2O (39.4 mg, 0.10 mmol, 5 mL, 1 equiv) was mixed with a solution of TOAB
in toluene (136.7 mg in 5 mL, 0.25 mmol, 2.5 equiv). The two-phase mixture was rapidly
stirred until Au3+ was all transferred to the organic phase to give a wine-red solution. The
[TOA][AuX4] was produced with a mix of Cl- and Br- ions. After removing the aqueous
phase, the organic phase was cooled to ~ 0 °C in the ice bath, followed by adding in
C8H17SH (70 µL, 0.40 mmol, 4 equiv). The solution was then slowly stirred for about 2
hours until the solution became clear. A freshly prepared ice-cold aqueous solution of
NaBH4 (2 mL, 0.5 mol/L, 1.0 mmol) was added to the reaction mixture with vigorous
stirring. After 3 h of reaction, the ice-bath was removed, and the solution was
continuously stirred for over 24 h. The long aging time will influences the purity and
yield of Au clusters markedly. After reaction was done, the organic phase was separated
36
out and dried under vacuum. The dry product was then washed with methanol and
ethanol three times respectively to remove excess thiols and byproducts. The pure
C8H17S-AuNPs were extracted with pure chloroform.
2.2.3 Synthesis of Glutathione-capped Gold Nanoparticles (GS-AuNPs)
In a typical synthesis, an aqueous solution of 20 mM HAuCl4 (5 mL) and 50 mM
glutathione (GSH, 3 mL). The mixture was then vigorously stirred for 2 min until the
yellowish solution turned cloudy. After being stirred, 1.0 mL 1.0 M NaOH was added to
the solution to adjust the pH to basic condition, which made the color of the solution turn
clear yellow. Thereafter, diluted 40 µL of NaBH4 (35 mM) was slowly added dropwise.
The solution slowly turned orange in the first several minutes. After stirring for 30 mins,
1.0 mL 1.0 M HCl was introduced to the solution to readjust the pH to acidic condition,
which quench the BH4- activity and stirred slowly (150 rpm) for overnight at room
temperature. The GS-AuNPs were purified from the raw product using water-IPA
mixtures. Typically, the raw solution was mixed with 12 mL IPA to induce the
precipitation of the byproducts. After that, the supernatant fluid was separated by
centrifugation and 2.0 mL of IPA was added to the supernatant to induce additional
precipitation. Then the GS-AuNPs were further extract from the precipitation using
ultrapure water.
2.2.4 TEM and UV-vis Spectroscopy Characterizations
TEM images were taken using CM200-FEG high-resolution transmission electron
microscope operating at a bias voltage of 200 kV. The TEM samples were prepared by
evaporating diluted nanoparticle solution on the carbon-coated copper grid. Images were
analyzed using ImageJ software. UV-vis and luminescence spectra were collected using
37
Vernier UV-vis spectrometer and 1.0 mL of diluted solution was used in each
experiment.
2.2.5 Thermal Gravimetric Analysis (TGA)
TGA experiments were performed on a TA2910 (TA Instrument Inc.) under N2
flow (40 mL/min for furnace and 30 mL/min for balance). The heating rate was 5 °C/min,
and 1-2 mg of the sample was used. Before experiment, the sample was kept under a N2
flow for 10 min to remove most of the physisorbed water and obtain a stable baseline.
2.2.6 NMR Spectroscopy
The gold nanoparticles and pure thiols were dissolved in deuterated solvents,
resulting in 600 µL solution for each sample. All NMR tubes were flame sealed to
prevent solvent evaporation and concentration change during the characterization.
Nuclear magnetic resonance (NMR) analysis was conducted on a Varian 500
spectrometer equipped with 1H/13C/15N 5 mm XYZ PFG triple-resonance probe using
standard VNMRJ software. All experiments were performed with 1H 90° pulse of 7.30 µs
13
and C 90° pulse of 31.0 µs at 298.15 K. One dimensional (1D) 1H spectra were
collected with a sweep width of 8012.8, an acquisition time of 2.045 s, a recycle delay of
2.5 s, and 128 scans and 8 scans for gold nanoparticles and pure thiols respectively. In the
quantitative NMR experiment, 64 scans and a recycle delay of 60 s were applied and the
1
measurement was repeated three times. H-1H gradient correlation spectroscopy
(gCOSY) were performed with a sweep width of 8012.8 Hz, an acquisition time of 150
ms, 128 points in the indirect dimension and 128 scans per increment, a recycle delay of
1 s. 1H-13C gradient heteronuclear single quantum correlation (gHSQC) spectroscopy was
performed with a sweep width of 8012.8 Hz, an acquisition time of 150 ms, 128 points in
38
the indirect dimension and 512 scans per increment, a recycle delay of 1 s. Diffusion
ordered NMR spectroscopy (DOSY) measurements were performed with DOSY bipolar
pulse pair stimulated echo (dbppse) pulse sequence [48]. A pulsed gradient duration δ of
2.0 ms incremented from 2.9 to 64.8 G cm-1 in 32 steps and a pulsed gradient separation
Δ of 50 ms were used in the measurements. The spectra were collected with a sweep
width of 8012.8 Hz, an acquisition time of 2.045 s, 32 scans, and a recycle delay of 10 s.
The reported spectra and diffusion coefficients were obtained using the DOSY toolbox in
VNMRJ software. A viscosity (η) value of 0.537 mPa and a self-diffusion coefficient
value of 2.23 × 10-9 m2 s-1 were used for chloroform and a viscosity (η) value of 0.890
mPa and a self-diffusion coefficient value of 2.300 × 10-9 m2 s-1 were used for water in
gradient field calibration and diffusion coefficient calculations for gold nanoparticles
samples at 298.15 K [52].
2.3 Results and Discussion
2.3.1 Synthesis of Gold Nanoparticles
In this work, two types of thiol-capped gold nanoparticles, C8H17S-AuNPs and
GS-AuNPs were prepared. However, their physical and chemical properties are
significantly different, C8H17S-AuNPs are organic solvent favorable AuNPs while GS-
AuNPs are water-soluble and biocompatible AuNPs because glutathione is a tri-peptide
(Gly-Cys-Glu). These two types of AuNPs were prepared in this work for better
demonstrating the general concept that NMR spectroscopy can be applied to characterize
gold nanoparticles regardless of their physical and chemical properties. More than that,
GS-AuNPs have great potential in biomedical imaging and drug delivery research due to
its biocompatibility and unique luminescent properties [53, 54]. However few NMR
39
investigations have been carried out on this material. In this work, we show some NMR
studies on the luminescent GS-AuNPs.
Figure 2.1. UV-Vis spectra of purified C8H17S-AuNPs and GS-AuNPs (solid lines).
Luminescence spectrum of GS-AuNPs (red dashed) with an excitation wavelength of 500 nm.
Inset shows the pictures of GS-AuNPs under room light (a) and UV light (365 nm) (b).
(a) (b)
Figure 2.2. TEM images of C8H17S-AuNPs (a) and GS-AuNPs (b) respectively.
40
Figure 2.3. 1H NMR spectra of (a) C8H17SH and C8H17S-AuNPs and (b) GSH and GS-AuNPs. *
notes the water (1.56 ppm) and isopropyl alcohol (1.1 ppm).
The as-prepared AuNPs were characterized with UV-vis/luminescent
spectroscopy, TEM and 1H NMR spectroscopy to verify the success of the synthesis and
the purity of the materials (Figure 2.1-2.3). From the UV-vis absorption spectra (Figure
41
2.1), C8H17S-AuNPs showed step-like multiple bands, indicating it is semiconducting and
no surface plasmon resonance (SPR) band can be observed clearly in the optical spectra
[49]. For GS-AuNPs, two absorption peaks were observed (450 nm and 515 nm) which is
in good agreement with the characteristic values for luminescent gold nanoclusters
reported in previous literatures [14, 17, 54]. Furthermore, the GS-AuNPs show red
emitting under UV light of 365 nm and the luminescent spectrum was collected in this
work. The emission wavelength was determined to be a broad band with a peak value of
700 nm. TEM was carried out to determine the size and investigate the morphologies of
the AuNPs as well. Both kinds of AuNPs have a well-defined spherical shape with
diameters of 2.50 ± 0.49 nm and 1.76 ± 0.47 nm for C8H17S-AuNPs and GS-AuNPs
respectively (Figure 2.2). The diameters of AuNPs were determined by analyzing 100
particles for each sample in corresponding TEM profiles. 1D 1H NMR spectroscopy was
applied to check the purity of the prepared AuNPs and further obtain some chemical shift
information of the ligands binding to the AuNPs (Figure 2.3). The results indicate that 1H
resonances of thiols binding to the AuNPs surfaces were significantly broadened or
shifted or even completely disappeared for both types of AuNPs. For C8H17S-AuNPs,
The 1H resonances of the protons attached to C1 completely disappeared and the 1H
resonances of the protons attached to C2 was shifted and seriously broadened. For other
protons, they showed little chemical shift changes but significant broadening comparing
to the pure thiol. The chemical shift change is due to the structure change of capping
groups, in which C8H17SH forms Au-S with interfacial gold atoms by eliminating one
proton. This affects protons attached to C1 and C2 primarily. The NMR resonance
broadening is primarily due to the distribution of the chemical environment (such as
42
C8H17S-Au bonding) and this has been studied and discussed in details in previous
research [40, 41]. For GS-AuNPs, besides the chemical shift changes for some protons
and resonance broadening, the protons attached to C8 showed significant resonance shift,
and multiple splitting peaks in a broad chemical shift range, indicating the multiple
chemical environment of the GS- ligands at the gold nanoparticles interfaces. The similar
results were observed in previous work for Au25(SG)18 nanoparticles and this is attributed
to the chirality of the ligands at the interfaces [53]. Briefly, after combining the optical
spectroscopy, TEM, and 1D 1H NMR spectroscopy, we conclude that small pure
spherical C8H17S-AuNPs and GS-AuNPs were successfully prepared.
2.3.2 Surface Chemistry and Ligand Structures at the Interfaces
In this work, 2D NMR spectroscopy was performed to investigate the surface
chemistry at the interfaces of the AuNPs. Figure 2.4 shows 1H-1H COSY and 1H-13C
HSQC NMR spectra of thiol-capped AuNPs. 1H 1D spectra indicated previously that 1H
resonances of ligands were broadened or shifted at the interfaces due to the chemical shift
anisotropic effect on AuNPs surfaces. Furthermore, for GS-AuNPs, some 1H resonances
show splitting patterns. To understand the splitting patterns and to obtain more chemical
shift information for other nucleus such as 13C, 2D NMR spectroscopies were utilized in
this work and the results are summarized in Table 2.1. For C8H17S-AuNPs, CH3 group
has a 1H resonance of 0.9 ppm and a 13C resonance of 14.2 ppm. Most CH2 groups (C3-
C7) have 1H resonances of around 1.3 ppm and 13C resonances varying from 22.9 ppm to
32.2 ppm (Table 2.1). 1H-1H COSY NMR experiment further showed a strong correlation
between CH3 group and CH2 groups of the thiol ligand. According to 1H-13C HSQC
NMR spectrum, the C2 group can also be detected, indicating a broad 1H resonance of
43
13
1.80 ppm and C resonance of 35.9 ppm. For GS-AuNPs, according to the results, C7
and C8 groups of the ligand have multiple 1H and 13

C resonances respectively. The
13
multiple C resonances for a single group indicate that the surface GS- ligands exhibit
multiple chemical environments and may have different types of chemically distinct
thiolate-gold binding modes. The 1H resonance splitting of the 3.3/3.6 (ppm) pair, 3.4/3.6
(ppm) pair, 2.9/3.2 (ppm) pair, and 3.2/3.4 (ppm) pair is caused by the nearby chiral
carbon (C8). Due to the size distribution of the synthesized gold nanoparticles and
complexity of the molecular structure of gold nanoclusters, the in-depth investigation of
the binding modes and chirality-induced splitting was not carried out in this work.
However, this idea has been demonstrated and investigated thoroughly on some well-
defined gold nanoparticles in previous reported literatures [29, 53].
The Au/thiol (Au/SR) ratio of AuNPs can be also obtained with NMR
spectroscopy. In this work, we used DMSO as the internal reference to determine the
Au/thiol ratio of purified C8H17S-AuNPs since the 1H resonances of DMSO (2.6 ppm)
and –SR ligand are well separated in the 1H NMR spectrum. The Au/SR ratio can be
determined according to following equations.
Table 2.1. Summary of 1H and 13C resonances of thiol-capped AuNPs

C8H17S-AuNPs GS-AuNPs
Group 1 13 1 13
H (ppm) C (ppm) H (ppm) C (ppm)
2 1.80 35.9 - -
a
3 1.30 29.9 a 3.91 53.2
4 1.30 a 29.9 a 2.20 25.9
a
5 1.30 29.9 a 2.61 31.5
6 1.27 32.2 - -
7 1.29 23.0 4.72 50.5, 52,5, 56.5
8 0.89 14.2 2.95 – 3.65 33.5, 35.5, 50.5
11 - - 3.99 41.3
a
. broad peak with multi groups.
44
(a) (b)
(c)
(d)
Figure 2.4. 1H-1H COSY NMR spectra of C8H17S-AuNPs (a) and GS-AuNPs (c), and 1H-13C
HSQC NMR spectra of C8H17S-AuNPs (b) and GS-AuNPs (d) respectively. In HSQC spectra, red
indicates CH2 group while black indicates the CH or CH3 groups. * notes for the water and
solvent peak.
𝑟𝑎𝑡𝑖𝑜 𝐴𝑢/𝑆𝑅 = 𝑀𝑤(𝑆𝑅)/𝑀(𝐴𝑢) ∗ (𝑚 𝐴𝑢𝑁𝑃 /𝑚(𝑆𝑅) − 1) Equation 2.1
𝑚 𝑆𝑅 = 2 ∗ 𝑟(𝐶𝐻! ) ∗ 𝑀𝑤 𝑆𝑅 ∗ 𝑚(𝐷𝑀𝑆𝑂)/𝑀𝑤 𝐷𝑀𝑆𝑂 Equation 2.2
In this work, 4.04 mg dried purified C8H17S-AuNPs and 2.44 mg DMSO were
used for preparing the NMR sample and 1D 1H NMR spectrum was collected after
sealing the NMR tube. The mass of the ligand on the surface can be calculated by using
45
the Eqn. 2.2. r CH! is the 1H ratio of the methyl group in C8H17S-AuNPs to that in the
DMSO and it was estimated to be 0.105 according to the 1H NMR spectrum (Figure 2.5).
So the Au/SR ratio was calculated to be 2.39. According to the TGA profile of purified
C8H17S-AuNPs (Figure 2.8a), the weight loss was calculated to be 23.2 wt %. Hence, the
Au/SR ratio is estimated to be 2.44, which is in good agreement with the quantitative
NMR result.
Figure 2.5. 1H NMR spectrum of purified C8H17S-AuNPs with DMSO at 298 K. * is the water
peak.
2.3.3 Determination of Hydrodynamic Size Using DOSY
The diffusion coefficient of nanoparticles in solution can be well determined by
measuring the diffusion coefficient of ligands that are covalently bonded to the surfaces
of nanoparticles by diffusion ordered NMR spectroscopy (DOSY). If the diffusion
coefficient is determined, the hydrodynamic size of the nanoparticles can be then
calculated by using the Stokes-Einstein equation. Diffusion is dependent on the size and
shape of individual objects by the well-known Debye-Einstein equation
𝐷 = 𝑘! 𝑇/𝑓! Equation 2.3
46
Where 𝑘! is the Boltzmann constant; 𝑇 is the Temperature in Kelvin; and 𝑓! is the
friction factor. For a spherical particle in a homogeneous solution, this equation can be
further simplified.
𝐷 = 𝑘! 𝑇/6𝜋𝜂𝑟! Equation 2.4
which is commonly known as the Stokes-Einstein equation. 𝑟! is the hydrodynamic
radius; and 𝜂 is the viscosity of the solvent used. So the hydrodynamic radius of the
particles can be calculated based on the following equation.
𝑟! = 𝑘! 𝑇/6𝜋𝜂𝐷 Equation 2.5
(a) (b)
Figure 2.6. 2D DOSY NMR spectra of C8H17S-AuNPs (a) and GS-AuNPs (b) with δ = 2 ms, Δ =
50 ms at 298 K.
Table 2.2. Summary of DOSY experiments on thiol-capped AuNPs.
Sample Da (m2 s-1) Dsolventb (m2 s-1) rH (nm) dH (nm) dTEM (nm)
C8H17S-AuNPs (2.45 ± 0.28) × 10-10 2.17 × 10-9 1.64 ± 0.19 3.28 ± 0.38 2.50 ± 0.49
C8H17SH 1.40 × 10-9 2.23 × 10-9 0.29 0.58 -
GS-AuNPs (1.32 ± 0.10) × 10-10 1.64 × 10-9 1.32 ± 0.10 2.63 ± 0.20 1.76 ± 0.47
GSH 3.44 × 10-10 1.26 × 10-9 0.39 0.78 -
47
In this work, the diffusion coefficients of C8H17S-AuNPs and GS-AuNPs were
determined by DOSY in CDCl3 and D2O respectively (Figure 2.6). The hydrodynamic
radiuses were then calculated according to the Stokes-Einstein equation, giving the
average hydrodynamic diameters of 3.28 ± 0.38 nm and 2.63 ± 0.20 nm for C8H17S-
AuNPs and GS-AuNPs, respectively (Table 2.2). However, TEM results show that the
diameters of C8H17S-AuNPs and GS-AuNPs are 2.50 ± 0.49 nm and 1.76 ± 0.47 nm
respectively, which are smaller than the values obtained by DOSY NMR spectroscopy.
This is because TEM is mainly focusing on measuring the physical size of the metal core
while the DOSY NMR spectroscopy measures the hydrodynamic size of AuNPs in
solution, taking the ligands binding to the surfaces into account. The hydrodynamic sizes
of octanethiol and glutathione were measured to be 0.58 nm and 0.78 nm in diameter
respectively. Combining this result with the TEM outcomes, the estimated hydrodynamic
diameters for AuNPs fall in the range of 3.08-3.66 nm for C8H17S-AuNPs and 2.54-3.32
nm for GS-AuNPs respectively. These values agree well with the hydrodynamic
diameters of AuNPs measured directly by the DOSY NMR spectroscopy, where the
hydrodynamic diameters are 3.28 ± 0.38 nm and 2.63 ± 0.20 nm for C8H17S-AuNPs and
GS-AuNPs respectively.
2.4 Conclusions
In this work, we demonstrate that NMR spectroscopy is a quite powerful
technique for investigating the AuNPs, providing useful and reliable structural and
dimensional information. It is shown that HSQC NMR spectroscopy is able to provide
chemical shift information that can be further used to study the structure of ligands on the
surfaces. Quantitative NMR provides a simple and cost-effective way to estimate the
48
Au/SR ratio. Furthermore, the hydrodynamic size of AuNPs can be determined using
DOSY NMR spectroscopy. For recent nanotechnology research, tools for nanomaterials’
qualitative and quantitative analysis are of extreme importance, not only for quality
control in production but also for applications in various research fields. Based on this
work, we have demonstrated the great potential of applying NMR spectroscopy in
nanomaterials characterizations and expect that NMR spectroscopy including
multidimensional NMR spectroscopy and DOSY NMR spectroscopy can be applied to
characterize a broad range of nanomaterials including quantum dots and oxide
nanoparticles in the future.
2.5 References
[1] P.L. Stiles, J.A. Dieringer, N.C. Shah, R.P. Van Duyne, Surface-enhanced raman
spectroscopy, Annual Rev. Anal. Chem., 1 (2008) 601-626.

Rev, 37 (2008) 2028-2019.
10413.
[6] M. Stratakis, H. Garcia, Catalysis by supported gold nanoparticles: Beyond aerobic

oxidative processes, Chem. Rev., 112 (2012) 4469-4506.
[7] B. Sharma, R.R. Frontiera, A.-I. Henry, E. Ringe, R.P. Van Duyne, SERS:
Materials, applications, and the future, Materials Today, 15 (2012) 16-25.
[8] J.N. Anker, W.P. Hall, O. Lyandres, N.C. Shah, J. Zhao, R.P. Van Duyne,
Biosensing with plasmonic nanosensors, Nat. Mater., 7 (2008) 442-453.
49
[9] D.-K. Lim, K.-S. Jeon, J.-H. Hwang, H. Kim, S. Kwon, Y.D. Suh, J.-M. Nam,
Highly uniform and reproducible surface-enhanced Raman scattering from DNA-
tailorable nanoparticles with 1-nm interior gap, Nat. Nanotechnol., 6 (2011) 452-
460.
[10] J.F. Li, Y.F. Huang, Y. Ding, Z.L. Yang, S.B. Li, X.S. Zhou, F.R. Fan, W. Zhang,
Z.Y. Zhou, D.Y. Wu, B. Ren, Z.L. Wang, Z.Q. Tian, Shell-isolated nanoparticle-
enhanced Raman spectroscopy, Nature, 464 (2010) 392-395.
[11] V. Marjomaki, T. Lahtinen, M. Martikainen, J. Koivisto, S. Malola, K. Salorinne,

M. Pettersson, H. Hakkinen, Site-specific targeting of enterovirus capsid by
functionalized monodisperse gold nanoclusters, Proc. Natl. Acad. Sci., 111 (2014)
1277-1281.
[12] B.V. Enustun, J. Turkevich, Coagulation of colloidal gold, J. Am. Chem. Soc., 85
(1963) 3317-3328.

[14] K. Pyo, V.D. Thanthirige, K. Kwak, P. Pandurangan, G. Ramakrishna, D. Lee,

Ultrabright luminescence from gold nanoclusters: rigidifying the Au(I)–thiolate
shell, J. Am. Chem. Soc., 137 (2015) 8244-8250.
[15] Z. Tang, B. Xu, B. Wu, M.W. Germann, G. Wang, Synthesis and structural
determination of multidentate 2,3-dithiol-stabilized Au clusters, J. Am. Chem. Soc.,
132 (2010) 3367-3374.
[16] J. Xie, Y. Zheng, J.Y. Ying, Protein-directed synthesis of highly fluorescent gold
nanoclusters, J. Am. Chem. Soc., 131 (2009) 888-889.
[17] Y. Yu, Z. Luo, D.M. Chevrier, D.T. Leong, P. Zhang, D.-e. Jiang, J. Xie,
Identification of a highly luminescent Au22(SG)18 nanocluster, J. Am. Chem. Soc.,
136 (2014) 1246-1249.
[18] N. Zheng, J. Fan, G.D. Stucky, One-step one-phase synthesis of monodisperse

noble-metallic nanoparticles and their colloidal crystals, J. Am. Chem. Soc., 128
(2006) 6550-6551.
[19] M. Zhu, E. Lanni, N. Garg, M.E. Bier, R. Jin, Kinetically controlled, high-yield
synthesis of Au25 clusters, J. Am. Chem. Soc., 130 (2008) 1138-1139.

Chem. Commun., (1994) 801-802.
50
[21] Z. Wu, J. Suhan, R. Jin, One-pot synthesis of atomically monodisperse, thiol-
functionalized Au25 nanoclusters, J. Mater. Chem., 19 (2009) 622-626.

Science, 298 (2002) 2176-2179.
[23] C. Yu, L. Zhu, R. Zhang, X. Wang, C. Guo, P. Sun, G. Xue, Investigation on the
mechanism of the synthesis of gold(I) thiolate complexes by NMR, J. Phys. Chem.
C, 118 (2014) 10434-10440.
[24] L. Zhu, C. Zhang, C. Guo, X. Wang, P. Sun, D. Zhou, W. Chen, G. Xue, New
insight into intermediate precursors of Brust–Schiffrin gold nanoparticles synthesis,
J. Phys. Chem. C, 117 (2013) 11399-11404.
[25] H. Häkkinen, The gold–sulfur interface at the nanoscale, Nature Chemistry, 4

(2012) 443-455.
[26] J.C. Azcárate, G. Corthey, E. Pensa, C. Vericat, M.H. Fonticelli, R.C. Salvarezza,
P. Carro, Understanding the surface chemistry of thiolate-protected metallic
nanoparticles, J. Phys. Chem. Lett., 4 (2013) 3127-3138.

nanoparticles by inductively coupled plasma–mass spectrometry, ACS Nano, 7
(2013) 1129-1136.

8608-8610.
Chem. Soc., 131 (2009) 6535-6542.

(2008) 3754-3755.
51
135 (2013) 10011-10013.
[35] C. Zeng, H. Qian, T. Li, G. Li, N.L. Rosi, B. Yoon, R.N. Barnett, R.L. Whetten, U.
Landman, R. Jin, Total structure and electronic properties of the gold nanocrystal
Au36(SR)24, Angew. Chem. Int. Ed., 51 (2012) 13114-13118.
[36] H. Qian, W.T. Eckenhoff, Y. Zhu, T. Pintauer, R. Jin, Total structure determination
of thiolate-protected Au38 nanoparticles, J. Am. Chem. Soc., 132 (2010) 8280-8281.
430.
[38] Z. Hens, J.C. Martins, A solution NMR toolbox for characterizing the surface
chemistry of colloidal nanocrystals, Chem. Mater., 25 (2013) 1211-1221.
[39] L.E. Marbella, J.E. Millstone, NMR techniques for noble metal nanoparticles,
Chem. Mater., 27 (2015) 2721-2739.
[40] B.S. Zelakiewicz, A.C. de Dios, Tong, 13C NMR spectroscopy of 13C1-labeled
octanethiol-protected Au nanoparticles: Shifts, relaxations, and particle-size effect,
J. Am. Chem. Soc., 125 (2003) 18-19.
[41] G.C. Lica, B.S. Zelakiewicz, Y.Y. Tong, Electrochemical and NMR
characterization of octanethiol-protected Au nanoparticles, J. Electroanal. Chem.,
554-555 (2003) 127-132.
[42] Z. Wu, R. Jin, Stability of the two Au-S binding modes in Au25(SG)18 nanoclusters
probed by NMR and optical spectroscopy, ACS Nano, 3 (2009) 2036-2042.
[43] A.M. Smith, L.E. Marbella, K.A. Johnston, M.J. Hartmann, S.E. Crawford, L.M.
Kozycz, D.S. Seferos, J.E. Millstone, Quantitative analysis of thiolated ligand
exchange on gold nanoparticles monitored by 1H NMR spectroscopy, Anal. Chem.,
87 (2015) 2771-2778.
[44] S.E. Lehman, Y. Tataurova, P.S. Mueller, S.V.S. Mariappan, S.C. Larsen, Ligand
characterization of covalently functionalized mesoporous silica nanoparticles: an
NMR toolbox approach, J. Phys. Chem. C, 118 (2014) 29943-29951.
52
[45] R. Sharma, G.P. Holland, V.C. Solomon, H. Zimmermann, S. Schiffenhaus, S.A.
Amin, D.A. Buttry, J.L. Yarger, NMR characterization of ligand binding and
exchange dynamics in triphenylphosphine-capped gold nanoparticles, J. Phys.
Chem. C, 113 (2009) 16387-16393.
[46] O.A. Wong, C.L. Heinecke, A.R. Simone, R.L. Whetten, C.J. Ackerson, Ligand
symmetry-equivalence on thiolate protected gold nanoclusters determined by NMR
spectroscopy, Nanoscale, 4 (2012) 4099-4094.
[47] R.L. Donkers, Y. Song, R.W. Murray, Substituent effects on the exchange dynamics
of ligands on 1.6 nm diameter gold nanoparticles, Langmuir, 20 (2004) 4703-4707.
[48] K.F. Morris, C.S. Johnson, Diffusion-ordered two-dimensional nuclear magnetic

resonance spectroscopy, J. Am. Chem. Soc., 114 (1992) 3139-3141.
[49] K. Salorinne, T. Lahtinen, J. Koivisto, E. Kalenius, M. Nissinen, M. Pettersson, H.

Häkkinen, Nondestructive size determination of thiol-stabilized gold nanoclusters in
solution by diffusion ordered NMR spectroscopy, Anal. Chem., 85 (2013) 3489-
3492.
[50] G. Canzi, A.A. Mrse, C.P. Kubiak, Diffusion-ordered NMR spectroscopy as a

reliable alternative to tem for determining the size of gold nanoparticles in organic
solutions, J. Phys. Chem. C, 115 (2011) 7972-7978.
[51] K. Salorinne, T. Lahtinen, S. Malola, J. Koivisto, H. Häkkinen, Solvation chemistry

of water-soluble thiol-protected gold nanocluster Au102 from DOSY NMR
spectroscopy and DFT calculations, Nanoscale, 6 (2014) 7823-7824.
[52] H. Kato, T. Saito, M. Nabeshima, K. Shimada, S. Kinugasa, Assessment of

diffusion coefficients of general solvents by PFG-NMR: Investigation of the
sources error, J. Magn. Reson., 180 (2006) 266-273.
[53] H. Qian, M. Zhu, C. Gayathri, R.R. Gil, R. Jin, Chirality in gold nanoclusters
probed by NMR spectroscopy, ACS Nano, 5 (2011) 8935-8942.
[54] N. Goswami, F. Lin, Y. Liu, D.T. Leong, J. Xie, Highly luminescent thiolated gold
nanoclusters impregnated in nanogel, Chem. Mater., 28 (2016) 4009-4016.
53
2.6 Supplementary Materials
(a) (b)
(c) (d)

Figure 2.7. 1H-1H COSY NMR spectra of C8H17SH (a) and GSH (c), and 1H-13C HSQC NMR
spectra of C8H17SH (b) and GSH (d) respectively.

54
(a) (b)

Figure 2.8. Thermogravimetric analysis (TGA) of the purified (a) C8H17S-AuNPs and (b) GS-
AuNPs.

(a) (b)

Figure 2.9. 2D DOSY NMR spectra of C8H17S-AuNP (a) and GS-AuNP (b) with δ = 2 ms, Δ =
50 ms at 298 K.

55
CHAPTER 3
Alanine Adsorption and Thermal Condensation at the Interface of Fumed Silica
Nanoparticles
3.1 Introduction
The interaction of biomolecules at the surface of inorganic materials has attracted
considerable attention in recent years since it has significant impact on drug delivery
research, bio-nanotechnology, and bio-nanocomposite materials [1-12]. Understanding
the adsorption and binding mechanism of biomolecules at the biomolecule-inorganic
interfaces is of major significance for developing novel bioinorganic composite systems
for biomedical applications [1, 10, 13-17]. Investigating the thermal condensation of
biomolecular species on inorganic oxide surfaces is of great interest since mineral
surfaces potentially served as catalysts for peptide bond formation in prebiotic chemistry
[18-23], and could have been involved in the synthesis of the first small oligopeptides
[21]. Considerable studies have demonstrated peptide bond formation occurs at relatively
low temperatures in the absence of any activating agents when gently heating amino
acids at the interface of inorganic oxides such as silica, alumina, and other minerals [19,
21, 23]. Among all the bioinorganic systems, the amino acid/silica system is the simplest
and most practical system and constitutes the first step in understanding more
complicated bioinorganic systems such as peptide/silica and protein/silica systems.
Silica (SiO2) is one of the most common inorganic materials on Earth and has
been extensively investigated for decades due to its diverse properties such as
polymorphism and biocompatibility [2, 24]. It has widespread applications ranging from
structural materials to catalysis and as a medium for drug delivery [2, 5, 8, 12, 16, 17, 24,
56
25]. Amorphous silica nanomaterials including fumed silica, mesoporous silica and
colloidal silica nanoparticles are one class of synthetic silica materials, featuring small
particle sizes and high surface areas [26, 27]. They have been studied extensively from
experimental synthesis to structural characterization to biomedical applications [2-4, 6, 7,
9, 11, 16, 17, 24-42]. The adsorption and thermal condensation of amino acids on silica
nanomaterials have attracted considerable attention during recent years to understand the
interaction between biomolecules and nanoparticles including the varying toxicity of
different silica nanoparticle types [1, 3-6, 13, 15, 19-21, 23, 43-60].
According to the literature review, considerable experimental studies have been
carried on mesoporous and colloidal silica nanomaterials [19, 21, 45, 47-54, 56, 58], but
very limited effort has been put on understanding the behaviors of amino acid on fumed
silica nanoparticles. Ben Shir et al investigated the binding and dynamics of glycine and
alanine on SBA-15 and MCM-41 surfaces by using rotational-echo double-resonance
(REDOR) [61] NMR [48, 50, 51]. They found that the adsorption of alanine on
amorphous silica surfaces occurs via direct interaction of its charged amine group with
silica surface silanols. Amitay-Rosen et al applied 2H MAS NMR to understand the
structural and dynamical state of water and alanine molecules adsorbed on SBA-15
surfaces at different degrees of surface hydration [47, 58]. It is found that two distinct
alanine populations were identified at the interface where one is dynamically undergoing
exchange between bound and free states while the other is statically bonded to the
surface. They also revealed that a small amount of interfacial water molecules made
alanine exhibit a solution-like environment with isotropic mobility. Comparing to
mesoporous and colloidal silica nanomaterials, fumed silica (Cab-O-Sil) nanoparticles are
57
another class of synthetic nanomaterials composed of amorphous structure with
extensively high surface area and nanoscale size [27]. They are prepared at high
temperature by hydrolyzing silicon tetrachloride vapor in a flame followed by rapid
quenching to room temperature. Many studies have been carried out on the surface
chemistry at fumed silica nanoparticle interfaces because of the considerable utility of
high surface area amorphous silicates [27, 29, 34, 35, 37, 38, 40, 41, 62, 63]. These
studies have shown that the surface chemistry of fumed silica nanoparticles is very
different from mesoporous and colloidal silica nanoparticles due to the presence of
metastable strained ring structures in fumed silica that result from the fast quenching
during the synthesis of the material in a flame. This unique property has been found to
closely relate with the high surface reactivity of the fumed silica nanoparticles [4, 18].
In the present work, a combination of multiple spectroscopic techniques,
including IR spectroscopy and NMR spectroscopy is implemented to investigate the
behavior of alanine molecules on fumed silica nanoparticles. The adsorption species of
alanine on fumed silica surfaces were observed directly by cross-polarization magic-
angle-spinning (CP-MAS) [64, 65] NMR spectroscopy in contrast with previous studies
for alanine adsorption on other silica surfaces [48, 50]. More than that, the adsorption of
alanine on the surfaces at high and low hydrated levels was thoroughly investigated and a
possible adsorption mechanism is proposed. Furthermore, it is found surprisingly that
adsorbed alanine on the fumed silica nanoparticles are able to form a cyclic peptide,
alanine anhydride during thermal condensation at ~ 170 °C with a high selectivity and a
yield of approximate 100 %. This significant finding indicates the great potential of
fumed silica nanoparticles in synthesizing peptides including cyclization reactions. To
58
further unravel the mechanism of such high-selectivity and high-yield peptide formation
on fumed silica nanoparticles, we carry out a comparative study of fumed and colloidal
silica nanoparticles, and the results indicate that the high efficiency is very likely related
to the intrinsic strained ring structures presenting at the interfaces of fumed silica
nanoparticles that are not abundant in colloidal forms. This is considered to be a first step
towards understanding the mechanism of the “surface-catalyzed” peptide bond formation
reactions on silica surfaces since relatively less effort has been put on this topic compared
to extensive investigations on adsorption of amino acids on silica surfaces.
3.2.1 Materials
Fumed silica nanoparticles (~7 nm) with BET (Brunauer, Emmett and Teller)
surface area of 395 ± 25 m2/g and natural abundance pure L-alanine (99 % purity) were
purchased from Sigma Aldrich. U-[13C/15N]-L-alanine were purchased from Cambridge.
Isotopes. Inc. All materials were used as received and stable isotope enrichment levels of
labeled compounds are 97-99 %. In the following, silica or SiO2 refers to fumed silica
13
nanoparticles unless otherwise specified. Ala/SiO2 and C/15N-Ala/SiO2 will refer to
13
natural abundance alanine and C/15N labeled alanine adsorbed on fumed silica
nanoparticles, respectively.
3.2.2 Adsorption of Alanine on Fumed Silica Nanoparticles
Fumed silica nanoparticles were initially heated to 500 °C overnight to remove
impurities on the surface. In a typical adsorption procedure, 75 mg fumed silica
nanoparticles were mixed with L-alanine in 5.0 mL DI water and the mixture was then
stirred at room temperature for over three hours to ensure the adsorption reached
59
equilibrium. Nanoparticles were then separated by centrifugation at 6000 rpm for one
hour and dried under vacuum at room temperature overnight. The samples prepared from
solutions of various concentrations and are noted as Ala/SiO2-xM, respectively, where x
refers to the alanine concentration in the adsorption solutions (in mol·L-1). In this work,
13
C/15N-Ala/SiO2-0.10M and 13
C/15N-Ala/SiO2-0.03M samples were prepared for solid-
state NMR investigations. Briefly, two sets of 120.0 mg fumed silica nanoparticles were
13
mixed with 21.6 mg and 72.0 mg C/15N-L-alanine in 8.0 mL DI water and the pH
values of both solutions were maintained at 6.7. The suspensions were then stirred for
over three hours to reach equilibrium. The two mixtures were then centrifuged at 6000
rpm for one hour and the remaining powders were allowed to vacuum dry at room
temperature overnight. The as-prepared samples were noted as ‘hydrated’ samples. To

13
prepare ‘dry’ samples, the C/15N-Ala/SiO2 samples were packed in NMR rotors and
then further vacuum dried with cap removed (0.001 mBar) for one month. The NMR
investigations on dried samples were carried out immediately after drying. No obvious
rehydration was found in this work during the course of solid-state NMR experiments
indicating that the rotor sealing is quite good.
3.2.3 Synthesis of Colloidal Silica Nanoparticles
Silica nanoparticles with a size of ~38 nm were synthesized using the well-
established method developed by Stöber et al [31]. Briefly, 0.17 M tetraethoxysilane was
hydrolyzed and condensed in a 100.0 mL ethanol solution with 0.5 M NH3 and 2.0 M
H2O for 12 hours at room temperature. The formed silica nanoparticles were then
dialyzed against ultrapure water and stored at a final concentration of 1.6 mg/mL.
60
3.2.4 Adsorption of Alanine on Colloidal Silica Nanoparticles
1.0 mg pure L-alanine was added to a 100 mL colloidal silica nanoparticles
solution ([SiO2 NPs] ~ 1.6 mg/mL) and the solution was stirred in excess of three hours
to reach equilibrium. The alanine adsorbed colloidal silica nanoparticles were obtained
after drying from the solution at room temperature.
3.2.5 Thermal Condensation
Dried alanine adsorbed silica nanoparticles were incubated at 170 °C for 3 hours.
After incubation, the peptides were washed with DI water followed by centrifugation at
14000 rpm for 30 min. The supernatant fluid was collected and dried to obtain the solid
powder. The resulting solid powder was dissolved in a 700 mL H2O/D2O (90/10) solution
and transferred to a 5 mm NMR tube.
In this work, solid-state NMR spectroscopy was used to probe alanine thermal
13
condensation on fumed silica nanoparticles. Briefly, C/15N-Ala/SiO2-0.03M was first
packed in the rotor followed by incubation at 170 °C for three hours with rotor cap
removed. The rotor was then cooled down to room temperature and sealed for solid-state
NMR characterization.
3.2.6 Thermal Analysis
Thermal gravimetric analysis (TGA) experiments were performed with a TA2910
(TA Instrument Inc.) instrument under dry N2 flow (30 mL/min for furnace and 30
mL/min for balance). For each experiment, 7-10 mg of sample was used and a heating
rate of 5 °C/min was applied. Before each experiment, the sample was kept under a
nitrogen flow for 10 min to remove any physisorbed water and obtain a stable baseline.
61
3.2.7 IR Spectroscopy
IR spectra were recorded using a PerkinElmer FTIR spectrometer with a diamond
ATR accessory. The instrument carries a MIR light source of 300-8000 cm-1, an OptKBr
beam splitter (400-7800 cm-1) and a LiTaO3 detector (370-15700 cm-1). The spectra were
collected with a spectral window of 400-4000 cm-1, a resolution of 4 cm-1, and 32 scans.
3.2.8 Raman Spectroscopy
Raman spectroscopy was performed on a homebuilt system utilizing a 532 nm
laser and triple-grating monochromator (SpectraPro 300i, Action Research). The laser
beam was focused onto the sample through a Mitutoyo M Plan Apo 50x objective with
0.42 N.A. All spectra were collected with a laser power of 50 mW, an exposure time of
30 s, and 10 scans.
3.2.9 Transmission Electron Microscopy (TEM)
The size and morphology of the silica nanoparticles were characterized using a
Philips CM200-FEG high-resolution transmission electron microscope (TEM) operating
at a bias voltage of 200 kV. The TEM samples were prepared by dipping carbon-coated
copper grids in silica nanoparticle solutions and drying them in air for a minimum of two
hours.
Solid-state NMR spectra were collected on a Varian VNMRS 400 MHz
spectrometer with a 1.6 mm triple-resonance probe operating in triple-resonance
(1H/13C/15N) mode at a MAS speed of 35 kHz. 1H high-speed MAS NMR with the
DEPTH sequence [66], 1Hà13C and 1Hà15N CP-MAS experiments, two-dimensional

1
(2D) H-13C heteronuclear correlation (HETCOR) and 1
H-15N HETCOR NMR
62
experiments were performed to characterize all samples. 1H MAS NMR experiments
were conducted with a 1.6 µs 1H π/2 pulse, a recycle delay of 5.0 s, and a 10 kHz sweep
width at a spinning speed of 35 kHz. The CP condition for 1Hà13C CP-MAS NMR
experiments consisted of a 1.6 µs 1H π/2 pulse, followed by a 1.0 ms ramped (13 %) 1H
spin-lock pulse of 75 kHz rf field strength. The experiments were performed with a 25
kHz sweep width, a recycle delay of 3.0 s and a two-pulse phase-modulated (TPPM) [67]
1
H decoupling level of 156 kHz for all samples. The CP condition for 1Hà15N CP-MAS
NMR experiments consisted of a 1.6 µs 1H π/2 pulse, followed by a 1.0 ms ramped (10
%) 1H spin-lock pulse of 95 kHz rf field strength. The experiments were performed with
a 25 kHz sweep width, a recycle delay of 3.0 s and a 1H decoupling level of 156 kHz. A
contact time of 1.0 ms was applied in 1Hà13C and 1Hà15N CP-MAS NMR experiments
13
for pure alanine and C/15N-Ala/SiO2-0.10M while a contact of 2.0 ms was applied for
13
C/15N-Ala/SiO2-0.03M.
2D 1H-13C HETCOR and 2D 1H-15N HETCOR NMR experiments were carried

13
out on two C/15N-Ala/SiO2 samples. The experimental CP condition and TPPM 1H
decoupling parameters for 2D HETCOR NMR experiments were identical to 1D CP-
MAS NMR experiments with the exception of contact times. 2D 1H-13C HETCOR NMR
experiments were performed with a contact time of 2.0 ms and 0.25 ms, a recycle delay
of 3.0 s, sweep widths of 25 kHz and 10 kHz for direct and indirect dimensions,
respectively, and 32 complex t1 points. 2D 1H-15N HETCOR NMR experiments were
performed with different contact times (0.25, 1 and 2 ms) a recycle delay of 3.0 s, a
sweep width of 10 kHz for both dimensions and 32 complex t1 points.
63
In all experiments, the chemical shifts of 1H, 13
C and 15
N were indirectly
referenced to adamantane 1H (1.63 ppm), adamantane 13

C (38.6 ppm) and glycine 15
N
(31.6 ppm), respectively [68, 69]. The line-broadening (lb) factor was set to 25 Hz for
1
Hà13C CP-MAS spectra, 1Hà15N CP-MAS spectra, 2D 1H-13C HETCOR spectra, and
50 Hz for 2D 1H-15N HETCOR spectra.
3.2.11 Solution NMR Spectroscopy
Solution NMR spectroscopy experiments were performed on a VNMRS 500 MHz
spectrometer with a 5 mm triple-resonance probe operating in triple-resonance
(1H/13C/15N) mode. 1H NMR spectra were collected with a sweep width of 8012.8Hz, an
acquisition time of 2.045s, a recycle delay of 5.0 s, and 512 scans. 1H-13C heteronuclear
single-quantum correlation (HSQC) spectra were collected with a recycle delay of 1.0 s,
sweep widths of 8012.8Hz and 25141.4Hz for direct and indirect dimensions,
respectively, and 256 complex t1 points.
3.3.1 Morphology and Structure of Amorphous Silica Nanoparticles
Amorphous silica nanoparticles are typically prepared by two main methods:
high-temperature flame pyrolysis to form so-called fumed silica nanoparticles and
condensation of silanol groups in aqueous solution or under hydrothermal conditions to
form colloidal (Stöber) or mesoporous silica nanoparticles. In this work, fumed silica
nanoparticles and colloidal silica nanoparticles are investigated to compare their
efficiency of surface catalysis in forming peptide bonds for alanine. Fumed silica
nanoparticles are obtained by hydrolyzing silicon tetrachloride vapor in a flame followed
by rapid quenching to room temperature. This process generates small particles with
64
diameters from a few nm to tens of nm that are best described as ramified, chainlike
aggregates (Figure 3.1A). The fumed silica nanoparticles used in this study possess an
average particle size ~7 nm according to the manufacturer. Colloidal silica nanoparticles
are synthesized according to the Stöber method and are non-aggregated, monodisperse
spherical nanoparticles (Figure 3.1B). The average size of the Stöber silica nanoparticles
is determined to be ~38 nm in diameter. Because of the much smaller size of primary
particles, fumed silica nanoparticles feature a high surface area up to ~390 m2/g
compared to the synthesized colloidal silica nanoparticle whose surface area is only ~60
m2/g.
Raman spectroscopy was applied to investigate the surface structures of the two
types of silica nanoparticles and the spectra are shown in Figure 3.1C. Based on the
comparison between fumed silica and Stöber silica nanoparticles, it indicates that their
surface properties are different. For fumed silica nanoparticles, two significant bands at
~490 cm-1 (D1) and 605 cm-1 (D2) are observed that can be attributed to four- and three-
membered siloxane rings, respectively [37]. There is also a broad band centered at ~450
cm-1 attributed to the five-member and larger siloxane rings. Stöber silica nanoparticles
have a prominent band at ~490 cm-1 but no well-resolved band at ~605 cm-1, indicating
the absence of three-membered rings at the surface. This result is in good agreement with
previous experimental finding [4]. The relatively large population of strained three-
membered rings at fumed silica nanoparticle surfaces is due to the rapid quenching of
silica materials at very high temperatures during the synthesis in a flame. This process
freezes the silica structure in some strained structures such as three-member rings and
this is also observed for Aerosil fumed silica with particle diameters of 14 nm and 40 nm
65
[37]. Stöber silica nanoparticles are synthesized in solution via continuous condensation
reactions and they are principally composed of unstrained four-membered and larger
rings that are more stable and less reactive. The absence of three-membered ring is also
characteristic of other solution-derived silica nanoparticles including mesoporous silica
and silica gels [4].
Fumed silica Colloidal silica O

Si O
D1 O
Si
O
nanoparticles nanoparticles Si
O
Si
490 cm-1 Si
O
Si
O
Si
D2
605 cm-1
Si O Si
(a) 800 cm-1
(b)
300 400 500 600 700 800 900

Wavenumber (cm-1)
A B C
Figure 3.1. TEM images of (A) fumed silica nanoparticles and (B) colloidal silica nanoparticles.
(C) Raman spectra of silica nanoparticles: (a) fumed silica and (b) colloidal silica nanoparticles.
3.3.2 Adsorption Behavior of Alanine on Fumed Silica Nanoparticles
The adsorption of alanine on silica surfaces has been well studied [21, 48, 50] and
some factors such as initial concentration of alanine in the adsorption solution and pH
values of adsorption solution has been shown to impact the amount of adsorbed alanine at
the silica interface. In this paper, we use TGA to investigate the influence of initial
aqueous alanine concentration on the content of adsorbed alanine at the surface of fumed
silica nanoparticles and the results are summarized in Table 3.1. The amount of adsorbed
alanine is calculated from the TGA weight loss curves between 100 °C and 600 °C since
the water contributes to the weight loss below 100 °C. Figure 3.2 shows the amount of
alanine adsorbed on fumed silica nanoparticles as a function of initial aqueous alanine
concentration. With increasing the initial concentration, the amount of adsorbed alanine
66
increases correspondingly. Furthermore, the adsorption of alanine fits well to a Langmuir
isotherm in the low initial concentration range (< 0.05 M). However, at high initial
concentrations (> 0.08 M), the adsorption behavior is linear and cannot be properly fit to
a Langmuir isotherm. This is probably due to the incomplete separation between liquid
and solid phases or the formation of multilayers on the surface similar to previous
observations for lysine adsorption on fumed silica nanoparticles.
Figure 3.2. The amount of alanine adsorbed on fumed silica nanoparticles as a function of initial
aqueous alanine concentration at room temperature and near neutral pH (pH = 6.7).
Table 3.1. Summary of TGA results for alanine adsorption on fumed silica nanoparticles.
[Alanine] (M) Weight Loss (%) Adsorbed Alanine (molecules/nm2)
0.01 2.8 0.50 ± 0.03
0.03 4.2 0.76 ± 0.05
0.05 5.0 0.91 ± 0.06
0.08 7.0 1.31 ± 0.08
0.10 8.7 1.65 ± 0.10
0.15 10.5 2.02 ± 0.13
3.3.3 Adsorption Mechanism of Alanine on Fumed Silica Nanoparticles
Solid-state NMR spectroscopy is a powerful tool for elucidating surface chemistry
at nanomaterial interfaces. Here, a combination of solid-state NMR experiments was
performed to investigate the adsorption mechanism of alanine on fumed silica

67
nanoparticles surfaces. In previous studies, the protonation state of the amine group of
15
alanine on mesoporous silica was investigated by using N(1H) separated local field
(SLF) [70] experiments combined with PMLG-5 homonuclear decoupling [71]. These
results indicated that the amine group of alanine was protonated (NH3+) with a 1H
resonance at ~7.4 ppm that facilitated the binding to silica surface groups [48]. In this
work, the bulk alanine and adsorbed alanine on fumed silica nanoparticles were found in
zwitterionic forms since the 1H resonance of NH3+ was clearly detected for all species
with a similar chemical shift observed (Supplementary, Figure 3.17). Furthermore, two
1
H resonances were seen for methyl protons in the “hydrated” state: one at 1.4 ppm
corresponding to alanine in an aqueous environment and one at 1.2 ppm corresponding to
alanine interacting with the silica nanoparticles (Supplementary, Figure 3.17). This was
confirmed in the “dry” state where the 1H resonance at 1.4 ppm disappears presumably
due to the restriction of reorientational motion of alanine at low hydration level [47, 48,
58]. The alanine molecules become less mobile when interacting with the dry
nanoparticle surface. A 1H resonance at 1.7 ppm was detected for dry samples that is due
to the isolated silanol group on the fumed silica nanoparticle surface [29]. This result is
consistent with those obtained by Amitay-Rosen et al [47].
In Figures 3.3, the 1Hà13C and 1Hà15N CP-MAS NMR spectra of bulk U-
[13C/15N]-L-alanine and 13
C/15N-Ala/SiO2 samples are displayed with all peak
assignments summarized in Table 3.2. Bulk U-[13C/15N]-L-alanine is in the crystalline

13
state exhibiting three sharp C resonances for the carboxyl group (177.8 ppm), α-CH
15
(51.1 ppm) and β-CH3 (20.6 ppm) and one N resonance for the NH3+ (42.3 ppm)
illustrating that alanine is in its zwitterionic form. The splitting for the carboxyl group is
68
13
due to the C-13C J-coupling with the neighboring α-13CH. These resonances were also
detected for 13C/15N-Ala/SiO2-0.10M, indicating crystalline alanine exists in the samples
at this surface coverage. In addition to crystalline alanine, three unique 13C resonances at
176.0 ppm, 51.8 ppm and 16.2 ppm and one additional 15N resonance is observed in the
spectra, and are assigned to the carboxyl group, α-CH, β-CH3 and NH3+ of the adsorbed
alanine, respectively. The resonances broaden considerably following drying since the
adsorbed alanine becomes more rigid. The broadening effect is due to chemical shift
heterogeneity and illustrates that the alanine-nanoparticle surface environment is
disordered. The large change (4.4 ppm) in 13C chemical shift to lower ppm observed for
the β-CH3 for adsorbed alanine is likely due to methyl packing differences between the
crystalline and adsorbed forms. Chemical shifts to higher ppm are typically observed for
more restricted methyl packing environments in alanine containing peptides [72]. The
methyl packing is expected to be less restrictive on the silica nanoparticle surface.
69
B
Figure 3.3. 1Hà13C CP-MAS NMR spectra (A) and 1Hà15N CP-MAS NMR spectra (B) of U-
[13C/15N]-L-alanine, 13C/15N-Ala/SiO2-0.10M (“hydrated” and ”dry”) and 13C/15N-Ala/SiO2-
0.03M (“hydrated” and ”dry”).
13 15
Table 3.2. Summary of C and N chemical shifts of U-[13C/15N]-L-alanine and 13
C/15N-
Ala/SiO2 samples a
13 15
C N
Samples -
-CO2 α-CH β-CH3 NH3+
U-[13C/15N]-L-alanine 177.8 51.1 20.6 42.3
13
C/15N-Ala/SiO2-0.10M (hydrated) 177.8, 176.3 51.1, 51.8 20.6, 16.2 42.3, 43.3
13
C/15N-Ala/SiO2-0.10M (dry) 177.8, 176.0 51.1, 51.8 20.6, 16.2 42.3, 43.3
13
C/15N-Ala/SiO2-0.03M (hydrated) 176.0 51.8 16.2 43.3
13
C/15N-Ala/SiO2-0.03M (dry) 176.0 51.8 16.2 43.3
a
Chemical shifts are reported in ppm from TMS.
The crystalline alanine was found to disappear when lowering the concentration
of alanine in the adsorption solution. In the 1Hà13C and 1Hà15N CP-MAS NMR spectra
of 13C/15N-Ala/SiO2-0.03M, the characteristic resonances of crystalline alanine were not
detected and only adsorbed alanine was observed at the silica nanoparticle interface. The
13
C and 15N resonances of adsorbed alanine appeared at identical isotropic chemical shifts
70
to those of 13C/15N-Ala/SiO2-0.10M. Furthermore, the broadening effect of the adsorbed
13
state was clearly observed for “dry” C/15N-Ala/SiO2-0.03M, demonstrating the
adsorbed species is highly disordered as evidenced by the heterogeneous line broadening.

1
Based on the results of Hà13C CP-MAS NMR and 1
Hà15N CP-MAS NMR
experiments, it is convincing to argue that at high surface coverage (13C/15N-Ala/SiO2-
0.10M), both crystalline and adsorbed states of alanine exist on the nanoparticle surfaces
while only the adsorbed alanine state is present at low surface coverage (13C/15N-
Ala/SiO2-0.03M).
(A)
(B)
Figure 3.4. 1Hà13C 2D HETCOR MAS NMR spectra of (A) “hydrated” 13C/15N-Ala/SiO2-
0.10M and (B) “dry” 13C/15N-Ala/SiO2-0.10M. Experiments were conducted with a 2.0 ms
contact time at 400 MHz and a MAS frequency of 35 kHz.

71
(A) (B)

Figure 3.5. 1Hà15N 2D HETCOR MAS NMR spectra of (A) “hydrated” 13C/15N-Ala/SiO2-
contact time at 400 MHz and a MAS frequency of 35 kHz.
(A)
(B)
Figure 3.6. 1Hà13C 2D HETCOR MAS NMR spectra of (A) “hydrated” 13C/15N-Ala/SiO2-
0.03M and (B) “dry” 13C/15N-Ala/ SiO2-0.03M. Experiments were conducted with a 2.0 ms
contact time at 400 MHz with MAS frequency of 35 kHz.
72
(A) (B)
Figure 3.7. 1Hà15N 2D HETCOR MAS NMR spectra of (A) “hydrated” 13C/15N-Ala/SiO2-
contact time at 400 MHz with MAS frequency of 35 kHz.
To further elucidate the interaction of alanine molecules with the surface groups
on fumed silica nanoparticles, 2D 1H-13C and 1H-15N HETCOR NMR experiments were
performed. The 2D 1H-13C and 1H-15N HETCOR NMR spectra of “hydrated” and “dry”
13
C/15N-Ala/SiO2-0.10M are shown in Figures 3.4 and 3.5 along with the 1H traces for
13 15 13
specific C and N chemical shifts. For both “hydrated” and “dry” C/15N-Ala/SiO2-
0.10M samples, the 1H-13C and 1H-15N correlations for crystalline alanine are identical.
13
The C resonances at 177.8 ppm, 51.1 ppm, and 20.6 ppm assigned to the carboxyl
group, α-CH, and β-CH3 of crystalline alanine clearly show three correlations with 1H
resonances at 0.9 ppm, 3.4 ppm, and 8.2 ppm corresponding to the β-CH3, α-CH and to
the NH3+ in interaction with the carboxylate of a neighboring molecule through COO-HN
15
hydrogen bonds in the crystalline state, respectively. The N resonance at 42.3 ppm
assigned to the NH3+ of crystalline alanine also shows three correlations with these 1H
resonances in the two states. However, some differences for 1H-13C and 1H-15N
correlations were found for adsorbed alanine after comparing the “hydrated” state and the
73
“dry” state, indicating that the binding behaviors of alanine molecules on the surfaces are
13
different in the two states. In the “hydrated” state, the C resonance at 176.3 ppm
assigned to the carboxyl group of adsorbed alanine shows two clear correlations with 1H
15
resonances at 4.8 ppm and 1.2 ppm. The N resonance at 43.3 ppm is assigned to the
NH3+ of adsorbed alanine and shows one strong correlation with a 1H resonance at 5.4
ppm. The 1H resonance at 4.8 ppm is assigned to surface bound water whose behavior is
similar to bulk, isotropic water and the 1H resonance at 1.2 ppm was assigned to β-CH3 of
adsorbed alanine. The 1H resonance at 5.4 ppm could be due to the interaction between
water and NH3+ of free alanine that are not bound to the silica nanoparticle surface. These
results indicate that the carboxyl group and the NH3+ of adsorbed alanine both interact
with the interfacial water via a fast-exchange process in the “hydrated” state. After
drying, the carboxyl group and the NH3+ of adsorbed alanine show distinguishable
correlations with the 1H resonance at 7.6 ppm that are assigned to the hydrogen bonding
protons that interacts with surface silanol groups [50]. This indicates that the protonated
amine group and the carboxyl group interact with silanol groups by forming hydrogen
bonds on the silica nanoparticle surfaces in the “dry” state.
2D 1H-13C and 2D 1H-15N HETCOR NMR of the “hydrated” and “dry” 13C/15N-
Ala/SiO2-0.03M are shown in Figure 3.6 and Figure 3.7. Since only adsorbed alanine was
13
detected on surfaces for C/15N-Ala/SiO2-0.03M, the 1H-13C and 1H-15N correlations
shown in the spectra can offer more insight into the adsorbed state of alanine on fumed
13
silica nanoparticle surfaces. Similar to the findings for C/15N-Ala/SiO2-0.10M, in the
“hydrated” state, the carboxyl group and the NH3+ interact with interfacial water at ~ 4.6
ppm, indicating the alanine molecules are mobile interacting with free water at the
74
interfaces. In the “dry” state, a clear strong 1H contact is observed at ~ 7.6 ppm for the
carboxyl group indicating they interact with the silanols. To further get a more detailed
picture of the adsorption on surfaces, we performed HETCOR NMR experiments with
variable mixing time and the results are shown in Figure 3.8. Since the 2D 1H-13C and 2D
1
H-15N HETCOR NMR experiments are dipolar-based experiments, in which
magnetization is transferred through space, long-range correlations can be detected by
applying a longer CP contact time. With a mixing time of 2.0 ms, all 1H-13C and 1H-15N
correlations can be seen clearly. However, applying a mixing time of 0.25 ms offers a
chance to see only short-distance correlations, which makes it possible to determine the
groups that interact with the NH3+ interacting at silanol groups (1H resonance at ~ 7.6
ppm). Based on the 2D 1H-13C and 2D 1H-15N HETCOR NMR spectra with a mixing
time of 0.25 ms, a weak correlation to 1H resonance at 7.6 ppm was detected for the
carboxyl group of alanine while a strong correlation to the same 1H resonance (7.6 ppm)
was seen clearly for the protonated amine groups of adsorbed alanine. This result
provides convincing evidence that both protonated amine group and the carboxyl group
of alanine interact with the silanol group directly via hydrogen bonds at low hydration
level (Figure 3.9).
75
(A)
(B)
76
Figure 3.8. 1Hà13C (A) and 1Hà15N (B) 2D HETCOR MAS NMR spectra of “dry” 13C/15N-
Ala/SiO2-0.03M with contact times of 2.0 ms and 0.25 ms. Experiments were conducted at 400
MHz with MAS frequency of 35 kHz.
Two Ala/SiO2 samples with different surface coverages ratios (13C/15N-Ala/SiO2-
0.03M and 13C/15N-Ala/SiO2-0.10M) were prepared and characterized in this work. At a
high surface coverage (0.10 M), both crystalline and adsorbed states of alanine were
observed on the fumed silica nanoparticle surfaces while only the adsorbed state was
detected at a low surface coverage (0.03 M). Furthermore, it is found that the carboxyl
group and protonated amine group of alanine were found to interact with interfacial water
via a fast-exchange process in the “hydrated” state. After thorough drying, adsorbed
alanine molecules become more restricted and show strong interactions with the surface
silanol groups via hydrogen bonding between silanol groups and the carboxyl groups and
protonated amine groups.
“hydrated” “dry”
Figure 3.9. Schematic representation of alanine adsorption on fumed silica nanoparticles surfaces
in “hydrated” and “dry” state.
77
4.5
0.08 0.15 M
bulk alanine 4.0
0.07 3.5
0.10 M
0.06 3.0
0.08 M 2.5
DTG (%/oC)
DTG (%/oC)
0.05
2.0
0.04
0.05 M 1.5
0.03 0.03 M
1.0
0.02 0.01 M
0.5
0.01 0.0
0.00 -0.5
100 150 200 250 300 350 400
Temperature (oC)
Figure 3.10. DTG curves of bulk alanine (dashed, red) and Ala/SiO2 (solid, black) samples as a
function of initial concentration of amino acids in the adsorption solutions.
3.3.4 Thermal Condensation of Alanine on Fumed Silica Nanoparticles
Amino acids can undergo thermal condensation at the interface of inorganic
materials such as silica, alumina and other minerals [19, 21, 22, 52, 73]. In this work,
differential thermal gravimetric (DTG) analysis and solid-state NMR spectroscopy were
used to investigate the thermal condensation of alanine at the interfaces of fumed silica
nanoparticles. Figure 3.10 shows the DTG profiles of bulk alanine and Ala/SiO2 as a
function of alanine concentration. For bulk alanine, no thermal event occurs below 200
°C while one broad peak was detected around 270 °C with a shoulder at 250 °C.
However, for alanine adsorbed on fumed silica nanoparticles, a well-distinguished peak is
observed around 170 °C at a heating rate of 5 °C/min, which is due to the thermal
condensation of alanine forming peptide bonds. The other peaks observed above 220 °C
correspond to a complicated thermal degradation process with the elimination of NH3 and
78
CO2 similar to bulk alanine and will not be further discussed in this paper. Table 3.3
summarizes the thermal condensation results as a function of initial alanine concentration
in the prepared solutions. With the increasing amount of alanine on the surface, the
temperature of thermal condensation had little change, indicating that the adsorbed
alanine is the main species contributing to the thermal condensation. In this study, we
focus primarily on Ala/SiO2-0.03M since it was shown to be a monolayer-adsorbed
sample. By lowering the heating rate from 5 °C/min to 1 °C/min, the thermal
condensation peak was detected at even lower temperature around 149 °C (Figure 3.11).
This finding indicates that the fumed silica nanoparticle surfaces are able to catalyze the
formation of peptide bond and lower dramatically the thermal condensation temperature
of alanine. For the product of thermal condensation, it is very likely alanine anhydride
since it has been proposed previously that a single-cycle heating yields primarily the
cyclic anhydrides (or diketopiperazines, DKP). The thermal analysis results of alanine
anhydride and alanine anhydride adsorbed on fumed silica nanoparticles support this
assumption since the onset temperature of alanine anhydride (~213°C) degradation agrees
well with the onset temperature of thermal degradation (~213°C) for alanine adsorbed on
fumed silica nanoparticles (Figure 3.11). However, thermal analysis is in fact difficult to
provide convincing evidence for resolving the product of thermal condensation. Due to
this, additional techniques such as NMR spectroscopy and IR spectroscopy were used in
this study to further characterize the product produced.
79
A 0.04
Ala/SiO2-0.03M B 0.04
Ala/SiO2-0.03M 5 oC/min
5 oC/min
Alanine anhydride Alanine anhydride
Alanine anhydride/SiO2-0.03M
0.03 173 0.03
DTG (%/oC) peptide bond formation
DTG (%/oC)
173 oC
0.02 0.02
213
1 oC/min
149
0.01 0.01
191 210 213

0.00 0.00
50 100 150 200 250 300 350 400 50 100 150 200 250 300 350 400
Temperature (oC) Temperature (oC)
Figure 3.11. DTG curves for (A) Ala/SiO2-0.03M (black) and alanine anhydride (red). (B)
Alanine anhydride/SiO2-0.03M (green). The experiments were carried out at two different heating
rates (5 °C/min and 1 °C/min).
Table 3.3. Summary of alanine adsorption and thermal condensation on fumed silica
nanoparticles from thermal analysis 29
DTG peak values (°C) Adsorbed Alanine
[Alanine]
Peak I Peak II (%)
C=0.01M - - 2.8
C=0.03M 171 280 4.2
C=0.05M 171 279 5.0
C=0.08M 172 269 7.0
C=0.10M 173 263 8.7
C=0.15M 171 253 10.5

Solid-state NMR spectroscopy and IR spectroscopy were applied as techniques
for investigating the thermal condensation of alanine catalyzed by the fumed silica
nanoparticle surfaces. 1Hà13C and 1Hà15N CP-MAS NMR experiments were performed
on isotope labeled samples (13C/15N-Ala/SiO2-0.03M) following thermal condensation
and the results are shown in Figure 3.12. After thermal incubation at 170 °C for three
13 15
hours, the intensities of the C resonance at 176.0 ppm and N resonance at 43.3 ppm
decreased and two new resonances appear due to peptide bond formation during thermal
13 15
condensation. The C resonance at 171.1 ppm and N resonance at 120.2 ppm were
assigned to the carbonyl group and the amide group of the formed peptide respectively
80
13 13
(Figure 3.12d). The C resonance of the carbonyl group is consistent with the C
resonance of the carbonyl group of alanine anhydride (Figure 3.12b), indicating the
formation of alanine anhydride. Furthermore, the intensity of carboxyl group decreased
after thermal condensation, showing a small broad peak overlaid with the strong carbonyl
peak assigned to the peptide. This indicates that the vast majority of adsorbed alanine was
15
converted to peptides. This point was further confirmed by N solid-state NMR
15
spectroscopy where a weak N signal for NH3+ groups and a strong signal for peptide
15
amide bond was observed following thermal condensation. The N resonance of the
15
amide group in the formed peptide shows ~3 ppm difference compared to the N
resonance of the amide group for crystalline alanine dipeptide (~123 ppm) [74].
15
However, it has a similar N resonance to the amide group of alanine residue in silk
protein (~120 ppm) that is involved in strong hydrogen-bonding [75]. This indicates that
the formed peptide may interact with the silica surfaces via amide groups forming
hydrogen bonds to the silica surface. According to 2D 1H-13C and 2D 1H-15N HETCOR
NMR spectra (Supplementary, Figure 3.18), the 1H resonance of amide group was
detected at 6.4 ppm while the 1H resonances of α-CH and β-CH3 is at 3.2 ppm and 1.2
ppm, respectively. This smaller 1H resonance of amide proton provides further evidence
that amide protons are involved in a hydrogen bond network with the silica surfaces [53].
81
Figure 3.12. 1Hà13C and 1Hà15N CP MAS spectra of (a) 13C, 15N-Alanine, (b) alanine
anhydride (c) 13C, 15N-Ala/SiO2-0.03M and (d) 13C, 15N-Ala/SiO2-0.03M incubated at 170 °C for
three hours.
Similar to the results from solid-state NMR spectroscopy, IR spectroscopy also
show alanine anhydride formed during thermal condensation (Figure 3.13). The band for
O=C-O stretching (1624 cm-1) disappeared and two well-defined bands appear at 1668
cm-1 and 1689 cm-1, which are assigned to the C=O stretching of amide I [76]. This result
indicates that alanine did undergo thermal condensation at the interfaces and form peptide
bond. Further, after comparing the spectra of Ala/SiO2-0.03M following thermal
incubation with the pure alanine anhydride it is compelling to find that vibrational bands
identified from two spectra are very similar, indicating the formed peptide is alanine
anhydride in agreement with the solid-state NMR interpretation.
82
Figure 3.13. FTIR spectra of (a) fumed silica nanoparticles, (b) alanine, (c) Ala/SiO2-0.03M, (d)
Ala/SiO2-0.03M incubated at 170 °C for three hours, (e) product washed off nanoparticles after
thermal incubation and (f) alanine anhydride.
Table 3.4. Assignments of FTIR band
Band position (cm-1) Assignments

1689 Amide I C=O stretching
1668 Amide I C=O stretching
1624, 1615 O=C-O stretching
1587 NH3+ stretching
1513 NH3+ stretching
1473 Ring-stretching
1455 CH3 stretching
1412 CH3 stretching
1362 O=C-O stretching

83
(d) peptide formed on colloidal silica nanoparticles
(c) peptide formed on fumed silica nanoparticles
β
O N
(b) alanine anhydride α

β N O
α
β O
β
alanine -
(a) H3N +
α
O
α
4.5 4.0 3.5 2.0 1.5 1.0

1
H Chemical Shift (ppm)
Figure 3.14. 1H NMR spectra of (a) alanine, (b) alanine anhydride, (c) the peptide formed on
fumed silica nanoparticles, and (d) peptide formed at colloidal silica nanoparticles.
The product formed during the thermal condensation was then washed off the
fumed silica nanoparticle surfaces and characterized with solution NMR spectroscopy.
According to the 1H NMR spectra shown in Figure 3.14, the 1H resonances and splitting
patterns of the formed peptide are identical to those of pure alanine anhydride (Figure
84
3.14a). This result provides strong evidence of that the peptide formed during the thermal
condensation is alanine anhydride and almost all of the adsorbed alanine was converted
to alanine anhydride, one of the simplest cyclic peptides. This finding was further
confirmed by 1H-13C HSQC NMR spectra that indicated the formed peptide is alanine
anhydride (Supplementary, Figure 3.19). In summary, this combination of experiments
has shown that fumed silica nanoparticle surfaces are able to serve as catalysts for
converting alanine to alanine anhydride via thermal condensation, significantly lowering
the reaction temperature (Figure 3.15). Moreover, the yield of the alanine anhydride is
estimated to be 98.8 % since only a very small amount of alanine was unreacted and left
on the surfaces after thermal condensation (Supplementary, Figure 3.20).
Figure 3.15. Schematic representation of thermal condensation of alanine at 170 °C forming

alanine anhydride on fumed silica nanoparticles.
3.3.5 A Comparative Study Between Fumed Silica Nanoparticles and Colloidal Silica
Nanoparticles on Surface-catalyzed Peptide Bond Formation Reaction
From the present study on thermal condensation of alanine on fumed silica
nanoparticles, it is illustrated that fumed silica nanoparticles have exceptionally high
efficiency for surface-catalyzed peptide bond formation reaction. Here, a simple question
then arises: do other types of amorphous silica nanomaterials have the same property? To
85
answer this question, we decided to investigate another type of the most common silica
nanomaterials, colloidal silica nanoparticles. The colloidal silica nanoparticles were
synthesized following the classical Stöber method and they possess a well-defined
spherical shape with an average diameter around 38 nm. The surface area is much lower
that of fumed silica nanoparticles, resulting in a much lower amount of adsorbed alanine
on the surfaces. The TGA profile indicates the adsorbed alanine is less than 2 wt% and
the DTG curves shows a small broad peak around 190 °C attributed to the peptide bond
formation reaction (Supplementary, Figure 3.21). Figure 3.14d shows the 1H NMR
spectrum of the product from the thermal condensation on Stöber silica nanoparticles.
The multiple small 1H resonances at 4.2 ppm indicate the formation of alanine anhydride
but the yield is very low. The vast majority amount of adsorbed alanine still remained
unreacted on the surface as indicated by the well-defined 1H resonance at 3.8 ppm. This
result is further supported by IR spectroscopy, showing that the alanine is the main
species present at the colloidal silica nanoparticle surfaces after thermal condensation
(Supplementary, Figure 3.22). Based on the comparison between fumed silica
nanoparticles and colloidal silica nanoparticles on surface-catalyzed peptide bond
formation reaction, it is concluded that fumed silica nanoparticles possess a better
efficiency than colloidal silica nanoparticles in forming peptide bond and it has an
efficiency of 98.8 % and high selectivity in the case of synthesizing alanine anhydride
from alanine.
3.3.6 Catalytic Property of Fumed Silica Nanoparticle Surfaces in Peptide Bond
Formation
86
It has been demonstrated that some amino acids are able to undergo thermal
condensation in the presence of inorganic oxides or mineral surfaces such as silica,
alumina, or clay since it was first discovered in 1978 [20-22, 77]. In this work, we
showed a good example where fumed silica nanoparticles are able to efficiently catalyze
the condensation of alanine, forming peptide bonds. More significantly, the formed
peptide is almost pure alanine anhydride, product of alanine dimerization. Though this
type of surface-catalyzed peptide bond formation reaction has been shown many times
during the past decades, the mechanism of the catalysis still remains unclear. However,
two things appear to be certain for the surface-catalyzed peptide bond formation reaction:
first, the peptide bond formation reaction from amino acids is thermodynamically
unfavored in aqueous solutions [78], however, it can be achieved in the adsorbed state on
surfaces where the water activity is low [20]. Second, keeping water activity low is the
driving force for the condensation reaction because it releases water as a product. In this
part of the discussion, we focus on understanding the role of the silica nanoparticle
surfaces in catalyzing the peptide bond formation by combining the findings from
previous research and the results we obtained in this work. Additionally, we also discuss
the possible reasons for the high efficiency for fumed silica nanoparticles. In the
discussion, diketopiperazine (DKP) will be used to stand for alanine anhydride since it is
frequently used to indicate the cyclized form of alanine.
Since the formation of alanine DKP involves the formation of two peptide bonds, a
possible two-step reaction mechanism has been proposed:

∆
Ala(ads) + Ala(ads) Ala-Ala (ads) + H2O (g) (I)
∆
Ala-Ala (ads) DKP (ads) + H2O (g) (II)
87
In step I, adsorbed alanine forms linear alanine dipeptide and it reacts further by
cyclisation to DKP in step II. Previous studies on glycine adsorbed on silica have shown
that the cyclization happens at a lower temperature than its initial linear dimerization. So
it might be the same case for alanine here in this work, where the linear alanine dipeptide
is formed and it would immediately undergo further cyclization to DKP at elevated
temperature.
According to the DTG results, the adsorbed alanine is the primary reservoir for DKP
formation at ~ 170 °C with a heating rate of 5 °C/min since the crystalline alanine shows
no significant thermal event below 200 °C. Furthermore, the little change in temperatures
of thermal condensation with the increasing amount of alanine on surfaces also indicates
that the adsorbed alanine is the main contribute for the condensation reaction. Based on
this finding, understanding the mechanism of DKP formation lies on unraveling the
interaction between adsorbed alanine and surface groups, and the behavior of adsorbed
alanine at the interfaces during thermal incubation. For the interaction between adsorbed
alanine and surface groups, solid-state NMR spectroscopy has provided some clues
regarding the binding state of alanine and more details can be found in our previous work
[48, 50, 58]. At very low water activity levels, the reorientation of adsorbed alanine is
restricted to some degree and it interacts with surface silanol groups via the carboxyl
group and protonated amine group (NH3+). This is likely the model describing the surface
chemistry at the very early stage of thermal incubation when the sample is being heated
up to 100 °C since most physisorbed water is removed from nanoparticles surfaces. In the
following thermal incubation with the temperature increasing up to 170 °C, the mobility
of the adsorbed alanine increases since the alanine-surface interaction may be broken and
88
reformed during this stage. This thermally driven reorientation makes alanine molecules
interact frequently with each other at the interfaces. Surface silanol groups are also able
to serve as the active sites for interacting with both carboxylate groups and amine groups,
which may lower the energy barrier for subsequent peptide bond formation reaction
between alanine molecules. When the temperature reaches the point of overcoming the
energy barrier of the peptide bond formation reaction (~170 °C with a heating rate of 5
°C in this work), the condensation is triggered and the peptide bond forms. This proposed
mechanism may give an explanation for forming peptide bond at lower temperature on
silica nanoparticle surfaces including fumed silica nanoparticles and colloidal silica
nanoparticles compared to that in bulk.
A O=C-OH B fumed nanoparticles

Si O Si alanine adsorbed fumed nanoparticles after
(d) thermal incubation at 170 oC for three hours
D1
(c)
D1
D2
(b) D2
Si O Si
(a)
300 400 500 600 700 800 900 300 400 500 600 700 800 900
Wavenumber (cm-1) Wavenumber (cm-1)
Figure 3.16. A) Raman spectra of (a) alanine adsorbed fumed silica nanoparticles (Ala/SiO2-
0.03M), (b) Ala/SiO2-0.03M after thermal incubation at 170 °C for three hours, (c) alanine
adsorbed colloidal silica nanoparticles, and (d) alanine adsorbed colloidal silica nanoparticles
after thermal incubation at 170 °C for three hours. B) Raman spectra of fumed silica (solid line)
and alanine adsorbed fumed silica nanoparticles after thermal incubation at 170 °C for three hours
(dashed line). The spectra were normalized using the peak area of the Si-O-Si band at ~800 cm-1.
To further understand the reason for the ultra high efficiency for fumed silica
nanoparticles, Raman spectra of alanine adsorbed fumed silica nanoparticles after
89
incubation at 170 °C for three hours was collected and compared to the fumed silica
nanoparticles (Figure 3.16B). Adsorbed alanine does not present in crystalline form since
there is no characteristic resonances were found for alanine in crystalline form
(Supplementary, Figure 3.23). More than that, the formed alanine anhydride was barely
detected in Raman spectrum due to the small amount at the interface and presence in
amorphous state, which is frequently found in surface chemistry studies without surface-
enhanced Raman scattering (SERS) techniques [79]. The spectra were normalized to the
amplitude of the band centered at ~800 cm-1 for carrying out a quantitative analysis. This
protocol is suitable and reliable because the band at ~800 cm-1 assigned to the symmetric
stretching vibration of the Si-O-Si group is very stable and this protocol has been proven
in previous studies [32, 80]. The intensity of D2 band showed a significant decrease while
the intensity of D1 band did not vary to any appreciable extent. Since D2 band is assigned
primarily to the three-membered siloxane ring structure, this result indicates that the
content of three-membered siloxane rings decreased during the processing and thermal
condensation. It has been suggested recently using electron paramagnetic resonance
(EPR) that the surface three-membered siloxane rings of fumed silica can form radicals
via hemolytic cleavage of siloxane bonds that can further generate hydroxyl radicals
when hydrolyzed [4]. Based on this finding, it may be reasonable to propose that surface
radicals may be generated on fumed silica nanoparticle surfaces during processing and
then catalyze the peptide bond formation reaction significantly. The contribution of
radicals to the high efficiency of fumed silica nanoparticles still remains uncertain and it
needs more investigation, however, it is important to note here that fumed silica
nanoparticles, in contrast to colloidal silica nanoparticles, has a considerable amount of
90
surface three-membered siloxane rings which can potentially serve as a reservoir for
surface radicals. In addition, a recent simulation study has proposed another mechanism
in which strained siloxane rings react first with carboxylic acids forming a -Si-O-C(=O)-
surface mixed anhydride, and the surface mixed anhydride undergo further reaction with
amines to from peptide bonds [18]. For more details on the mechanism, we will continue
our investigation and address more details in our future work.
3.4 Conclusions
Alanine adsorption and thermal condensation on fumed silica nanoparticles were
thoroughly investigated with 1H, 13

C and 15
N multi-nuclear, multi-dimensional MAS
solid-state NMR spectroscopy. Three species of alanine were detected on fumed silica
nanoparticles under different conditions: 1) crystalline and adsorbed alanine at high
surface coverage; 2) freely rotating and re-orientating adsorbed alanine interacting with
mobile interfacial water at high hydration level; 3) reorientation restricted adsorbed
alanine interacting with surface silanol groups via the carboxyl group and protonated
amine group (NH3+) under dry conditions at low surface. Furthermore, we carried out a
thorough investigation on thermal condensation of alanine on fumed silica nanoparticles
with a combination of thermal analysis and multiple spectroscopic techniques. It is found
that the adsorbed alanine can undergo a surface-catalyzed thermal condensation at round
170 °C, forming alanine anhydride with a very high yield of 98.8 %. This finding
suggests a potential way of synthesizing peptides with high efficiency and high
selectivity at inorganic nanostructured interfaces. After comparing to the study of
surface-catalyzed peptide bond formation on colloidal silica nanoparticles, it is found that
the high efficiency for fumed silica nanoparticles is likely related to the intrinsic strained
91
ring structures present at the nanoparticles surfaces. Furthermore, in this work, NMR
spectroscopy, IR spectroscopy and Raman spectroscopy have been successfully applied
for characterizing the alanine-silica interfaces, which demonstrates that these techniques
can be used in the future for more bio-inorganic interface research.
3.5 References
[1] F.S. Emami, V. Puddu, R.J. Berry, V. Varshney, S.V. Patwardhan, C.C. Perry, H.
Heinz, Prediction of specific biomolecule adsorption on silica surfaces as a function
of pH and particle size, Chem. Mater., 26 (2014) 5725-5734.
Res., 46 (2013) 792-801.
[3] A. Rimola, D. Costa, M. Sodupe, J.-F. Lambert, P. Ugliengo, Silica surface features
and their role in the adsorption of biomolecules: Computational modeling and
experiments, Chem. Rev., 113 (2013) 4216-4313.
[5] A.A. Shemetov, I. Nabiev, A. Sukhanova, Molecular interaction of proteins and

peptides with nanoparticles, ACS Nano, 6 (2012) 4585-4602.
[6] S.V. Patwardhan, F.S. Emami, R.J. Berry, S.E. Jones, R.R. Naik, O. Deschaume, H.
Heinz, C.C. Perry, Chemistry of aqueous silica nanoparticle surfaces and the
mechanism of selective peptide adsorption, J. Am. Chem. Soc., 134 (2012) 6244-
6256.
[7] M.A. Malfatti, H.A. Palko, E.A. Kuhn, K.W. Turteltaub, Determining the
pharmacokinetics and long-term biodistribution of SiO2 nanoparticles in vivo using
accelerator mass spectrometry, Nano Lett., 12 (2012) 5532-5538.
303-305.
92
397.
[10] G.A. Somorjai, H. Frei, J.Y. Park, Advancing the frontiers in nanocatalysis,
biointerfaces, and renewable energy conversion by innovations of surface
techniques, J. Am. Chem. Soc., 131 (2009) 16589-16605.
Nano Today, 4 (2009) 165-179.

Int. Ed., 47 (2008) 8582-8594.
[13] N.F. Breen, T. Weidner, K. Li, D.G. Castner, G.P. Drobny, A solid-state deuterium
NMR and sum-frequency generation study of the side-chain dynamics of peptides
adsorbed onto surfaces, J. Am. Chem. Soc., 131 (2009) 14148-14149.
[14] O. Mermut, D.C. Phillips, R.L. York, K.R. McCrea, R.S. Ward, G.A. Somorjai, In
situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic
polystyrene and hydrophilic silica surfaces using quartz crystal microbalance,
atomic force microscopy, and sum frequency generation vibrational spectroscopy, J.
Am. Chem. Soc., 128 (2006) 3598-3607.
[15] J.M. Gibson, J.M. Popham, V. Raghunathan, P.S. Stayton, G.P. Drobny, A solid-
state NMR study of the dynamics and interactions of phenylalanine rings in a
statherin fragment bound to hydroxyapatite crystals, J. Am. Chem. Soc., 128 (2006)
5364-5370.
[16] C. Graf, Q. Gao, I. Schütz, C.N. Noufele, W. Ruan, U. Posselt, E. Korotianskiy, D.

Nordmeyer, F. Rancan, S. Hadam, A. Vogt, J. Lademann, V. Haucke, E. Rühl,
Surface functionalization of silica nanoparticles supports colloidal stability in
physiological media and facilitates internalization in cells, Langmuir, 28 (2012)
7598-7613.
[17] J. Lu, M. Liong, J.I. Zink, F. Tamanoi, Mesoporous silica nanoparticles as a

delivery system for hydrophobic anticancer drugs, Small, 3 (2007) 1341-1346.
[18] A. Rimola, M. Sodupe, P. Ugliengo, Amide and peptide bond formation: Interplay
between strained ring defects and silanol groups at amorphous silica surfaces, J.
Phys. Chem. C, 120 (2016) 24817-24826.
[19] J.-F. Lambert, M. Jaber, T. Georgelin, L. Stievano, A comparative study of the

catalysis of peptide bond formation by oxide surfaces, Phys. Chem. Chem. Phys.,
15 (2013) 13371-13310.
[20] J.-F. Lambert, L. Stievano, I. Lopes, M. Gharsallah, L. Piao, The fate of amino
acids adsorbed on mineral matter, Planet. Space Sci., 57 (2009) 460-467.
93
[21] J.-F. Lambert, Adsorption and polymerization of amino acids on mineral surfaces:
A review, Orig. Life Evol. Biosph., 38 (2008) 211-242.
[22] D.A.M. Zaia, A review of adsorption of amino acids on minerals: Was it important
for origin of life?, Amino Acids, 27 (2004) 113-118.
[23] L.E. Orgel, Polymerization on the rocks: Theoretical introduction, Orig. Life Evol.
Biosph., 28 (1998) 227-234.
[24] R.S. McDonald, Surface functionality of amorphous silica by Infrared spectroscopy,

J. Phys. Chem., 62 (1958) 1168-1178.


[28] M. Lelli, D. Gajan, A. Lesage, M.A. Caporini, V. Vitzthum, P. Miéville, F.

Héroguel, F. Rascón, A. Roussey, C. Thieuleux, M. Boualleg, L. Veyre, G.
Bodenhausen, C. Coperet, L. Emsley, Fast characterization of functionalized silica
materials by silicon-29 surface-enhanced NMR spectroscopy using dynamic nuclear
polarization, J. Am. Chem. Soc., 133 (2011) 2104-2107.
Soc., 118 (1996) 5103-5119.
[30] C.C. Perry, X. Li, Structural studies of gel phases. Part 1.-Infrared spectroscopic
study of silica monoliths; the effect of thermal history on structure, J. Chem. Soc.,
Faraday Trans., 87 (1991) 761-766.

[32] D.D. Goller, R.T. Phillips, I.G. Sayce, Structural relaxation of SiO2 at elevated
temperatures monitored by in situ Raman scattering, J. Non-Cryst. Solids, 355
(2009) 1747-1754.
[33] B.A. Morrow, A.J. McFarian, Surface vibrational modes of silanol groups on silica,
J. Phys. Chem., 96 (1992) 1395-1400.
94
[34] A. Aboshi, N. Kurumoto, T. Yamada, T. Uchino, Influence of thermal treatments
on the photoluminescence characteristics of nanometer-sized amorphous silica
particles, J. Phys. Chem. C, 111 (2007) 8483-8488.
[35] V.A. Bakaev, C.G. Pantano, Inverse reaction chromatography. 2.

Hydrogen/deuterium exchange with silanol groups on the surface of fumed silica, J.
Phys. Chem. C, 113 (2009) 13894-13898.
[36] G. Hartmeyer, C. Marichal, B. Lebeau, S. Rigolet, P. Caullet, J. Hernandez,

Speciation of silanol groups in precipitated silica nanoparticles by 1H MAS NMR
spectroscopy, J. Phys. Chem. C, 111 (2007) 9066-9071.
[37] G. Vaccaro, S. Agnello, G. Buscarino, F.M. Gelardi, Thermally induced structural

modification of silica nanoparticles investigated by Raman and Infrared absorption
spectroscopies, J. Phys. Chem. C, 114 (2010) 13991-13997.
[39] A. Burneau, O. Barres, A. Vidal, H. Balard, G. Ligner, E. Papirer, Comparative

study of the surface hydroxyl groups of fumed and precipitated silicas. 3. DRIFT
characterization of grafted n-hexadecyl chains, Langmuir, 6 (1990) 1389-1395.

[42] C.T. Kresge, M.E. Leonowicz, W.J. Roth, J.C. Vartuli, J.S. Beck, Ordered
mesoporous molecular sieves synthesized by a liquid-crystal template mechanism,
Nature, 359 (1992) 710-712.
[43] A. Rimola, S. Tosoni, M. Sodupe, P. Ugliengo, Does silica surface catalyse peptide
bond formation? New insights from first-principles calculations, Chem. Phys.
Chem, 7 (2006) 157-163.
[44] N.N. Vlasova, L.P. Golovkova, The adsorption of amino acids on the surface of
highly dispersed silica, Coll. J., 66 (2004) 657-662.
[45] L. Stievano, L. Yu Piao, I. Lopes, M. Meng, D. Costa, J.-F. Lambert, Glycine and
lysine adsorption and reactivity on the surface of amorphous silica, Eur. J. Mineral.,
19 (2007) 321-331.
95
[46] N. Kitadai, T. Yokoyama, S. Nakashima, ATR-IR spectroscopic study of L-lysine
adsorption on amorphous silica, J. Colloid Interface Sci., 329 (2009) 31-37.
[47] T. Amitay-Rosen, S. Kababya, S. Vega, A dynamic magic angle spinning NMR

study of the local mobility of alanine in an aqueous environment at the inner surface
of mesoporous materials, J. Phys. Chem. B, 113 (2009) 6267-6282.
[48] I. Ben Shir, S. Kababya, T. Amitay-Rosen, Y.S. Balazs, A. Schmidt, Molecular

level characterization of the inorganic-bioorganic interface by solid state NMR:
Alanine on a silica surface, a case study, J. Phys. Chem. B, 114 (2010) 5989-5996.
[49] Q. Gao, W. Xu, Y. Xu, D. Wu, Y. Sun, F. Deng, W. Shen, Amino acid adsorption
on mesoporous materials: Influence of types of amino acids, modification of
mesoporous materials, and solution conditions, J. Phys. Chem. B, 112 (2008) 2261-
2267.
[50] I. Ben Shir, S. Kababya, A. Schmidt, Binding specificity of amino acids to

amorphous silica surfaces: solid-state NMR of glycine on SBA-15, J. Phys. Chem.
C, 116 (2012) 9691-9702.
[51] I. Ben Shir, S. Kababya, A. Schmidt, Molecular details of amorphous silica surfaces
determine binding specificity to small amino acids, J. Phys. Chem. C, 118 (2014)
7901-7909.
[52] M. Bouchoucha, M. Jaber, T. Onfroy, J.-F. Lambert, B. Xue, Glutamic acid

adsorption and transformations on silica, J. Phys. Chem. C, 115 (2011) 21813-
21825.
[53] N. Folliet, C. Gervais, D. Costa, G. Laurent, F. Babonneau, L. Stievano, J.-F.

Lambert, F. Tielens, A molecular picture of the adsorption of glycine in
mesoporous silica through NMR experiments combined with DFT-D calculations,
J. Phys. Chem. C, 117 (2013) 4104-4114.
[54] I. Lopes, L. Piao, L. Stievano, J.-F. Lambert, Adsorption of amino acids on oxide
supports: A solid-state NMR study of glycine adsorption on silica and alumina, J.
Phys. Chem. C, 113 (2009) 18163-18172.
[55] A. Rimola, M. Sodupe, P. Ugliengo, Affinity scale for the interaction of amino
acids with silica surfaces, J. Phys. Chem. C, 113 (2009) 5741-5750.
[56] M. Meng, L. Stievano, J.-F. Lambert, Adsorption and thermal condensation

mechanisms of amino acids on oxide supports. 1. Glycine on silica, Langmuir, 20
(2004) 914-923.
[57] J. Bujdák, B.M. Rode, Silica, alumina and clay catalyzed peptide bond formation:
enhanced efficiency of alumina catalyst., Orig. Life Evol. Biosph., 29 (1999) 451-
461.
96
[58] T. Amitay-Rosen, S. Vega, A deuterium MAS NMR study of the local mobility of
dissolved methionine and di-alanine at the inner surface of SBA-15, Phys. Chem.
Chem. Phys., 12 (2010) 6763-6711.
[59] J. Bujdák, B.M. Rode, Glycine oligomerization on silica and alumina, React. Kinet.
Catal. Lett., 62 (1997) 281-286.
[60] A.J. O’Connor, A. Hokura, J.M. Kisler, S. Shimazu, G.W. Stevens, Y. Komatsu,
Amino acid adsorption onto mesoporous silica molecular sieves, Sep. Purif.
Technol., 48 (2006) 197-201.
[61] T. Gullion, J. Schaefer, Rotational-echo double-resonance NMR, J. Magn. Reson.,

81 (1989) 196-200.
[62] V.M. Gunko, E.F. Voronin, E.M. Pakhlov, V.I. Zarko, V.V. Turov, N.V. Guzenko,
R. Leboda, Features of fumed silica coverage with silanes having three or two
groups reacting with the surface, Coll. Surf. A, 166 (2000) 187-201.
[63] V.V. Brei, 29Si solid-state NMR study of the surface structure of Aerosil silica, J.
Chem. Soc., Faraday Trans., 90 (1994) 2961-2964.
Rev., 128 (1962) 2042-2053.
[66] D.G. Cory, W.M. Ritchey, Suppression of signals from the probe in Bloch decay
spectra, J. Magn. Reson., 80 (1988) 128-132.
[67] A.E. Bennett, C.M. Rienstra, M.l. Auger, K.V. Lakshmi, R.G. Griffin,
Heteronuclear decoupling in rotating solids, J. Chem. Phys., 103 (1995) 6951-6958.
[68] S. Hayashi, K. Hayamizu, Chemical shift standards in high-resolution solid-state

NMR (2) 15N nuclei, Bull. Chem. Soc. Jpn., 64 (1991) 688-690.

NMR (1) 13C, 29Si, and 1H nuclei, Bull. Chem. Soc. Jpn., 64 (1991) 685-687.
[70] R.K. Hester, J.L. Ackerman, B.L. Neff, J.S. Waugh, Separated local field spectra in
NMR: Determination of structure of solids, Phys. Rev. Lett., 36 (1976) 1081-1083.
[71] E. Vinogradov, P.K. Madhu, S. Vega, High-resolution proton solid-state NMR

spectroscopy by phase-modulated Lee–Goldburg experiment, Chem. Phys. Lett.,
314 (1999) 443-450.
97
[72] M. Yang, Y. Nakazawa, K. Yamauchi, D. Knight, T. Asakura, Structure of model
peptides based on Nephila clavipes dragline silk spidroin (MaSp1) studied by 13C
cross polarization/magic angle spinning NMR, Biomacromolecules, 6 (2005) 3220-
3226.
[73] S. Fuchida, H. Masuda, K. Shinoda, Peptide formation mechanism on

montmorillonite under thermal conditions., Orig. Life Evol. Biosph., 44 (2014) 13-
28.
[74] M.P. Bhate, J.C. Woodard, M.A. Mehta, Solvation and hydrogen bonding in
alanine- and glycine-containing dipeptides probed using solution- and solid-state
Chem. Commun., 46 (2010) 6714-6713.
[76] A.R. Garcia, R.B. de Barros, J.o.P. Lourenço, L.M. Ilharco, The infrared spectrum
of solid L-alanine: Influence of pH-induced structural changes, J. Phys. Chem. A,
112 (2008) 8280-8287.
[77] N. Lahav, D. White, S. Chang, Peptide formation in the prebiotic era: Thermal
condensation of glycine in fluctuating clay environment, Science, 201 (1978) 67-69.
[78] A. Brack, From interstellar amino acids to prebiotic catalytic peptides: a review.,
Chem. Biodivers., 4 (2007) 665-679.
[80] R. Hemley, H. Mao, P. Bell, Raman spectroscopy of SiO2 glass at high pressure,
Phys. Rev. Lett., 57 (1986) 747-750.
98
Figure 3.17. 1H MAS NMR spectra of (a) dried fumed silica nanoparticles (b) 13C/15N-alanine,
(c) “hydrated” 13C/15N-Ala/ SiO2-0.03M, (d) “hydrated” 13C/15N-Ala/ SiO2-0.10M, (e) “dry”
13 15
C/ N-Ala/ SiO2-0.03M, (f) “dry” 13C/15N-Ala/ SiO2-0.10M. Experiments were conducted at 400
MHz and a MAS speed of 35 kHz for all samples with exception of dried fumed silica
nanoparticles (20 kHz). The resonance at -0.05 ppm is due to a silicone rubber impurity following
the vacuum drying process.
99
Figure 3.18. 1Hà13C (left) and 1Hà15N (right) 2D HETCOR NMR spectra of (a) 13C, 15N-Ala/
SiO2-0.03M (b) 13C, 15N-Ala/ SiO2-0.03M incubated at 170 °C for three hours.
0
Alanine anhyride
10 Peptide formed after thermal condensation
C Chemical Shift (ppm)
20
30
40
50
60
13
70
80
4.5 4.0 3.5 3.0 2.5 2.0 1.5 0.5
1
Figure 3.19. 1H-13C HSQC NMR spectra of alanine anhydride (black) and peptide formed at
fumed silica nanoparticles surfaces by incubating Ala/SiO2-0.03M at 170 °C for three hours (red).
100
(b) alanine
(a)
3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0
1
Figure 3.20. 1H NMR spectra of (a) alanine and (b) peptide formed at fumed silica nanoparticles
surfaces by incubating Ala/SiO2-0.03M at 170 °C for three hours. The spectra presented here
were scaled up 100x compared to the spectra in Figure 3.14a & 3.14b.
Figure 3.21. TGA (black) and DTG (red) curves for alanine adsorbed colloidal silica
nanoparticles. Heating rate is 5 °C/min.
101
Figure 3.22. FTIR spectra of (a) colloidal silica nanoparticles, (b) alanine, (c) alanine adsorbed
colloidal silica nanoparticles, (d) alanine adsorbed colloidal silica nanoparticles incubated at 170
°C for three hours, and (e) alanine anhydride.
Figure 3.23. Raman spectra of (a) alanine and (b) alanine anhydride in crystalline form.

102
CHAPTER 4
Investigating Lysine Adsorption on Fumed Silica Nanoparticles
4.1 Introduction
The nature of interactions between biomolecules and the surfaces of inorganic
materials has attracted considerable attention in recent years since it is of great
significance in many research fields, such as prebiotic chemistry [1-4], bio-
nanotechnology [5-10] and drug delivery [11-15]. For instance, some minerals have been
shown to catalyze peptide synthesis, providing a new explanation on the origin of life [1,
16]. In addition, some biomolecules with pharmaceutical properties can be adsorbed at
specific inorganic material interfaces and then released in vivo in a well-controlled way
[12, 14]. Adsorption of amino acids and peptides on silica surfaces is one specific class of
such bioorganic-inorganic interface systems. Silica was extensively studied and utilized
for various applications such as catalysis, solar cells and even cancer therapy [5, 10, 17].
Fumed silica is one class of synthetic silica materials with a high surface area [18]. It is
produced at high temperature by hydrolyzing silicon tetrachloride vapor in a flame
followed by rapid quenching to room temperature. Due to this, it acquires some unique
characteristics such as amorphous structure, nanoscale size and an extensively high
surface area. The studies of surface chemistry at fumed silica interfaces have been carried
out for decades because of the considerable utility of high surface area amorphous
silicates [18-32].
Lysine (Lys) has been used in a large number of studies due to its unique
structure, where there is one single side chain amine group. It is this side chain amine
group that makes lysine the simplest basic amino acid compared with the other two basic
103
amino acids, arginine and histidine. Due to this, lysine has been used in synthesizing a
range of nanomaterials [33-35]. Recently, Yokoi et al discovered that lysine is a good
ligand in synthesizing ultra-small silica nanoparticles (<10 nm) that show great potential
in future nanotechnology applications [35]. Figure 4.1 shows the summary of different L-
lysine forms and the charged state of a silica surface at variable pH values [36-39]. The
structure of bulk lysine has not been clearly defined but the structure of lysine mono-
hydrochloride dihydrate was determined by X-ray diffraction (XRD) and neutron
scattering techniques [40, 41].
pI=9.90
pK1 =2.20 pK2=9.10 pK3 =10.80
L-Lysine
LysH2 2+
LysH +
LysH Lys -
0 14
Silica
PZC=1.7 ~ 3.5
O
2+ +
LysH2 : H 3N (Dicationic Form)
OH
NH 3+
O
LysH + : +
H3 N (Cationic Form)
O
NH3 +
O
+
H 3N (Zwitterionic Form A)
O
NH 2
LysH :
O
H 2N (Zwitterionic Form B)
O
NH 3+
O
Lys - : H 2N (Anionic Form)
O
NH 2
Figure 4.1. Summary of different L-lysine forms and charged states of silica surface as a function
of pH values.
104
Considerable work has been done to understand the interaction between amino
acids and silica surfaces where the primary focus was on alanine and glycine adsorption
[42-54]. Less work has been performed to study lysine adsorption on silica surfaces [37,
38, 42, 52]. The main techniques that have been used to study the adsorption behavior of
lysine on surfaces of inorganic materials are IR spectroscopy and thermal analysis. By
using IR spectroscopy, it is easy to determine the protonation state of lysine on surfaces
at variable pH values [37, 38]. Thermal analysis, including thermal-gravimetric analysis
(TGA) and differential scanning calorimetry (DSC), has also been applied to study the
thermal transformation of lysine molecules on surfaces [42]. To date, solid-state NMR
techniques have not been used to study lysine adsorption and thermal transformation of
lysine molecules at silica interfaces. Compared with thermal analysis, solid-state NMR is
able to provide molecular and atomic level details [55]. Many solid-state NMR methods
and techniques have been developed and applied to study the interaction between amino
acids and silica surfaces [43, 44, 46-48, 51, 56, 57]. Shir et al. used rotational-echo
double-resonance (REDOR) NMR [58] to investigate the adsorbed state and the local
dynamics of alanine molecules on silica surface and they proposed that alanine is more
mobile when hydrated [44]. Also, Amitay-Rosen et al. used 2H NMR to study the
dynamics of water and alanine molecules adsorbed on silica surfaces [43].
In the present work, we applied solid-state NMR techniques including ultrafast
magic-angle-spinning [59, 60] (MAS) 1H NMR and multi-nuclear and multi-dimensional

13 15
C and N NMR to determine the structure of bulk lysine and investigate lysine
adsorbed on fumed silica nanoparticles. Furthermore, in order to elucidate the interaction
between lysine and silanol groups on silica surfaces, we performed density functional
105
theory [61, 62] (DFT) calculations for 1H, 13
C, 15
N chemical shifts of lysine-silanol
complex species and geometries. The combination of NMR experiments with DFT
calculations allows us to propose structural models for lysine interacting at silica
nanoparticle interfaces.
4.2.1 Materials
Fumed silica nanoparticles (~7 nm) with BET (Brunauer, Emmett and Teller)
surface area of 395 ± 25 m2/g and pure L-lysine (99% purity) were purchased from
Sigma-Aldrich. U-[15N,13C]-L-lysine·2HCl was purchased from Cambridge. Isotopes.
Inc. All materials were used as received and the stable isotope enrichment levels of
labeled compounds are 98%. U-[15N,13C]-L-lysine·2NaCl was prepared by crystallizing
in DI water after adjusting to pH~10 with 1.0 M NaOH.
4.2.2 Sample Preparation
Fumed silica nanoparticles used in the study were initially heated up to 500 °C for
24 hours to remove free water and impurities on the surface. In a typical adsorption
procedure, 150 mg of fumed silica nanoparticles was immersed in a 10.0 mL aqueous
solution of L-lysine with varying concentrations and the solution was stirred at room
temperature for 3 hours to ensure the adsorption reached equilibrium. The solid was then
separated by centrifugation and carefully dried under vacuum at 30 °C for over 15 hours.
The samples prepared from solutions of various concentrations were noted as Lys/SiO2-
xM, where x refers to the lysine concentration in the adsorption solution (in mol·L-1).
Since all experiments were carried out in pure DI water, the pH values of all the solutions
106
are around 10.0 (10.0 ± 0.3), corresponding to the isoelectric pH value of lysine in pure
water.
To prepare 13C, 15N-L-lysine adsorbed fumed silica samples, 60.0 mg fumed silica
nanoparticles and 9.0 mg 13C, 15N-L-lysine·2HCl (0.01 M) were mixed in DI water and
the solution pH was adjusted to 10.0 with 1.0 M NaOH. The volume of the final solution
is 4.0 mL and the suspension was then stirred for 3 hours to reach equilibrium. The
mixture was then centrifuged and the remaining powder was allowed to vacuum dry at 30
°C for over 15 hours.
4.2.3 Thermal-gravimetric Analysis (TGA)
TGA experiments were performed on Lys/SiO2-xM samples with a TA2910 (TA
Instrument Inc.) under N2 flow (60 mL/min for furnace and 40 mL/min for balance). The
heating rate was 5 °C/min and for each experiment, 7-10 mg of sample was used. Before
each experiment, the sample was kept under a N2 flow for 10 min to remove most of the
physisorbed water and obtain a stable baseline.

1
Hà13C and 1Hà15N cross-polarization magic-angle-spinning [63, 64] (CP-
MAS) NMR experiments, two-dimensional (2D) 13C-13C through-space correlation NMR

13
experiments with dipolar-assisted rotational resonance [65, 66] (DARR), 2D C-13C
through-bond double-quantum (DQ)/single-quantum (SQ) refocused incredible natural
abundance double quantum transfer NMR experiments [67, 68] (INADEQUATE) and 2D
15
N-13C heteronuclear correlation (HETCOR) NMR experiments were performed on
Varian VNMRS 400 MHz spectrometer. For the U-[15N,13C]-L-lysine·2HCl and natural
13
abundance Lys/SiO2 samples, 2D C-13C correlation experiments and CP-MAS NMR
107
experiments were collected with a 4.0 mm triple resonance probe operating in triple
resonance (1H/13C/15N) mode at a MAS speed of 10 kHz. The CP condition for 1Hà13C
CP-MAS NMR experiments consisted of a 4.0 µs 1H π/2 pulse, followed by a 2.0 ms
ramped (8 %) 1H spin-lock pulse of 62.5 kHz radio frequency (rf) field strength. The
experiments were performed with a 50 kHz sweep width, a recycle delay of 3.0 s and
two-pulse phase-modulated [69] (TPPM) 1H decoupling level of 65 kHz. The CP
condition for 1Hà15N CP-MAS NMR experiments consisted of a 3.25 µs 1H π/2 pulse,
followed by a 1.0 ms ramped (10 %) 1H spin-lock pulse of 75 kHz rf field strength. The
experiments were performed with a 50 kHz sweep width, a recycle delay of 3.0 s and 1H
decoupling level of 80 kHz. For the 13C, 15N-labeled Lys/SiO2 samples, CP-MAS NMR
experiments and 2D 15N-13C HETCOR NMR experiments were collected with a 3.2 mm
triple resonance probe operating in triple resonance (1H/13C/15N) mode at a MAS speed of
10 kHz. For 1Hà13C CP-MAS NMR experiments, the CP condition consisted of a 2.5 µs
1
H π/2 pulse, followed by a 1.0 ms ramped (8 %) 1H spin-lock pulse of 100 kHz rf field
strength. The experiments were performed with a 50 kHz sweep width, a recycle delay of
3.0 s and 1H decoupling level of 90 kHz. For 1Hà15N CP-MAS NMR experiments, the
CP condition consisted of 2.5 µs 1H π/2 pulse, followed by a 1.0 ms ramped (12 %) 1H
spin-lock pulse of 55 kHz rf field strength. The experiments were performed with a 25
kHz sweep width, a recycle delay of 5.0 s and 1H decoupling level of 90 kHz. 2D 13C-13C
through-space correlation NMR experiments, 2D 13C-13C INADEQUATE and 2D 15N-13C
HETCOR NMR experiments were used for assigning resonances (see supplementary for
experimental details).
108
1
H MAS NMR experiments and 2D 1H-13C HETCOR NMR experiments were
carried out on Varian VNMRS 800 MHz with a 1.6 mm triple resonance probe operating
in double resonance (1H/13C) mode. 1H MAS NMR experiments were collected with 2.0
µs 1H π/2 pulse, 25 kHz sweep width at 30 kHz MAS. 2D 1H-13C HETCOR NMR
experiments were done at spinning speed of 35 kHz. The 1Hà13C CP condition consisted
of a 2.6 µs 1H π/2 pulse, followed by a ramped (10 %) 1H spin-lock pulse of 100 kHz rf
field strength of variable contact time (0.25 ms, 2.0 ms). The sweep widths of direct
dimension and indirect dimension are 50 kHz and 25 kHz respectively with 32 complex t1
points. The recycle delay was 3.0 s and TPPM 1H decoupling with a rf field strength of
110 kHz was used during acquisition.
Ultrafast 1H MAS NMR experiments and 1H-1H back-to-back [70, 71] (BABA)
dipolar DQ/SQ correlation NMR experiments were carried out on a Bruker AVIII 850
MHz spectrometer equipped with a 1.3 mm double resonance probe (1H/13C) at a MAS
speed of 67 kHz. The experiments were done with one rotor period BABA for excitation
and reconversion, a 1.5 µs 1H π/2 pulse, relaxation delay of 5.0 s and 128 complex t1
points. In all experiments, the chemical shifts of 1H, 13

C and 15
N were indirectly
referenced to adamantane 1H (1.63 ppm), adamantane 13

C (38.6 ppm) and glycine 15
N
(31.6 ppm), respectively [72, 73].
4.2.5 DFT Calculation
Both geometry optimization and NMR chemical shifts calculation were
performed with B3LYP DFT method using 6-31G+(d, p) basis sets in Gaussian09 [74,
75]. It is well demonstrated that the B3LYP/6-31G+(d, p) level can provide reliable NMR
chemical shift results [49, 76, 77]. To reduce the computational cost, we used a silanol
109
(HOSiH3) molecule instead of using a complete silica surface model. In the calculations,
bulk lysine and lysine/silanol complex species (Figure 4.2A, 4.2B.) were geometrical
optimized first and then the optimized structures were used in NMR chemical shift
calculations. NMR chemical shifts calculations were performed using the Gauge-
Including Atomic Orbital [75, 78] (GIAO) method and 1H, 13

C and 15
N chemical shift
13 15
values were analyzed. In C and N data analysis, we applied two extrapolated curves
13 15
for C and N respectively to transform all calculated chemical shielding values to
chemical shift values. This method is demonstrated to be more accurate than simply using
a standard reference [76, 77]. For 1H, the calculated chemical shifts were referenced to 1H
chemical shift of tetramethylsilane (TMS) calculated with the same basis set.
(A) (B)
Figure 4.2. Bulk lysine and lysine/silanol complex models used in DFT calculations: A) Lysine,
B) Lys-H-OSiH3.
4.3.1 Protonation State of L-Lysine in the Solid State
The 1H MAS NMR spectra of natural abundance bulk lysine at different MAS
speeds are shown in Figure 4.3A and the 2D 1H-1H dipolar DQ/SQ NMR spectrum
110
collected with the BABA pulse sequence is shown in Figure 4.3B. With increasing
spinning speed, the resolution of the 1H MAS NMR spectrum for bulk lysine is improved
dramatically with well-resolved resonances observed for the spectrum collected with a 67
kHz MAS speed. According to 2D 1H-13C HETCOR NMR spectrum (Supplementary,
Figure 4.10), the resonances at 3.6 ppm and 2.2 ppm were assigned to α-CH and ε-CH2
respectively and that the broad component at 1.5 ppm is a combination of β-CH2, γ-CH2,
δ-CH2. It is difficult to determine the protonation states of the amine groups in bulk
lysine from the 1H MAS and 2D 1H-13C HETCOR NMR experiments. 1H-1H DQ/SQ
NMR experiment with a MAS speed of 67 kHz was applied to better assign the 1H
spectrum. In the 1H-1H DQ/SQ NMR spectrum, the resonance at 3.6 ppm was assigned to
α-CH since it has no on diagonal resonance and this result is consistent with the 2D 1H-
13
C HETCOR NMR result. The resonance at 8.4 ppm was assigned to α-NH3+ because
only strong DQ correlation to α-CH was observed with no obvious DQ correlation to ε-
CH2 (dashed circle). The result indicates that ε-NH2 is not protonated in bulk lysine and it
has a relatively small resonance that is probably convoluted by the broad component in
the 0-2 ppm region of the spectrum. DFT calculation further suggests that the ε-NH2
resonance probably appears around 0.7 ppm. The protonation state of lysine in its bulk
form is as proposed in Figure 4.3A and takes on the zwitterion form observed for most
amino acids such as alanine and glycine.
4.3.2 Adsorption Behavior of Lysine on Fumed Silica Nanoparticles
TGA is an accurate technique to measure the amount of adsorbed lysine on fumed
silica nanoparticles. Since all adsorbed amino acids will completely decompose when
heating to 600 °C under nitrogen gas flow, we can quantify the amount of lysine on the
111
surface from TGA curves. Figure 4.4A shows the TGA curves for lysine/silica samples
prepared from solutions with different initial lysine concentrations. Clearly, with
increasing initial concentration, the surface coverage increases. The weight loss between
100 °C and 600 °C is attributed to the amount of lysine on the surfaces and the
quantitative results are presented in Table 4.1. As expected, it shows that the adsorbed
amount of lysine increases when increasing the initial lysine concentration in solution.
The adsorption behavior of lysine molecules is described in Figure 4.4B. We applied
Langmuir isotherm to fit the data, showing that at low concentration, the adsorption
behavior of lysine fits well to a Langmuir isotherm but the fitting deviates at high
concentration (Figure 4.4B). This is mostly because at high concentration, the adsorption
behavior of lysine not only depends on the state of the surface, but also on the interaction
between lysine molecules. Based on the Langmuir isotherm fitting result for the first 5
points, ideally, the maximum amount of adsorbed lysine is 2.3 ± 0.2 molecule/nm2 and
the equilibrium constant is 32.2 ± 8.6 M-1.
(A)
β-CH2, δ-CH2, γ-CH2, ε-NH2
{
{
{
NH2
ε δ ε-CH2
α-NH3+
γ β
+ O α-CH
H3N
α 67 kHz
O-
30 kHz
14 12 10 8 6 4 2 0 -2 -4
1
112
β-CH2, δ-CH2, γ-CH2, ε-NH2
(B)
{
{
{
α-NH3+ ε-CH2
α-CH
-2
H Double Quantum Chemical Shift (ppm)

2
4
6
8
10
12
14
16
18
1
20
11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 -4
1
H Single Quantum Chemical Shift (ppm)
Figure 4.3. (A) 1H NMR spectra of natural abundance bulk lysine at MAS speeds of 30 kHz and
67 kHz at a 1H Larmor frequency of 800 MHz and 850 MHz, respectively. (B) 1H-1H BABA
NMR spectrum of natural abundance bulk lysine at spinning speed of 67 kHz at a 1H Larmor
frequency of 850 MHz.
1.05 2.50
Adsorbed lysine (molecules/nm2)
1.00 0M 2.00
Weight Percentage
0.95 0.01 M
1.50
0.03 M
0.90
0.05 M
0.08 M 1.00
0.85
0.15 M
0.80 0.10 M 0.50 Langmuir isotherm fitting for all points
0.12 M Langmuir isotherm fitting for first 5 points
0.75 0.00
100 200 300 400 500 600 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16
Temperature (oC) [Lysine] (mol/L)
(A) (B)
Figure 4.4. (A) TGA curves of lysine/silica samples as a function of initial concentration of
lysine in the adsorption solution. (B) The amount of lysine adsorbed on silica as a function of
initial concentration of lysine at room temperature and pH=10.0.
113
Table 4.1. Effect of initial concentration of solution on lysine adsorption on fumed silica
nanoparticles from TGA
Free water Total adsorbed Surface coverage
[Lysine] Lysine (wt %)
(wt %) amount (wt %) (molecules/nm2)
0.01 M 1.16 5.85 7.01 0.7
0.03 M 1.39 9.06 10.45 1.1
0.05 M 1.13 11.85 12.98 1.4
0.08 M 1.34 13.51 14.85 1.7
0.10 M 1.63 14.62 16.25 1.8
0.12 M 1.48 15.99 17.47 2.0
0.15 M 2.12 17.74 19.86 2.3

4.3.3 Adsorption State of Lysine on Fumed Silica Nanoparticles

1
Hà13C and 1Hà15N CP-MAS NMR experiments were applied in this work to
investigate the adsorption state of lysine at interfaces of nanoparticles. Figure 4.5 shows
the 1Hà13C and 1Hà15N CP-MAS NMR spectra of three different lysine samples and
two lysine/silica samples. The 13C and 15N resonance assignments are shown in Table 4.2.
Based on the carbon and nitrogen NMR spectra and Table 4.2, several conclusions can be
drawn. First, lysine has three different protonation states. For natural abundance lysine, it
was shown from the 2D 1H-1H dipolar DQ/SQ NMR spectrum that the ε-NH2 is
deprotonated and the α-NH2 is protonated (Figure 4.3). The 13C resonances at 177 ppm,
55 ppm, 35 ppm, 22 ppm, 32 ppm and 44 ppm were assigned to carboxyl group, a-CH, b-
CH, g-CH, d-CH and ε-CH2 respectively and the 15N resonances at 39 ppm and 24 ppm
were assigned to α-NH3+ and ε-NH2 respectively based on INADEQUATE and 2D 15N-
13
C HETCOR NMR experiments, respectively (Supplementary, Figure 4.11, Figure
13 15 15
4.12). For C, N-Lysine·2HCl, the N resonances of both amine groups have large
downfield shifts due to protonation and hydrogen bonding interaction with Cl-. In
13
addition, the C resonance of the carboxyl group shifts upfield by 6 ppm to 171 ppm,
13 15 13
indicating that the carboxyl group is protonated. For C, N-Lysine·2NaCl, the C
114
resonances of the carboxyl group and α-CH are identical to those of natural abundance
lysine, indicating the carboxyl group is deprotonated and the α-NH2 is protonated.
However, two resonances are found in ε-CH2 region with one at 40 ppm and the other at
43 ppm, indicating that there is a mixture of protonated and deprotonated components.
The proposed structure of each sample is presented in Table 4.2.
Comparing the lysine spectra with Lysine/SiO2 spectra, it is found that for both
13 15 13
the Lys/SiO2-0.01M and the C, N-Lysine/SiO2 the C resonances of the carboxyl
group and α-CH and the 15N resonance of α-NH2 are almost identical to those of natural
abundance lysine, indicating the carboxyl group is deprotonated and the α-NH2 is
protonated (α-NH3+). However, the 13C resonance of ε-CH2 has an identical value as that
13 15 15
of C, N-Lysine·2HCl and N resonance of ε-NH2 has an 8 ppm downfield shift
compared with that of natural abundance lysine, indicating that the ε-NH2 is protonated,
forming a strong hydrogen bonding interaction with the surface silanol groups. To prove
this hypothesis, we further applied 2D 1H-13C HETCOR NMR experiment to investigate
the correlation between silanol groups and adsorbed lysine. The results of the HETCOR
NMR experiment are discussed in the following paragraph. It is also worth mentioning
13 15
that one extra resonance at 182 ppm is found in carboxyl group region for C, N-
Lysine/SiO2. This is probably due to a small amount of NaCl present in the sample,
forming a lysine/NaCl complex [57].
115
(A)
ε-CH2 β-CH2 δ-CH2
C=O α-CH γ-CH2
(a) Bulk Lysine
(b) 13
C, 15N - Lysine*2HCl
(c) 13
C, 15N - Lysine*2NaCl
(d) Lysine/SiO2-0.01M
(e) 13
C, 15N - Lysine/SiO2
200 180 160 60 40 20 0

13
(B)
α-NH3+ ε-NH2
(a) Bulk Lysine
(b) 13
C, 15N - Lysine*2HCl
ε-NH3+
(c) 13
C, 15N - Lysine/SiO2
140 120 100 80 60 40 20 0 -20 -40

15
N Chemical Shift (ppm)
Figure 4.5. Top: 1Hà13C CP-MAS NMR spectra of (a) bulk lysine, (b) 13C, 15N-Lysine·2HCl, (c)
13
C, 15N-Lysine·2NaCl, (d) Lys/SiO2-0.01M and (e) 13C, 15N-Lysine/SiO2 (0.01 M). The spectra
were collected with a MAS speed of 10 kHz, a relaxation delay time of 3 s and a contact time of
1.0 ms. Bottom: 1Hà15N CP-MAS NMR spectra of (a) bulk lysine, (b) 13C, 15N-lysine·2HCl and
(c) 13C, 15N-Lysine/SiO2 (0.01 M).
116
Table 4.2. 13C and 15N chemical shifts of lysine and lysine/SiO2 samples a
Natural 13
C, 15N- 13
C, 15N- Lys/ SiO2- 13
C, 15N-
Sample Abundance
Lysine·2HCl Lysine·2NaCl 0.01M Lysine/SiO2
Lysine
C=O 177 171 177 176 176, 182
Cα 55 53 55 55 55
Cβ 35 26, 31 33 30 30
Cγ 22 21, 23 22 23 23
Cδ 32 25, 26 31, 25 27 27
Cε 44 40 43, 40 40 40
α-NH2 /α-NH 3+ 39 45 - - 39
ε-NH2 /ε-NH3+ 24 40 - - 32
a
Chemical shifts are reported in ppm.

β-CH2, δ-CH2, γ-CH2
ε-CH2
ε-NH2-H-OSi-
α-CH
(b) α-NH3+
(a)
14 12 10 8 6 4 2 0 -2 -4
1
Figure 4.6. 1H NMR spectra of (a) natural abundance bulk lysine and (b) 13C, 15N-Lysine/SiO2 at
a MAS speed of 67 kHz.

The 1H MAS spectra of natural abundance pure lysine and 13
C, 15
N-Lysine/SiO2
at a MAS speed of 67 kHz are shown in Figure 4.6. It is found that 1H resonances of α-
CH and ε-CH2 for adsorbed lysine have small offsets compared to those of bulk lysine.
This is probably due to the change of structure and protonation state during the
adsorption. The broad resonance at around 7.0 ppm is assigned to the protonated amine
groups (ε-NH3+) interacting with the surface silanol groups at the silica nanoparticle
interface. Figure 4.7 shows the 2D 1H-13C HETCOR NMR spectrum of 13

C, 15
N-
117
Lysine/SiO2 with different CP contact times (0.25 ms, 2.0 ms). Since 2D 1H-13C
HETCOR NMR experiment is a dipolar-based experiment, where magnetization is
transferred through space, long-range correlations can be detected by applying a
relatively long CP contact time. In addition to seeing the expected direct 1H-13C
correlations, the correlation shown around 7.4 ppm in 1H dimension of the spectrum with
a 2.0 ms contact time is assigned to the correlation between silanol group and the ε-CH2
of adsorbed lysine. This result provides strong evidence that the side-chain amine group
of adsorbed lysine interacts with silanol groups on silica surfaces and gets protonated.
α-CH ε-CH2 β-CH2 δ-CH2 γ-CH2
-6
0.25 ms 2.0 ms
-4
-2
6
1
10
65 60 55 50 45 40 35 30 25 20 65 60 55 50 45 40 35 30 25 20
13
Figure 4.7. H- C 2D HETCOR NMR spectrum of 13C, 15N-Lysine/SiO2 with different mixing
1 13
times (0.25 ms, 2.0 ms). Experiments were done at a 1H Larmor frequency of 800 MHz with a 1.6
mm triple resonance probe operating in double resonance (1H/13C) mode and a MAS speed of 35
kHz.

118
The 13C, 15N-Lysine/SiO2 sample prepared in this work involves sodium chloride
introduced by the pH adjustment process with NaOH during adsorption. The NaCl is
difficult to remove since the solubility of lysine in water is similar to that of sodium
chloride. To understand if sodium chloride will impact the lysine adsorption on silica
surfaces, we carried out several experiments with natural abundance lysine that is initially
salt free and applied 1Hà13C CP-MAS NMR spectroscopy to characterize the state of
adsorbed lysine. The results are shown in Figure 4.8 for (a) Lys/SiO2-0.01M sample, (b)
Lys/SiO2-0.10M sample and (c) the sample prepared in a similar way as Lys/SiO2-0.10M
sample with 0.20 M NaCl (Lys/SiO2-0.10M/NaCl). Based on the NMR results, it is easy
to determine that salt free Lys/SiO2-0.10M sample shows a small peak at 164 ppm that
was not observed in the spectra of both Lys/SiO2-0.01M sample and Lys/SiO2-
0.10M/NaCl sample. This is because lysine molecules form a monolayer on silica
nanoparticles for Lys/SiO2-0.01M/NaCl and Lys/SiO2-0.10M/NaCl samples while they
form multilayers for Lys/SiO2-0.10M sample. The resonance at 164 ppm is assigned to
carbonyl carbon of carbamates since primary amines are known to be able to react with
CO2 to form alkyl-ammonium and alkylcarbamates according to a reaction shown in
Figure 4.8 [77, 79, 80]. The formed carbamates can interact with adsorbed lysine in the
salt free system by forming hydrogen bonding with protonated α-NH3+, making it
detectable by 1Hà13C CP-MAS NMR spectroscopy. As a result, all amine groups of
adsorbed lysine are protonated in the former case, preventing them from reacting with
CO2 in air. After introducing NaCl into Lys/SiO2-0.10M, both sodium ions and chloride
ions can break the hydrogen bonding system formed by carbamates and α-NH3+ of
adsorbed lysine, making the small peak disappear. This point also agrees with the TGA
119
result where the surface coverage of sample (c) is about 1.60 molecules/nm2. This is
lower than the surface coverage of Lys/SiO2-0.10M sample (1.82 molecules/nm2).
According to these two results, we conclude that sodium chloride decreased the amounts
of adsorbed lysine and it prevented lysine from forming multilayers on silica surfaces.
The sample (c) is in fact a monolayer sample, where the surface coverage of lysine
reaches the maximum for monolayer adsorption (~1.60 molecules/nm2).
H H
H
H CO2 N R -H+ N R
N R
O C O C
H
O O
β-CH2
ε-CH2
α-CH δ-CH2
C=O
γ-CH2
(c) Lys/SiO2-0.10M/NaCl
(b) Lys/SiO2-0.10M
(a) Lys/SiO2-0.01M
200 180 160 60 40 20 0

13
C chemical shift (ppm)
Figure 4.8. 1Hà13C CP-MAS NMR spectra of (a) Lys/SiO2-0.01M, (b) Lys/SiO2-0.10M, and (c)
Lys/SiO2-0.10M/NaCl.

120
4.3.4 DFT Calculation
To elucidate the adsorption state of lysine on silica surfaces and to determine the
exact complex structure lysine forms with surface silanol group, we applied DFT
calculations. Generally, we optimized structures and calculated the chemical shifts for
bulk lysine and a possible lysine/silanol complex determined from NMR experiments
(Figure 4.9). In this work, we extrapolated curves for 13C and 15N to convert all calculated
13
chemical shielding values to chemical shift values. The extrapolated equation for C
chemical shifts was derived based on the experimental and calculated 13C chemical shift
values of bulk lysine since the structure of bulk lysine was elucidated in this work.
15
Extrapolated equation of N chemical shift was obtained from Alexandra Dos et al’s
15
work [76, 77] since they studied the structure of poly-L-lysine systematically by N
13 15
NMR and DFT calculations. The extrapolated equations of C and N chemical shifts
are as follows:
13
C: δExtrapolate= -1.123* σCal +206.1 ppm
15
N: δExtrapolate= -0.778* σCal +212.9 ppm
13 15
By using these equations, all calculated C and N chemical shielding values were
converted to chemical shift values and are presented in Table 4.3. It is found that the N-H
distance in hydrogen bonding system is 1.807 Å for Lys-H-OSiH3, corresponding to
hydrogen bonding energy of 42.01 kJ/mol (Supplementary, Table 4.4). From chemical
shift calculations, the α-NH3+ protons of bulk lysine have three different chemical shifts
at 11.6, 1.9, 0.5 ppm due to the asymmetry of the protons. After averaging the calculated
chemical shifts, the average value (4.7 ppm) is still far off the experiment result. This is
probably due to the fact that the α-NH3+ group may interact with other groups like
121
carboxylate group and Cl- ions or the model used for calculation is not reliable enough to
get reliable α-NH3+ 1H chemical shifts [81]. For other groups, the 1H chemical shifts are
very close to the experimental results. For Lys-H-OSiH3, ε-NH3+ group shows calculated
chemical shifts of 7.5, 1.1, 0.7 ppm where the hydrogen bonding proton has a chemical
shift of 7.5 ppm. Considering the slow free rotation of the ε-NH3+ group due to the strong
hydrogen binding, the calculation result is consistent with experimental result (7.4 ppm).
Moreover, the calculated 13C chemical shift of ε-CH2 for Lys-H-OSiH3 is only 1 ppm off
the experimental data and the calculated 15N chemical shift of α-NH3+ and ε-NH3+ have 2
ppm and 4 ppm off the experimental data respectively for Lys-H-OSiH3. Combining the
calculated and experimental results, it is convincing to argue that the lysine side-chain
amine group is the dominant hydrogen-bonding interaction with surface silanol groups at
silica nanoparticles and Lys-H-OSiH3 complex is the most probable structure. Combined
with NMR experiment results, the proposed favorable model for lysine adsorption on
fumed silica nanoparticles surfaces was presented in figure 4.9.
11.6
O H 1.9
H
N
O H H 0.6
C
2.8
H2C
1.8 1.2
CH2
H2C
1.6 2.7
CH2
7.5 N
H
0.7
H
H
O
1.1
Si
H
H
H
122
Table 4.3. Calculated 1H, 13C and 15N chemical shielding values and extrapolated chemical shifts
for lysine and lysine/silanol complex from DFT calculations a
Lysine LysH+…-OSiH3
Nucleus Group
Calculated Experimental Calculated Extrapolated Experimental
C=O 26.0 177 26.0 177 177
α-CH 133.0 55 133.2 57 55
13 β-CH2 154.3 35 154.5 33 30
C
γ-CH2 162.2 22 162.4 24 23
δ-CH2 155.4 32 158.4 28 27
ε-CH2 145.3 44 146.7 41 40
15 α-NH3+ 226.3 39 226.7 37 39
N
ε-NH2/ ε-NH3+ 232.7 24 227.8 36 32
α-CH 2.8 3.6 2.8 - 3.6
β-CH2 1.8 1.8 1.8 - 1.5
γ-CH2 1.2 1.5 1.2 - 1.2
1
H δ-CH2 1.5 2.0 1.6 - 1.5
ε-CH2 2.8 2.2 2.7 - 2.6
+
α-NH3 11.6, 1.9, 0.5 8.4 11.6, 1.9, 0.6 - 8.4
ε-NH2/ ε-NH3+ 0.7 - 7.5, 1.1, 0.7 - 7.4
a
Chemical shifts are reported in ppm.
Figure 4.9. Schematic representation of the favorable model for lysine adsorption on fume silica
nanoparticles surfaces.
123
4.4 Conclusions
Structure of bulk lysine and lysine adsorbed on fumed silica nanoparticles were
thoroughly investigated by ultrafast MAS 1H, 13

C and 15
N NMR spectroscopy. Bulk L-
lysine has protonated α-NH3+ and deprotonated ε-NH2. Lysine adsorbed on fumed silica
nanoparticles from solution interacts with silica surfaces through hydrogen bonding
between side-chain amine groups and surface silanol groups. Combined with DFT
calculations, we further proposed that the Lys-H-OSiH3 complex is the favorable model
for the lysine adsorption state on silica surfaces. The proposed model can be used to
elucidate the mechanism of synthesizing ultra-small silica nanoparticles (~10 nm) with
lysine as capping ligands and it is of use to researchers interested in surface
functionalization and modification. Also, the agreement of DFT calculations of NMR
chemical shifts with the corresponding experimental values is good in general, which
means the combination of solid-state NMR and DFT chemical shift calculations can
indeed be used to study surface chemistry at the interface of biomolecules and
nanoparticles.
4.5 References

15 (2013) 13371-13310.
124
Res., 46 (2013) 792-801.
6256.

7598-7613.
[10] C.E. Ashley, E.C. Carnes, K.E. Epler, D.P. Padilla, G.K. Phillips, R.E. Castillo,
D.C. Wilkinson, B.S. Wilkinson, C.A. Burgard, R.M. Kalinich, J.L. Townson, B.
Chackerian, C.L. Willman, D.S. Peabody, W. Wharton, C.J. Brinker, Delivery of
small interfering rna by peptide-targeted mesoporous silica nanoparticle-supported
lipid bilayers, ACS Nano, 6 (2012) 2174-2188.
303-305.
397.
[13] J. Liu, A. Stace-Naughton, X. Jiang, C.J. Brinker, Porous nanoparticle supported

lipid bilayers (protocells) as delivery vehicles, J. Am. Chem. Soc., 131 (2009)
1354-1355.

125
[15] M. Vallet-Regi, A. Rámila, R.P. del Real, J. Pérez-Pariente, A new property of
MCM-41: Drug delivery system, Chem. Mater., 13 (2001) 308-311.
Biosph., 28 (1998) 227-234.
[19] F. Tielens, C. Gervais, J.F.o. Lambert, F. Mauri, D. Costa, Ab initio study of the
hydroxylated surface of amorphous silica: A representative model, Chem. Mater.,
20 (2008) 3336-3344.
Soc., 118 (1996) 5103-5119.

J. Phys. Chem., 62 (1958) 1168-1178.
J. Phys. Chem., 96 (1992) 1395-1400.


Phys. Chem. C, 113 (2009) 13894-13898.
126

[31] K. Saalwächter, M. Krause, W. Gronski, Study of molecular interactions and

dynamics in thin silica surface layers by proton solid-state NMR spectroscopy,
Chem. Mater., 16 (2004) 4071-4079.

[33] T. Yokoi, J. Wakabayashi, Y. Otsuka, W. Fan, M. Iwama, R. Watanabe, K.

Aramaki, A. Shimojima, T. Tatsumi, T. Okubo, Mechanism of formation of
uniform-sized silica nanospheres catalyzed by basic amino acids, Chem. Mater., 21
(2009) 3719-3729.
[34] M.M. Tomczak, D.D. Glawe, L.F. Drummy, C.G. Lawrence, M.O. Stone, C.C.
Perry, D.J. Pochan, T.J. Deming, R.R. Naik, Polypeptide-templated synthesis of
hexagonal silica platelets, J. Am. Chem. Soc., 127 (2005) 12577-12582.
[35] T. Yokoi, Y. Sakamoto, O. Terasaki, Y. Kubota, T. Okubo, T. Tatsumi, Periodic

arrangement of silica nanospheres assisted by amino acids, J. Am. Chem. Soc., 128
(2006) 13664-13665.
[36] S.H. Behrens, D.G. Grier, The charge of glass and silica surfaces, J. Chem. Phys.,
115 (2001) 6716-6721.
[37] N. Kitadai, T. Yokoyama, S. Nakashima, In situ ATR-IR investigation of L-lysine

adsorption on montmorillonite, J. Colloid Interface Sci., 338 (2009) 395-401.

[39] A.D. Roddick-Lanzilotta, P.A. Connor, A.J. McQuillan, An in situ infrared

spectroscopic study of the adsorption of lysine to TiO2 from an aqueous solution,
Langmuir, 14 (1998) 6479-6484.
127
[40] T.F. Koetzle, M.S. Lehmann, J.J. Verbist, W.C. Hamilton, Precision neutron
diffraction structure determination of protein and nucleic acid components. VII. The
crystal and molecular structure of the amino acid L-lysine monohydrochloride
dihydrate, Acta Cryst., B28 (1972) 3207-3214.
[41] D.A. Wright, R.E. Marsh, The crystal structure of L-lysine monohydrochloride
dihydrate, Acta Cryst., 15 (1962) 54-64.
19 (2007) 321-331.


level characterization of the inorganic−bioorganic interface by solid state NMR:
2267.

C, 116 (2012) 9691-9702.
7901-7909.
Phys. Chem. C, 113 (2009) 18163-18172.

(2004) 914-923.
Chem. Phys., 12 (2010) 6763-6711.
128
Technol., 48 (2006) 197-201.

21825.
4300.
[56] P.A. Mirau, J.L. Serres, M. Lyons, The structure and dynamics of poly(L-lysine) in
templated silica nanocomposites, Chem. Mater., 20 (2008) 2218-2223.
[57] R. Manríquez, F.A. López-Dellamary, J. Frydel, T. Emmler, H. Breitzke, G.

Buntkowsky, H.-H. Limbach, I.G. Shenderovich, Solid-state NMR studies of
aminocarboxylic salt bridges in L-lysine modified cellulose, J. Phys. Chem. B, 113
(2009) 934-940.

81 (1989) 196-200.
[60] S.P. Brown, Probing proton–proton proximities in the solid state, Prog. Nucl. Magn.
Reson. Spectrosc., 50 (2007) 199-251.
[61] P. Hohenberg, W. Kohn, Inhomogeneous electron gas, Phys. Rev., 136 (1964) 864-
871.
[62] W. Kohn, L.J. Sham, Self-consistent equations including exchange and correlation
effects, Phys. Rev., 140 (1965) 1133-1138.
Rev., 128 (1962) 2042-2053.
[65] K. Takegoshi, S. Nakamura, T. Terao, 13C-1H dipolar-assisted rotational resonance

in magic-angle spinning NMR, Chem. Phys. Lett., 344 (2001) 631-637.
129
[66] K. Takegoshi, S. Nakamura, T. Terao, 13C–1H dipolar-driven 13C–13C recoupling
without 13C rf irradiation in nuclear magnetic resonance of rotating solids, J. Chem.
Phys., 118 (2003) 2325-2341.
[67] A. Lesage, C. Auger, S. Caldarelli, L. Emsley, Determination of through-bond

carbon-carbon connectivities in solid-state NMR using the INADEQUATE
experiment, J. Am. Chem. Soc., 119 (1997) 7867-7868.
[68] A. Lesage, M. Bardet, L. Emsley, Through-bond carbon−carbon connectivities in

disordered solids by NMR, J. Am. Chem. Soc., 121 (1999) 10987-10993.
[70] M.e.a. Feike, Broadband multiple-quantum NMR spectroscopy, J. Magn. Reson.,

122 (1996) 214-221.
[71] I. Schnell, H.W. Spiess, High-resolution 1H NMR spectroscopy in the solid state:
Very fast sample rotation and multiple-quantum coherences, J. Magn. Reson., 151
(2001) 153-227.


[74] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J.
Chem. Phys., 98 (1993) 5648-5652.
[75] K. Wolinski, J.F. Hinton, P. Pulay, Efficient implementation of the gauge-

independent atomic orbital method for NMR chemical shift calculations, J. Chem.
Soc., Chem. Commun., 112 (1990) 8251-8260.
[76] A. Dos, V. Schimming, M.C. Huot, H.-H. Limbach, Acid-induced amino side-chain
interactions and secondary structure of solid poly-L-lysine probed by 15N and 13C
solid state NMR and ab initio model calculations, J. Am. Chem. Soc., 131 (2009)
7641-7653.
[77] A. Dos, V. Schimming, S. Tosoni, H.-H. Limbach, Acid−base interactions and

secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR and
ab initio model calculations, J. Phys. Chem. B, 112 (2008) 15604-15615.
[78] G. Schreckenbach, T. Ziegler, Calculation of NMR shielding tensors using gauge-

including atomic orbitals and modern density functional theory, J. Phys. Chem., 99
(1995) 606-611.
130
[79] V. Schimming, C.-G. Hoelger, G. Buntkowsky, I. Sack, J.-H. Fuhrhop, S.
Rocchetti, H.-H. Limbach, Evidence by 15N CPMAS and 15N−13C REDOR NMR
for fixation of atmospheric CO2 by amino groups of biopolymers in the solid state,
J. Am. Chem. Soc., 121 (1999) 4892-4893.
[80] S. Maeda, S. Oumae, S. Kaneko, K.-K. Kunimoto, Formation of carbamates and

cross-linking of microbial poly(ε-L-lysine) studied by 13C and 15N solid-state NMR,
Polym. Bull., 68 (2011) 745-754.
[81] J. Schmidt, A. Hoffmann, H.W. Spiess, D. Sebastiani, Bulk chemical shifts in

hydrogen-bonded systems from first-principles calculations and solid-state-NMR, J.
Phys. Chem. B, 110 (2006) 23204-23210.
4.6 Supplementary
Figure 4.10. 1H-13C HETCOR NMR spectrum of bulk lysine.
The experiment was done at 800 MHz with a 1.6 mm triple resonance probe
operating in double resonance (1H/13C) mode and a MAS speed of 35 kHz. The 1Hà13C
CP condition consisted of a 2.6 µs 1H π/2 pulse, followed by a ramped (5 %) 1H spin-lock
pulse of 100 kHz rf field strength of variable contact time (0.25 ms, 2.0 ms). The sweep
131
widths of direct dimension and indirect dimension are 50 kHz and 25 kHz respectively
with 32 complex t1 points. The recycle delay was 3.0 s and TPPM 1H decoupling with a
rf field strength of 110 kHz was used during acquisition.
Figure 4.11. 2D 13C-13C through-bond double-quantum (DQ)/single-quantum (SQ) refocused

INADEQUATE NMR spectrum of 13C, 15N-Lysine·2HCl.
13
In 2D C-13C through-bond double-quantum (DQ)/single-quantum (SQ)
refocused INADEQUATE1,2 NMR experiments, the 1Hà13C CP condition consisted of a
4.0 µs 1H pulse, followed by a 2.0 ms ramped (3 %) 1H spin-lock pulse of 62.5 kHz rf
field strength. The experiments were collected with a 50 kHz sweep width in both
dimensions, a recycle delay of 3.0 s, 256 t1 complex points, a τ delay of 3.0 ms and
TPPM 1H decoupling with a rf field strength of 70 kHz.
132
Figure 4.12. 15N-13C 2D HETCOR NMR spectrum of 13C, 15N-lysine/SiO2.
The spectrum was collected with a 3.2 mm triple resonance probe operating in
triple resonance (1H/13C/15N) mode and a contact time of 1.0 ms at a MAS speed of 10
kHz. In 2D 15N-13C HETCOR NMR experiments were performed with a double cross
polarization sequence, where the magnetization was first transferred from 1H to 15N, and
then transferred further to 13C from 15N. The CP condition for 1Hà15N consisted of 2.5
µs 1H π/2 pulse, followed by a 1.0 ms ramped (6%) 1H spin-lock pulse of 55 kHz rf field
strength and the CP condition for 15Nà13C consisted of a 1.0 ms ramped (3 %) 15N spin-
lock pulse of 45 kHz rf field strength with continuous wave (CW) 1H decoupling of 90
kHz. The sweep widths of direct dimension and indirect dimension were 50 kHz and 10
kHz respectively with 64 complex t1 points. The recycle delay was 3 s and TPPM 1H
decoupling with a rf field strength of 90 kHz was used during acquisition.
133
Figure 4.13. 2D 13C-13C through-space correlation NMR spectrum of 13C, 15N-Lysine·2HCl.
13
In 2D C-13C through-space correlation experiments, the 1Hà13C cross-polarization
condition consisted of a 4.0 µs 1H pulse, followed by a 2.0 ms ramped (3 %) 1H spin-lock
pulse of 62.5 kHz rf field strength. The sweep widths of direct dimension and indirect
dimension were 50 kHz and 25 kHz respectively with 256 t1 complex points. The recycle
delay was 3.0 s and TPPM 1H decoupling with a rf field strength of 70 kHz was used
during acquisition. During the dipolar-assisted rotational resonance (DARR)3,4 mixing
period, continuous wave (CW) irradiation was applied on the 1H channel at a rotary
resonance condition (ωτ=ωrf) of 10 kHz and the mixing time was set at 5.0 ms.
134
Figure 4.14. Extrapolated curve of 13C chemical shift derived from the experimental and
calculated 13C chemical shift values of bulk lysine (δExtrapolate= -1.123* σCal +206.1 ppm).
Table 4.4. Summary of lysine/silanol complex from DFT calculations
Model E /a.u. r (OH) / Å r (ε-NH) / Å E (ε-NH) / kJ/mol

H 3SiOH -367.161 0.963 --- ---
H3SiO- -366.582 --- --- ---
Lys -497.079 --- 1.017 ---
LysH+ -497.437 --- 1.026 ---
Lys-H-OSiH3 -864.256 0.992 1.807 42.01

135
CHAPTER 5
Secondary Structure of the Gly-Gly-X (X=Leu, Tyr, Gln) motif in Spider Dragline Silk
Investigated by Solid-state NMR Spectroscopy
5.1 Introduction
Spider silk is a natural high-performance fiber featuring outstanding mechanical
properties and biocompatibility in comparison to other natural and man-made materials.
It has been used by human long before it appeared as a focus of materials science. In
ancient Greece, people used natural cobwebs to seal bleeding wounds, and in Australia,
spider silk was used for fishing. In recent decades, spider silk has attracted considerable
attention in textile, materials science, and biomedical research due to its excellent
mechanical properties that outperform most other natural and man-made fibers. In
particular, dragline silk is five times tougher than steel by weight and even three times
tougher than man-made synthetic fibers, such as Kevlar 49 [1-3]. Besides the classical
mechanical properties, dragline silk possesses a unique property called
‘supercontraction’, which can be described as an increase in diameter and a shrinking in
length up to 40% when a native dragline silk is exposed to humidity great than 60 % [4-
7].
Dragline silk produced from the major ampullate gland is used by spiders to
construct the frame and radii of an orb and it is thought to be one of the toughest
biopolymers known [8]. From the standpoint of the molecular structure of silk, the
excellent mechanical properties are thought to originate from the secondary and tertiary
structures of silk proteins. Dragline silk is composed almost entirely of two proteins,
major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and the typical repetitive amino
136
acid sequences of MaSp1 and MaSp2 are present in Figure 5.1 [9, 10]. Generally, it is
proposed that the silk proteins have the ability to form β-sheet nanocrystalline domains
that provide spider silk extraordinary strength, and amorphous domains composed of
disordered helical, β-turn, and random coil structures that are responsible for the excellent
extensibility (Figure 5.2). Overall, it is this unique combination of β-sheet, helical and
turn-like protein structures that imparts spider silk its extremely high toughness.
Nephila clavipes MaSp1:

AGAAAAAAGGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAGGAGQGGY
GGLGGQGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAA
Nephila clavipes MaSp2:
SAAAAAAAASGPGQQGPGGYGPGQQGPGGYGPGQQGPSGPGSAAAAAAAAS
GPGQQGPGGYGPGQQGPGGYGPGQQGLSGPGSAAAAAAA
Figure 5.1. Typical repetitive amino acid sequences of silk proteins for N. clavipes. The amino
acid residues in red indicate motifs that are recognized in the literature as being involved in β-
sheets. Other colors differentiate motifs that are assumed to compose the amorphous regions of
the silk including GGX (X=A, L, Q, Y) and GPGXX (X=Q, Y) motifs.

Crystalline Domain Amorphous Domain
Figure 5.2. Illustration of a microstructural model for spider silk.
137
The secondary structure of the spider silk proteins in silk fibers has been studied
extensively with a variety of techniques including X-ray diffraction (XRD) [11-22],
solid-state nuclear magnetic resonance (NMR) [4, 6, 7, 19, 23-52], Infrared (IR) [53-60]
and Raman spectroscopy [5, 17, 61-69]. The combination of these structural
characterization techniques has illustrated that the poly(Ala) motif and poly(Gly-Ala)
motif flanking the poly(Ala) runs in the protein sequence form β-sheet structures [11, 25-
27, 30, 31, 33, 38, 39] while the Gly-Gly-Ala motif primarily in the MaSp1 and the Gly-
Pro-Gly-X-X motif in the MaSp2 protein take on 31-helical structures and elastin-like
type II β-turn structures, respectively [24, 30, 37, 38, 43]. However, for other types of
Gly-Gly-X (X=Leu, Tyr, Gln) repetitive amino acid sequences in the MaSp1, their
secondary structures have not been well understood yet. A very first study conducted by
Michal and Jelinski in 1998 has shown that LGGQ sequence doesn’t form β-sheet
structure or 31-helical structure based on the rotational-echo double-resonance (REDOR)
NMR results [29]. Up to date, no clear structural model has been identified for the Gly-
Gly-X motif and a complete picture of molecular structure of spider dragline silk is still
lacking. The Gly-Gly-X motif is of particular interest because it has a relatively large
content in the MaSp1 and it is hypothesized to make the primary contribution to the
extensibility of the silk [70]. A clear understanding of its structure in spider silk could
lead us to reveal the relationship between the protein structure and the excellent
mechanical properties for spider silks. In this work, we isotopically label the Leu, Tyr,
Gln amino acid residues in spider dragline silk and study the secondary structure of Gly-
Gly-X (X=Leu, Tyr, Gln) motif using solid-state NMR spectroscopy and structural
dependent chemical shift analysis. The chemical shifts of X residues (Leu, Tyr, Gln)
138
obtained from solid-state NMR spectroscopy indicate that the structure of Gly-Gly-X
motif in dragline silk protein is not β-sheet or α-helix and is best interpreted as a
disordered structure with evidence for 31-helix, β-turn, and/or random coil conformations.
Additionally, the chemical shifts of the Gly residues that are adjacent to X residues
obtained from two-dimensional (2D) 13C-13C correlation NMR indicate that the Gly-Gly-
X (X=Leu, Tyr, Gln) motif is very likely associated with 31-helical structures in spider
dragline silk.
5.2.1 Materials
U-[13C, 15N]-L-alanine, U-[13C, 15N]-L-leucine, U-[13C, 15N]-L-glutamine, and U-
[13C, 15
N]-L-phenylalanine were purchased from Cambridge Isotopes Laboratories, Inc.
and used as received.
5.2.2 Isotopic enrichment of spider dragline silk
Mature female N. clavipes spiders were fed with tap water and crickets once per
week. Spiders were forcibly silked at a speed of 2 cm/s for 1 h every other day. The
major ampullate silk (dragline silk) was separated from the minor ampullate silk under an
optical microscope (Olympus, Waltham, MA, USA). To prepare isotope enriched
dragline silk, the spiders were fed a 200 µL saturated solution of U-[13C, 15N]-L-alanine,
U-[13C, 15N]-L-leucine, U-[13C, 15N]-L-glutamine, and U-[13C, 15N]-L-phenylalanine over
four feedings during silk collection.
5.2.3 Scanning Electron Microscopy (SEM)
Scanning electron microscopy (SEM) images of N. Clavipes dragline silk were
collected using an XL-30 Environmental FEG (FEI) scanning electron microscope
139
operating at a voltage of 10 kV in Hi-Vac mode with a vacuum of 8×10-5 mbar. The
spider silk fibers were mounted onto a sample stub with the use of double-sided carbon
tape and then was sputter deposited with 3 nm of gold to prevent charging during
imaging.
5.2.4 Solid-state NMR Measurement
spectrometer equipped with a 1.6 mm triple-resonance cross polarization magic angle
spinning (CP-MAS) probe operating in triple resonance mode (1H/13C/15N). One-
dimensional (1D) 1Hà13C CP-MAS and two-dimensional (2D) 13

C–13C through-space
correlation NMR experiments with dipolar-assisted rotational resonance (DARR)
experiments [38, 71, 72] were performed at a spinning speed of 35 kHz. The CP
condition consisted of a 1.6 µs 1H π/2 pulse, followed by a 1.0 ms ramped (6%) 1H spin-
lock pulse with a radio frequency (rf) field strength of 156 kHz at the ramp maximum.
The experiments were performed with a 25 kHz sweep width, a recycle delay of 3.0 s,
and two-pulse phase-modulated (TPPM) 1H decoupling during acquisition with a level of

13
132 kHz. In 2D C–13C through-space correlation experiments, the spectra were
collected with 1024 points in the direct dimension, 320 t1 complex points in the indirect
dimension, and 32 scans averages with spectral widths in the direct and indirect
dimension of 25 and 35 kHz, respectively. During the DARR mixing period, continuous
wave (CW) irradiation was applied on the 1H channel at n = 1 (ωR = ω1) rotary
resonance condition with mixing times (τm) of 50, 150 ms, and 1 s. The 2D spectra were
processed with an exponential line broadening of 100 Hz in the direct dimension and a
Gaussian function of the form exp(-(t/gf)2) in the indirect dimension with the constant, gf,
140
equal to 0.0025. The 13C isotropic chemical shift was indirectly referenced to adamantane
(38.56 ppm).
5.3.1 Morphology of Spider Dragline Silk
A SEM image of N. Clavipes dragline silk is present in Figure 5.3. It is found that
the dragline silk is a uniform cylindrical fiber along the length of the fibers. The average
diameter of the dragline silk is estimated to be 3.9 ± 0.2 um.
Figure 5.3. SEM image of N. Clavipes dragline silk.
5.3.2 Solid-state NMR spectroscopy
The N. clavipes silk is a MaSp1-rich silk with a low MaSp2 content (~80:20,
MaSp1:MaSp2). Thus, when investigating this silk it is the MaSp1 protein that is
primarily characterized. The consensus primary amino acid sequence for MaSp1 is shown
in Figure 5.4a and it is found that Leu, Gln, and Tyr are present in the Gly-Gly-X motif.
However, it should be noted here that although the N. clavipes dragline silk is primarily
MaSp1 protein, minor contributions from residues present in the MaSp2 protein cannot
be discounted. According to the primary amino acid sequence for the MaSp2 [10], Leu is
141
entirely absent from the MaSp2 and Tyr is present in the same Gly-Gly-X motif in the
MaSp2, so the contribution from the MaSp2 is believed to be negligible for Leu and
similar to that from the MaSp1 for Tyr residues. For Gln, it is present in a non-Gly-Gly-X
Gln-Gln motif in the MaSp2 and it could adopt other secondary structures different from
Gln in the MaSp1. However, due to the low ratio of the MaSp2 in N. clavipes dragline
silk and low content of Gln in the MaSp2, the contribution from the MaSp2 for Gln is
13
very little and mostly negligible. In this study, the N. clavipes dragline silk is C
enriched for the Ala, Gly, Leu, Gln, and Tyr amino acid residues, and the 1Hà13C CP-
13
MAS NMR spectrum of the C-labeled N. clavipes dragline silk is present in Figure
5.4b. The Ala methyl, Cβ, resonances were found at 17.4 and 20.9 ppm and this result
agrees well with the previous studies, in which the Ala residues are thought to be
heterogeneous in silk with a minimum of two components at 17.4 and 20.9 ppm assigned
to the Ala residues present in disordered 31-helix structures similar to poly-glycine II and
ordered β-sheet structures, respectively [38]. Furthermore, it is presumed that the Ala
residues in 31-helix structures are located in the repetitive Gly-Gly-X motif while, the Ala
residues in β-sheet structures are located in the poly(Ala) and flanking poly(Gly-Ala)
motifs in the primary amino acid sequence. Besides the Ala residues, other X residues
(Leu, Tyr, Gln) found in Gly-Gly-X are thought to be present in the amorphous domains
of silk protein. By isotopically labeling the X residues (Leu, Tyr, Gln) in spider dragline
silk, we have been able to probe the secondary structures adopted by them and further to
understand the molecular structure of the amorphous domains in spider silk. According to
the 1Hà13C CP-MAS NMR spectrum of the 13C-labeled N. clavipes dragline silk (Figure
5.4b), the Leu, Tyr, and Gln residues are found to be 13C enriched well and this permits
142
13
further NMR analysis by 2D C–13C correlation experiments to extract the
conformation-dependent 13C chemical shifts for the Leu, Tyr, and Gln residues.
(A) MaSp1: GGAGQGGYGGLGSQGAGRGGLGGQGAGAAAAAA
(B)
Figure 5.4. (A) Consensus primary amino acid sequence for N. clavipes MaSp1 and (B) 1Hà13C
CP-MAS NMR spectrum of 13C-labeled N. clavipes dragline silk.
13
2D C–13C correlation experiments with dipolar assisted rotational resonance
13
(DARR) were conducted in this study to extract the conformation-dependent C
chemical shifts for the amino acid residues that are found in the Gly-Gly-X motif. This
13
2D method is particularly useful for extracting all C chemical shifts for each site
including the CO chemical shifts that are completely overlapped in the 1Hà13C CP-MAS
13
spectrum (see Figure 5.4b). The C chemical shifts are summarized in Table 1. The
results for the Ala and Gly residues are similar to previously reported solid-state NMR
studies where the components for β-sheet and 31-helix were observed with nearly
13
identical chemical shifts. For the other C labeled amino acid residues including Leu,
Tyr, and Gln, the chemical shifts indicate that these residues are present in non-β-sheet
13
structures. More than that, the observed C shifts for CO, Cα, and Cβ do not match α-
143
helical structures but show similarity to those observed for the Ala residues in a model
31-helix for the most part. Specifically, the observed shifts for CO and Cα locate in a
chemical shift range between the β-sheet and the random coil, and the observed shifts for
Cβ are present at the higher ppm side of the random coil. However, the similarity to the
Ala residues in a model 31-helix is not convincing enough to identify that Gly-Gly-X
motif is in 31-helical structures since this trend is also very similar to the β-turn chemical
shift trends depending on the residue position in turn structures [73]. Overall, the
chemical shifts of X residues (Leu, Tyr, Gln) obtained from solid-state NMR
spectroscopy indicate that the structure of Gly-Gly-X motif in dragline silk protein is not
β-sheet or α-helix and is best interpreted as a disordered structure with evidence for 31-
helix, β-turn, and/or random coil conformations.

13
2D C-13C correlation experiments with a long DARR mixing time (1s) allows
observation of relatively long range correlations between adjacent residues in proteins.
This technique is very useful in identifying the location of amino acid residues in
different motifs. Specifically, for Gly-Gly-X motif, correlations of X residue (Leu, Tyr,
13
Gln) to the adjacent Gly residue can be identified with 2D C-13C correlation
experiments and further used for obtaining chemical shifts of the Gly residue that is
present in the Gly-Gly-X motif. For Leu, Tyr, and Gln residues, long-range correlations
to Gly Cα are observed at 42.5, 41.2, and 42.1 ppm, respectively, which is consistent with
Gly present in 31-helix (41.4-42.5 ppm) and inconsistent with Gly present in β-turn
(Figure 5.5, Table 5.1). This result indicates that the Gly-Gly-X motif in silk protein is
very likely in a 31-helical structure.
144
Table 5.1. 13C chemical shifts (in ppm from TMS) for N. clavipes dragline silk, random coil, and
polypeptides with defined secondary structures [37, 74-84].
residue N. clavipes α-helix β-sheet random coil 31-helix a β-turn
Gly Cα 43.2 46.0 43.2-44.3 43.4 41.4-42.5 43.8-44.1

Gly CO 169.7 174.9 168.4-169.7 173.2 171.2-173.1 170.6-170.7
Ala Cα 49.3 52.3-52.8 48.2-49.3 50.8 48.9 50.8-51.7
Ala Cβ 17.4, 20.9 14.8-16.0 19.9-20.7 17.4 17.4 16.5-17.4
Ala CO 172.8 176.2-176.8 171.6-172.2 176.1 174.6 176.8-177.5
Gln CO 172.6 175.4-175.9 171.9-172.2 174.3 - -
Gln Cα 52.7 56.4-57.0 51.0-51.4 54.0 - -
Gln Cβ 29.6, 25.8 25.6-26.3 29.0-29.9 27.7 - -
Gln Cγ 32.2, 30.6 29.7-29.8 29.7-29.9 32.0 - -
Gln Cδ 176.6 - - 178.8 - -
Tyr CO 172.7 176.7 169.7 174.2 - -
Tyr Cα 55.1 54.8-58.6 52.1 56.2 - -
Tyr Cβ 37.7 36.1 39.3 37.1 - -
Tyr Cγ 128.6 129.7 128.0 128.9 - -
Tyr Cδ 130.9 129.7 128.0 131.6 - -
Tyr Cε 116.1 116.1 115.0 116.5 - -
Tyr Cζ 156.2 154.2 155.2 155.6 - -
Leu CO 176.0 175.7 170.5-171.3 175.9 - -
Leu Cα 53.8 55.7 50.5-51.2 53.4 - -
Leu Cβ 41.6 39.5 43.3 40.7 - -
Leu Cγ 24.0 - - 25.2 - -
Leu Cδ 23.3 24.4 24.9 24.3 - -
a
(GGA)10 model peptide with (φ, ψ) values near (-90°, 150°).
145
(A) (B)
(C)
Figure 5.5. 2D 13C-13C correlation spectra of 13C labeled N. clavipes spider dragline silk collected
with a long dipolar assisted rotational resonance (DARR) mixing period of 1 s: correlation of
Leu, Gln, and Tyr to Gly are noted in red in (A), (B), and (C), respectively.
146
5.3.3 Molecular Structure of Dragline Spider Silk
In the past decades, a combination of structural characterization techniques
including X-ray diffraction, NMR spectroscopy, vibrational spectroscopy, and electron
microscopy has been used to understand the molecular structure of dragline spider silk
[4-7, 11-69]. Due to the complexity of hierarchical structures in spider silk as a natural
protein-based biopolymer, obtaining a clear picture of the molecular structure of spider
silk is not an easy job and it requires advanced techniques and considerable effort. In this
paragraph, achievements researchers have made in the past a few years, together with the
accomplishment present in the current work are summarized briefly. The molecular
structure of silk includes the protein sequences, secondary structures, tertiary structures,
and nano/micro-structures. The protein sequences of the MaSp1 and MaSp2 proteins in
spider dragline silk were first studied and identified by Randy et al in the early 1990s [9,
10]. Solid-state NMR spectroscopy and X-ray diffraction were then applied to investigate
the secondary structures of silk proteins and the protein self-assembly (nano/micro-
structure) in fibers, respectively. By combining these two techniques, the poly(Ala) motif
and poly(Gly-Ala) motif flanking the poly(Ala) runs in the amino acid sequence were
found to be predominant in β-sheet structures and to organized into nanocrystalline
domains with sizes of at least 2×5×7 nm [11, 12, 25, 26, 30, 38]. Additionally, Gly-Gly-
X (X=Ala, Leu, Tyr, Gln) motif in MsSp1 and Gly-Pro-Gly-X-X motif in MaSp2 have
been characterized by solid-state NMR spectroscopy and were found to be predominant
in disordered structures forming amorphous domains in silk fibers. Specifically, Gly-Gly-
X (X=Ala, Leu, Tyr, Gln) motif in MsSp1 was found to take on 31-helical structure based
on previous reports and this work [24, 30, 37, 38, 52], while Gly-Pro-Gly-X-X motif in
147
MaSp2 was found to be in elastin-like type II β-turn structures [43]. In comparison to the
relatively rich understanding on the secondary structure of spider silk proteins, very
limited progress has been made towards understanding the tertiary structures and
nano/micro-structures in silk. In recently years, molecular dynamics simulation has been
developed as a powerful and primary tool for unraveling the relationship between
hierarchy structures and excellent mechanical properties of silk fibers [85-87]. However,
to achieve the ultimate goal of unraveling the mystery of spider silk, more experimental
effort and advanced characterization techniques are needed.
5.4 Conclusions
Solid-state NMR spectroscopy was applied as a primary tool to investigate the
secondary structures adopted by the Gly-Gly-X (X=Leu, Tyr, Gln) motif in the spider
dragline silk and the results show that this type of motif is very likely in 31-helical
structures. This found is consistent with the previous finding that Gly-Gly-X motif does
13
not form α-helical and β-sheet structures. More than that, 2D C-13C correlation
experiments with a long mixing time provide a way to obtain more chemical shift
information for the Gly-Gly-X motif. Specifically, the correlation of X amino acid
residues to the adjacent Gly residue was properly identified, so 13C chemical shifts of the
adjacent Gly residues were obtained. The Gly chemical shifts indicate that the Gly-Gly-X
motif in the silk protein is very likely in 31-helical structures other than β-turn structures.
5.5 References
[1] M. Heim, D. Keerl, T. Scheibel, Spider silk: From soluble protein to extraordinary
fiber, Angew. Chem. Int. Ed., 48 (2009) 3584-3596.
[2] R.W. Work, Force-elongation behavior of web fibers and silks forcibly obtained
from orb-web-spinning spiders., Text. Res. J., 46 (1976) 485-492.
148
[3] F. Vollrath, D.P. Knight, Liquid crystalline spinning of spider silk., Nature, 410
(2001) 541-548.
[4] L.W. Jelinski, A. Blye, O. Liivak, C.A. Michal, G. LaVerde, A. Seidel, N. Shah, Z.
Yang, Orientation, structure, wet-spinning, and molecular basis for supercontraction
of spider dragline silk, Int. J. Biol. Macromol., 24 (1999) 197-201.
[5] Z. Shao, F. Vollrath, J. Sirichaisit, R.J. Young, Analysis of spider silk in native and
supercontracted states using Raman spectroscopy, Polymer, 40 (1999) 2493-2500.
[6] J.D. van Beek, J. Kummerlen, F. Vollrath, B.H. Meier, Supercontracted spider
dragline silk: a solid-state NMR study of the local structure, Int. J. Biol. Macromol.,
24 (1999) 173-178.
[7] Z. Yang, O. Liivak, A. Seidel, G. LaVerde, D.B. Zax, L.W. Jelinski,

Supercontraction and backbone dynamics in spider silk: 13C and 2H NMR studies,
J. Am. Chem. Soc., 122 (2000) 9019-9025.
(2006) 377-385.
Natl. Acad. Sci., 87 (1990) 7120-7124.
392-398.

[14] C. Riekel, B. Madsen, D. Knight, F. Vollrath, X-ray diffraction on spider silk

during controlled extrusion under a synchrotron radiation X-ray beam,
[15] C. Riekel, F. Vollrath, Spider silk fibre extrusion: combined wide- and small-angle
X-ray microdiffraction experiments, Int. J. Biol. Macromol., 29 (2001) 203-210.
149
[17] G.R. Plaza, J. Pérez-Rigueiro, C. Riekel, G.B. Perea, F. Agulló-Rueda, M.
Burghammer, G.V. Guinea, M. Elices, Relationship between microstructure and
mechanical properties in spider silk fibers: identification of two regimes in the
microstructural changes, Soft Matter, 8 (2012) 6015-6012.
[18] S. Sampath, T. Isdebski, J.E. Jenkins, J.V. Ayon, R.W. Henning, J.P.R.O. Orgel, O.
Antipoa, J.L. Yarger, X-ray diffraction study of nanocrystalline and amorphous
structure within major and minor ampullate dragline spider silks, Soft Matter, 8
(2012) 6713-6711.
[19] J.E. Jenkins, S. Sampath, E. Butler, J. Kim, R.W. Henning, G.P. Holland, J.L.
Yarger, Characterizing the secondary protein structure of black widow dragline silk
using solid-state NMR and X-ray diffraction, Biomacromolecules, 14 (2013) 3472-
3483.
[20] S. Sampath, J.L. Yarger, Structural hysteresis in dragline spider silks induced by
supercontraction: an X-ray fiber micro-diffraction study, RSC Adv., 5 (2014) 1462-
1473.
[21] R. Madurga, T.A. Blackledge, B. Perea, G.R. Plaza, C. Riekel, M. Burghammer, M.

Elices, G. Guinea, J. Pérez-Rigueiro, Persistence and variation in microstructural
design during the evolution of spider silk, Sci. Rep., 5 (2015) 1-11.
[22] C. Riekel, M. Burghammer, T.G. Dane, C. Ferrero, M. Rosenthal, Nanoscale

structural features in major ampullate spider silk, Biomacromolecules, 18 (2017)
231-241.
[23] A. Simmons, E. Ray, L.W. Jelinski, Solid-state 13C NMR of Nephila clavipes
dragline silk establishes structure and identity of crystalline regions,
Macromolecules, 27 (1994) 5235-5237.
[24] J. Kummerien, J.D. van Beek, F. Vollrath, B.H. Meier, Local structure in spider
dragline silk investigated by two-dimensional spin-diffusion nuclear magnetic
resonance Macromolecules, 29 (1996) 2920-2928.
[25] A. Simmons, C.A. Michal, L.W. Jelinski, Molecular orientation and two-component
nature of the crystalline fraction of spider dragline silk, Science, 271 (1996) 84-87.
1480-1481.
[27] O. Liivak, A. Flores, R.V. Lewis, L.W. Jelinski, Conformation of the polyalanine
repeats in minor ampullate gland silk of the spider Nephila clavipes ,
Macromolecules, 30 (1997) 7127-7130.
[28] A.D. Parkhe, S.K. Seeley, K. Gardner, L. Thompson, R.V. Lewis, Structural studies
of spider silk proteins in the fiber, J. Mol. Recogn., 10 (1997) 1-6.
150
[29] C.A. Michal, L.W. Jelinski, Rotational-echo double-resonance in complex
biopolymers: a study of Nephila clavipes dragline silk, J. Biomol. NMR, 12 (1998)
231-241.
[30] J.D. van Beek, S. Hess, F. Vollrath, B.H. Meier, The molecular structure of spider
dragline silk: Folding and orientation of the protein backbone, Proc. Natl. Acad.
Sci., 99 (2002) 10266-10271.
[31] T. Asakura, M. Yang, T. Kawase, Structure of characteristic sequences in Nephila

clavipes dragline silk (Masp1) studied with 13C solid state NMR, Polym. J., 36
(2004) 999-1003.
[32] P.T. Eles, C.A. Michal, Strain dependent local phase transitions observed during
controlled supercontraction reveal mechanisms in spider silk, Macromolecules, 37
(2004) 1342-1345.
[33] G.P. Holland, R.V. Lewis, J.L. Yarger, WISE NMR characterization of nanoscale
heterogeneity and mobility in supercontracted Nephila clavipes spider dragline silk,
J. Am. Chem. Soc., 126 (2004) 5867-5872.

peptides based on Nephila clavipes dragline silk spidroin (Masp1) studied by 13C
3226.
[35] B. Bonev, S. Grieve, M.E. Herberstein, A.I. Kishore, A. Watts, F. Separovic,

Orientational order of Australian spider silks as determined by solid-state NMR,
Biopolymers, 82 (2006) 134-143.
[36] J.D. van Beek, B.H. Meier, A DOQSY approach for the elucidation of torsion angle
distributions in biopolymers: Application to silk, J. Magn. Reson., 178 (2006) 106-
120.
[37] I. Marcotte, J.D. van Beek, B.H. Meier, Molecular disorder and structure of spider
dragline silk investigated by two-dimensional solid-state NMR spectroscopy,
Macromolecules, 40 (2007) 1995-2001.
[39] G.P. Holland, J.E. Jenkins, M.S. Creager, R.V. Lewis, J.L. Yarger, Quantifying the
fraction of glycine and alanine in β-sheet and helical conformations in spider
dragline silk using solid-state NMR, Chem. Commun., (2008) 5568-5563.
151
[40] G.P. Holland, J.E. Jenkins, M.S. Creager, R.V. Lewis, J.L. Yarger, Solid-state
NMR investigation of major and minor ampullate spider silk in the native and
hydrated states, Biomacromolecules, 9 (2008) 651-657.
[41] M.S. Creager, J.E. Jenkins, L.A. Thagard-Yeaman, A.E. Brooks, J.A. Jones, R.V.
Lewis, G.P. Holland, J.L. Yarger, Solid-state NMR comparison of various spiders’
dragline silk fiber, Biomacromolecules, 11 (2010) 2039-2043.
[42] T. Izdebski, P. Akhenblit, J.E. Jenkins, J.L. Yarger, G.P. Holland, Structure and
dynamics of aromatic residues in spider silk: 2D carbon correlation NMR of
dragline fibers, Biomacromolecules, 11 (2010) 168-174.
Chem. Commun., 46 (2010) 6714-6713.
[44] J.E. Jenkins, M.S. Creager, R.V. Lewis, G.P. Holland, J.L. Yarger, Quantitative
correlation between the protein primary sequences and secondary structures in
spider dragline silks, Biomacromolecules, 11 (2010) 192-200.
[45] J.E. Jenkins, G.P. Holland, J.L. Yarger, High resolution magic angle spinning NMR
investigation of silk protein structure within major ampullate glands of orb weaving
spiders, Soft Matter, 8 (2012) 1947-1954.
[46] G.P. Holland, Q. Mou, J.L. Yarger, Determining hydrogen-bond interactions in

spider silk with 1H-13C HETCOR fast MAS solid-state NMR and DFT proton
chemical shift calculations, Chem. Commun., 49 (2013) 6680-6683.
[47] X. Shi, J.L. Yarger, G.P. Holland, 2H-13C HETCOR MAS NMR for indirect
detection of 2H quadrupole patterns and spin-lattice relaxation rates, J. Magn.
Reson., 226 (2013) 1-12.
[48] X. Shi, J.L. Yarger, G.P. Holland, Elucidating proline dynamics in spider dragline
silk fibre using 2H-13C HETCOR MAS NMR, Chem. Commun., 50 (2014) 4856-
4854.
[49] D. Xu, J.L. Yarger, G.P. Holland, Exploring the backbone dynamics of native
spider silk proteins in Black Widow silk glands with solution-state NMR
spectroscopy, Polymer, 55 (2014) 3879-3885.
[50] X. Shi, G.P. Holland, J.L. Yarger, Molecular dynamics of spider dragline silk fiber
investigated by 2H MAS NMR, Biomacromolecules, 16 (2015) 852-859.
[51] D. Xu, X. Shi, F. Thompson, W.S. Weber, Q. Mou, J.L. Yarger, Protein secondary
structure of Green Lynx spider dragline silk investigated by solid-state NMR and
X-ray diffraction, Int. J. Biol. Macromol., 81 (2015) 171-179.
152
[52] G. Gray, A. van der Vaart, C. Guo, J. Jones, D. Onofrei, B. Cherry, R. Lewis, J.
Yarger, G. Holland, Secondary structure adopted by the Gly-Gly-X repetitive
regions of dragline spider silk, International Journal of Molecular Sciences, 17
(2016) 2023-2013.

Vib. Spectrosc., 46 (2008) 63-68.
[54] R. Ene, P. Papadopoulos, F. Kremer, Combined structural model of spider dragline

silk, Soft Matter, 5 (2009) 4568-4567.
[55] P. Papadopoulos, R. Ene, I. Weidner, F. Kremer, Similarities in the structural

organization of major and minor ampullate spider silk, Macromol. Rapid Commun.,
30 (2009) 851-857.
[56] P. Papadopoulos, J. Sölter, F. Kremer, Hierarchies in the structural organization of

spider silk-a quantitative model, Colloid Polym Sci, 287 (2009) 231-236.
[57] R. Ene, P. Papadopoulos, F. Kremer, Partial deuteration probing structural changes

in supercontracted spider silk, Polymer, 51 (2010) 4784-4789.
[58] R. Ene, P. Papadopoulos, F. Kremer, Quantitative analysis of infrared absorption

coefficient of spider silk fibers, Vib. Spectrosc., 57 (2011) 207-212.
[59] S. Ling, Z. Qi, D.P. Knight, Z. Shao, X. Chen, Synchrotron FTIR microspectroscopy
of single natural silk fibers, Biomacromolecules, 12 (2011) 3344-3349.

[61] Z. Shao, R.J. Young, F. Vollrath, The effect of solvents on spider silk studied by
mechanical testing and single-fibre Raman spectroscopy, Int. J. Biol. Macromol., 24
(1999) 295-300.
[62] J. Sirichaisit, R.J. Young, F. Vollrath, Molecular deformation in spider dragline silk
subjected to stress, Polymer, 41 (2000) 1223-1227.

[64] T. Lefèvre, M.-E. Rousseau, M. Pézolet, Protein secondary structure and orientation
in silk as revealed by Raman spectromicroscopy, Biophys. J., 92 (2007) 2885-2895.
153
[65] P. Colomban, H.M. Dinh, J. Riand, L.C. Prinsloo, B. Mauchamp, Nanomechanics
of single silkworm and spider fibres: a Raman and micro-mechanical in situ study
of the conformation change with stress, J. Raman Spectrosc., 39 (2008) 1749-1764.
[66] M.-E. Rousseau, T. Lefèvre, M. Pézolet, Conformation and orientation of proteins

in various types of silk fibers produced by Nephila clavipes spiders,
[67] P. Colomban, H.M. Dinh, A. Bunsell, B. Mauchamp, Origin of the variability of the
mechanical properties of silk fibres: 1-The relationship between disorder, hydration
and stress/strain behaviour, J. Raman Spectrosc., 43 (2011) 425-432.
[68] T. Lefèvre, F. Paquet-Mercier, J.-F. Rioux-Dubé, M. Pézolet, Structure of silk by

raman spectromicroscopy: From the spinning glands to the fibers, Biopolymers, 97
(2011) 322-336.
[69] T. Lefèvre, M. Pézolet, Unexpected β-sheets and molecular orientation in

flagelliform spider silk as revealed by Raman spectromicroscopy, Soft Matter, 8
(2012) 6350-6358.
[70] S.L. Adrianos, F. Teulé, M.B. Hinman, J.A. Jones, W.S. Weber, J.L. Yarger, R.V.
Lewis, Nephila clavipes flagelliform silk-like GGX motifs contribute to
extensibility and spacer motifs contribute to strength in synthetic spider silk fibers,
[71] K. Takegoshi, S. Nakamura, T. Terao, 13C–1H dipolar-assisted rotational resonance

[72] K. Takegoshi, S. Nakamura, T. Terao, 13C-1H dipolar-driven 13C-13C recoupling

Phys., 118 (2003) 2325-2341.
[73] Y. Shen, A. Bax, Identification of helix capping and β-turn motifs from NMR
chemical shifts, J. Biomol. NMR, 52 (2012) 211-232.

2654-2657.
680-688.
154
266.

Am. Chem. Soc., 124 (2002) 8794-8795.
[78] T. Asakura, J. Yao, 13C CP/MAS NMR study on structural heterogeneity in Bombyx
mori silk fiber and their generation by stretching, Protein Sci., 11 (2002) 2706-
2713.

2

(2001) 291-305.
[81] D.S. Wishart, C.G. Bigam, A. Holm, R.S. Hodges, B.D. Sykes, 1H, 13C and 15N
random coil NMR chemical shifts of the common amino acids. I. Investigations of
nearest-neighbor effects., J. Biomol. NMR, 5 (1995) 67-81.
[82] H. Saito, Conformation‐dependent 13C chemical shifts: A new means of

conformational characterization as obtained by high‐resolution solid‐state 13C NMR,
Magn. Reson. Chem., 24 (1986) 835-852.

resolution 13C NMR study of silk fibroin in the solid state by the cross-polarization-
magic angle spinning method. Conformational characterization of silk I and silk II
type forms of Bombyx mori fibroin by the conformation-dependent 13C chemical
shifts, Macromolecules, 17 (1984) 1405-1412.
[84] H.R. Kricheldorf, Secondary structure of peptides. 3. 13C NMR cross

polarization/magic angle spinning spectroscopic charaterization of solid
polypeptides, Macromolecules, 16 (1983) 615-623.
367.
155
[86] A. Nova, S. Keten, N.M. Pugno, A. Redaelli, M.J. Buehler, Molecular and
nanostructural mechanisms of deformation, strength and toughness of spider silk
fibrils., Nano Lett., 10 (2010) 2626-2634.
[87] S.W. Cranford, A. Tarakanova, N.M. Pugno, M.J. Buehler, Nonlinear material
behaviour of spider silk yields robust webs, Nature, 482 (2012) 72-76.
156
CHAPTER 6
Protein Secondary Structure in Bombyx mori Silk Investigated by Solid-state NMR
Spectroscopy
6.1 Introduction
Silkworm silk has been used commercially in textile production for centuries and
it is recognized as one of the most studied protein-based materials in history. Over the
past 70 years, researchers from a wide range of fields including chemistry, physics,
biology, and engineering have been attracted by silkworm silk and considerable effort has
been put into understanding the properties, structures, and applications of silkworm silk
as evidenced by the booming publications on silk related topics [1-9]. Among all studies,
a majority of work focuses on B. mori silk since it is a domesticated silkworm with a
large annual production and a popular use in textile industry for centuries.
The Bombyx mori cocoon is composed of a raw silk fiber with 700-1500 m in
length and 10-20 µm in diameter. The raw silk fiber consists of two parallel fibroin fibers
held together with a layer of sericin on their surfaces [11]. Upon degumming the cocoon
and removing the sericin, soft and shiny fibroin fibers can be obtained and this is the
primary material used in the textile industry. Moreover, it is worthy to note here that most
research has been carried on fibroin fibers rather than raw cocoons.
Silk fibroin consists of a heavy (H) chain of 390 kDa [12] and a light (L) chain of
26 kDa [13] connected by a disulfide bond at the C-terminus of H-chain [14], forming a
H-L complex (see supplementary). The H-L complex also binds a glycoprotein named
P25 (30 kDa) in a ratio of 6:1 via hydrophobic interactions to form an elementary
micellar unit [15]. In silk fibroin, the H-chain serves as the main structural component
157
responsible for the excellent mechanical properties due to its ability to form different
kinds of secondary structures, which could be further organized into nano/micro
hierarchical structures via molecular self-assembly [16]. L-chain plays little mechanical
role overall as its size is much smaller than H-chain, and additionally, no significant
secondary structures were identified for L-chain. The amino acid composition of the
heavy chain (in mol%) is dominated by four amino acids: Gly (46%), Ala (30%), Ser
(12%), and Tyr (5.3 %) [17]. The amino acid sequence of the heavy chain contains a
large number of repetitive sequences that are organized into 12 domains. According to
the literature, the repetitive sequences can be roughly classified into three primary motifs:
(1) a highly repetitive GAGXGA (X=S, Y, V) sequence that makes up the bulk of the
crystalline/semi-crystalline regions [18, 19]; (2) a relatively less repetitive GAAS
sequence, which may serve as a “sheet-breaking” motif [20]; (3) a sequence used to
separate the domains and form the amorphous regions in fibroin fibers, e.g.,
TGSSGFGPYVANGGYSGYEYAWSSESDFGT [21]. It is presumed that the excellent
mechanical properties of silk fibers are closely related to the secondary structures of these
characteristic sequences in silk fibroin, which are further organized into hierarchical
structures. So obtaining a clear molecular picture of the secondary structures in silk
fibroin is very critical for interpreting the superior nature of silk fibroin properly.
Silk I and silk II are reported as dimorphs of B. mori silk fibroin on the basis of
extensive investigations from X-ray fiber diffraction [16, 22-29], electron diffraction [27,
30], vibrational spectroscopy [22, 31-38], and solid-state NMR spectroscopy [2, 5, 6, 18-
21, 39-58]. Silk I is the structure of B. mori silk fibroin in the solid state obtained from
the middle silk gland after drying. Silk II is the structure of B. mori silk fibroin fiber after
158
spinning. It is feasible to obtain silk fibroin in the silk I or silk II form under non-native
conditions [57]. For example, if the silk fibroin is dissolved in 9.4 M LiBr solution and
dialyzed against distilled water, the resulting structure will be the silk I form, and if the
silk I sample is treated with formic acid and dried, it will give the silk II form. NMR
spectroscopy, particular solid-state NMR spectroscopy has been primarily used to
differentiate the two distinct dimorphs based on the characteristic conformation-
dependent chemical shifts and the line shape of resonances.
The secondary structures of silk fibroin in the silk I and silk II have been studied
extensively with a variety of techniques including X-ray diffraction, solid-state NMR
spectroscopy, and vibrational spectroscopy over the past decades [2, 10, 16, 18-20, 22-29,
31, 32, 34-38, 42, 46-52, 54, 56, 58, 59]. The combination of these structural
characterization techniques has illustrated that in the silk II, the GAGAGS motif and the
GAGAGY motif flanking the GAGAGS repetitive sequences form β-sheet structures and
are further organized to form β-sheet nanocrystallites [16, 18], while the GAGAGX
(X=S, Y, V) was hypothesized to be in type II β-turn structures in the liquid silk (silk I)
before spinning [10, 60]. According to the review on previously reported studies, it is
found that very limited effort has been put on elucidating the structural features of the
Tyr residues in comparison to the extensive studies on the Gly, Ala, and Ser residues
which are frequently found in the (GA)n and GAGAGS repetitive sequences [10, 37, 41,
47, 50, 53, 61]. Tyrosine is very likely to play important roles in the structure and
dynamics of silk fibroin because of its unique side chain structures. Previous work has
shown that the Tyr residues contribute primarily to the solubility of silk fibroin in water
and form bends and turns in Silk I [41, 47]. NMR studies on model peptides further
159
showed that the Tyr residues are very likely involved in forming β-sheet structures in silk
II with presenting at the edges of the nanocrystalline regions [47, 50, 53]. Although these
interpretations from the early studies on tyrosine provide some structural insights towards
understanding the function of the Tyr residues in silk fibroin, the molecular picture of the
Tyr residues in silk fibroin still remains ambiguous. So a thorough revision is necessary
to get a clear structural understanding on the Tyr residues with advanced characterization
techniques. In this work, two-dimensional (2D) 13C-13C correlation NMR spectroscopy
was applied to understand the structural role of the Tyr residues in solid-state silk fibroin,
together with a brief structural analysis on the Ala and Ser residues.
6.2.1 Materials
Medium size B. mori silkworms were purchased from Mulberry Farms Inc. and
kept in an incubator in the lab for them to grow. [U-13C]-glucose, [U-2H/13C/15N]-
phenylalanine, and [U-2H/13C/15N]-serine were purchased from Cambridge Isotopes
Laboratories, Inc. and used as received. Sodium carbonate and lithium bromide were
purchased from Sigma-Aldrich and used as received.
6.2.2 Isotopic Enrichment of Silk Fibroin
100 mg of [U-13C]-glucose or [U-2H/13C/15N]-serine or [U-2H/13C/15N]-
phenylalanine was dissolved in 4 mL distilled water at a concentration of 25 mg/mL. The
solution was used during the 3rd through the 7th day of the fifth instar with the following
amounts: 0.8 mL for the first day, 1.2 mL for the second and third days, and 0.8 mL for
the fourth day. At each day, the solution was pipetted onto 120 mg dehydrated food and
160
then the food was fed to two silkworms. Each worm was fed 50 mg of isotopes with this
labeling protocol.
6.2.3 Preparation of degummed silk
Silk cocoons were cut into small pieces and washed with DI water. A solution of
0.5 wt% of Na2CO3 and 1 wt% of Marseille soap in distilled water was made. The
solution was heated until boiling and the cocoons were placed in the solution for 30 mins.
After 30 mins, the silk was washed with DI water until all soap was removed. This
degumming process was repeated one more time to obtain the silk fibroin. The wet silk
fibroin was then dried at room temperature.
6.2.4 Preparation of lyophilized silk
25 mg degummed silk (silk fibroin) was dissolved in 0.5 mL of 9.3 M LiBr
aqueous solution at 60 °C for 4 hours then the centrifugation was used to remove
insoluble solids. The supernatant fluid was then dialyzed for 3 days at 4 °C in a 3500 Da
MW cut-off dialysis cassette to eliminate the LiBr. The water was changed five times
during the dialysis. After dialysis, the silk fibroin solution was frozen and further
lyophilized to obtain the lyophilized silk.
6.2.5 Solid-state NMR spectroscopy
spectrometer equipped with 1.6 mm triple-resonance MAS probe operating in triple
resonance mode (1H/13C/15N). 1Hà13C cross-polarization magic-angle-spinning (CP-
MAS) NMR experiments were performed at 20 kHz magic-angle-spinning (MAS) with a
1.5 µs 1H π/2 pulse and a 1.0 ms ramped (10 %) 1H spin-lock pulse with a radio
frequency (rf) field strength of 97 kHz at the ramp maximum during the CP transfer. The
161
experiments were performed with a 25 kHz sweep width, a recycle delay of 3 s, 4096
scans and a two-pulse phase-modulated (TPPM) 1H decoupling with a rf field strength of
120 kHz.
13
Two-dimensional (2D) through-bond C-13C double-quantum/single-quantum
(DQ/SQ) refocused incredible natural-abundance double-quantum transfer experiments
(INADEQUATE) [62] were performed at 35 kHz MAS and 1H decoupling with a rf field
strength of 120 kHz. The CP condition composed of a 1.5 µs 1H π/2 pulse and a 1.0 ms
ramped (10 %) 1H spin-lock pulse with a radio frequency (rf) field strength of 97 kHz at
the ramp maximum during the CP transfer. The spectra were collected with 50 kHz
sweep widths in both dimensions, a recycle delay of 2 s, 128 t1 points and a τ delay of 2
ms.
2D through-space 13C-13C dipolar assisted rotational resonance (DARR) [63, 64]
NMR experiments were performed at 20 kHz MAS and 1H decoupling with a rf field
strength of 120 kHz. The CP condition consisted of a 1.5 µs 1H π/2 pulse and a 1.0 ms
ramped (10 %) 1H spin-lock pulse with a radio frequency (rf) field strength of 97 kHz at
the ramp maximum during the CP transfer. The spectra were collected with a recycle
delay of 2 s, 512 t1 points, a DARR mixing time of 1s, and a 50 kHz sweep width and a
25 kHz sweep width for direct and indirect dimensions, respectively.
6.3.1 Isotopic labeling of silk fibroin in fibers
The enrichment of silk spun from the B. mori silkworm fed with isotopically
labeled materials was characterized using 1Hà13C CP-MAS NMR spectroscopy (Figure
6.1). It is found that the silk exhibits modest 13C-enrichment (~ 5 %) for the Ala, Gly, and
162
Figure 6.1. 1Hà13C CP-MAS spectra of degummed silkworm silk collected from B. mori
silkworms that were fed with (a) [U-2H/13C/15N]-serine, (b) [U-2H/13C/15N]-phenylalanine, (c) [U-
13
C]-glucose enriched food, and (d) regular food that was not isotopically enriched. The spectra
were collected at 20 kHz MAS and a 1 ms CP contact time. The spectra were collected with the
same number of scans (4096 scans) and processed with the same exponential line broadening (lb
= 100 Hz).
Ser residues if the silkworm was fed with [U-13C]-glucose enriched food (Figure 6.1c).
However, no obvious enrichment was found for the aromatic groups in the Tyr residues
in this case after comparing with the spectrum of natural abundance silk (Figure 6.1d)
13
because of the weak C resonance intensities of the Tyr residues (110-160 ppm). This
13
result agrees well with the previous C NMR studies on silkworm silks and major
ampullate spider silks where the [U-13C]-glucose protocol resulted in no significant 13C-
enrichment of the aromatic Tyr residues [65]. When B. mori silkworm was fed with [U-
163
2
H/13C/15N]-phenylalanine, a significant 13
C-enrichment (~ 17 %) for the Tyr residues
was observed (Figure 6.2b). More notably, the appearance of a distinct shoulder at 136.7
ppm is consistent with the Cγ resonance of the Phe residues, whose percentage in silk
fibroin is only 0.7 %. Further evidence will be provided by the INADEQUATE NMR
spectroscopy. Additionally, when B. mori silkworm was fed with [U-2H/13C/15N]-serine,
the 13C-enrichment for the Ser residues was found to be at a level of ~10 % that is double
of the enrichment with [U-13C]-glucose. Overall, the NMR results illustrate that silk
exhibits decent 13C-enrichments for the Ser (~10 %) and Tyr (~17 %) residues when the
silkworms were fed with [U-2H/13C/15N]-serine and [U-2H/13C/15N]-phenylalanine
respectively, while silk exhibits modest 13C-enrichment (~ 5 %) for the Ala, Gly, and Ser
residues if the silkworm was fed with [U-13C]-glucose enriched food.
6.3.2 Conformation of Ala residues in silk fibroin
Alanine is the most abundant amino acid other than glycine in silk fibroin and it is
primarily found in GAGAGX motifs. The conformation of the Ala residues in silk I and
silk II has been extensively studied by solid-state NMR spectroscopy and X-ray
diffraction in recent years [10, 18, 19, 25, 30, 46]. 13C NMR studies on the Ala Cβ group
has illustrated that the Ala residues have heterogeneous chemical environments in the silk
II since the Ala Cβ shows a broad and asymmetric resonance in the 1Hà13C CP-MAS
NMR spectrum (Figure 6.1d). The resonance at 17.3 ppm is attributed to the Ala residues
present in the amorphous domain composed of disordered structures, while the resonance
at 20.8 ppm is assigned to the Ala residues present in the crystalline domain with the
primary sequence (GAGAGS)n. With a quantitative solid-state NMR study on the [3-
13
C]-Ala labeled B. mori silk fibroin fibers, Asakura et al. have further indicate that 56 %
164
of the Ala residues are present in crystalline domain with 18% distorted β-turn structures
and 38% β-sheet structures, and 44% of the Ala residues are present in amorphous
domain with 22% distorted β-turn and 22% β-sheet structures [6, 18]. For the Ala
residues in silk I, a recent solution NMR study on the liquid silk in B. mori silkworm
gland has shown that it is primarily in type II β-structures [10].
6.3.3 Conformation of Ser residues in silk fibroin
Serine is primarily found in the GAGAGS motif in the H-chain of B. mori silk
fibroin and the GAGAGS motif is illustrated to form the crystalline domains in the silk
fibers. According to the previous studies on natural silk materials and synthetic model
peptides with a combination of X-ray diffraction and NMR spectroscopy, it is concluded
that the GAGAGS motif is in repeated type II β-turn structures in the silk I [6, 10, 48,
52], while it is found to be in β-sheet structures in the silk II [25, 41, 51]. In the present
work, the conformation of the Ser residues in silk I and silk II were reinvestigated with
2D 13C-13C correlation NMR spectroscopy. It is found that in the degummed silk (silk II),
13
the Ser CO, Cα, and Cβ have C resonances of 170.9 ppm, 55.3 ppm and 63.8 ppm
respectively (Figure 6.2). These chemical shifts are consistent with the chemical shifts of
the Ser residues present in β-sheet structures (Table 1). The degummed silk was then
dissolved in the LiBr solution and the lyophilized silk was obtained after dialysis,
centrifugation, and lyophilization. The lyophilized silk is assumed to have the similar
structure to silk fibroin in the liquid state (silk I) since crystallization is not allowed for
silk fibroin during lyophilization. The NMR results show that the Ser CO, Cα, and Cβ
13
have C resonances of 171.8 ppm, 56.2 ppm and 61.6 ppm respectively (Figure 6.3).
This result agrees well with the chemical shifts reported for the Ser residues in the
165
GAGSGA motif in the liquid silk [10] and indicate that the Ser residues are in disordered
type II β-turn structures in silk I.
Figure 6.2. 2D through-bond 13C-13C DQ/SQ refocused INADEQUATE NMR correlation

spectrum of degummed silk collected from B. mori silkworms that were fed with [U-2H/13C/15N]-
serine.
6.3.4 Conformation of Tyr residues in silk fibroin
Tyr is typically found in the GAGAGY and the GAGYGA motifs in silk fibroin.
Although the structure of silk fibroin has been studied for half a century, the
conformation of the Tyr residues has not been clearly understood. In this work, the
conformation of the Tyr residues in silk fibroin was investigated primarily by 2D 13C-13C
166
13
correlation NMR spectroscopy. Specifically, 2D through-space C-13C DARR NMR
13
experiments and 2D through-bond C-13C DQ/SQ refocused INADEQUATE NMR
experiments were applied to extract the chemical shifts of the Tyr residues in the silk
13
fibroin. Importantly, decent C enrichment for the Tyr residues in the fibroin provides
additional fingerprinting chemical shift information for the Tyr residues due to the
significant enhancement of signal-to-noise ratio in NMR experiments. The 13C chemical
shifts of the amino acid residues in silk fibroin were summarized in Table 6.1.

spectrum of lyophilized silk collected from B. mori silkworms that were fed with [U-2H/13C/15N]-
serine.
167
Table 6.1. 13C chemical shifts (in ppm from TMS) of amino acid residues in degummed B. mori
silk and lyophilized B. mori silk, solid polypeptides with known secondary structures, and
random coil conformations [18, 19, 42, 44, 45, 47, 48, 56, 58, 66-68].
B. mori Silk
random
Residue Degummed Lyophilized α-helix β-sheet 31-helix β-turn
coil
silk silk
Gly Cα 43.3 43.3 46.0 43.2-44.3 43.4 41.4-42.5 43.8-44.1
Gly CO 169.5 171.0 174.9 168.4-169.7 173.2 171.2-173.1 170.6-170.7
Ala Cα 49.6 50.7 52.3-52.8 48.2-49.3 50.8 48.9 50.8-51.7
17.3
Ala Cβ 16.6 14.8-16.0 19.9-20.7 17.4 17.4 16.5-17.4
20.8
Ala CO 172.8 173.9 176.2-176.8 171.6-172.2 176.1 174.6 176.8-177.5
Ser Cα 55.3 56.2 59.2 54.4-55.0 58.3 - 58.0
Ser Cβ 63.8 61.6 60.7 62.3-63.9 63.8 - 60.7
Ser CO 170.9 171.8 - 170.0-171.2 174.6 - 173.7
Tyr Cα 54.7 56.4 54.8-58.6 52.1 56.2 - -
36.3
Tyr Cβ 37.4 36.3 36.1 39.3 37.1 - -
40.5
Tyr Cγ 127.4 127.3 129.7 128.0 128.9 - -
Tyr Cδ 130.3 130.6 129.7 128.0 131.6 - -
Tyr Cε 115.3 115.4 116.1 115.0 116.5 - -
Tyr Cζ 155.8 156.0 154.2 155.2 155.6 - -
Tyr CO 171.2 173.3 176.7 169.7 174.2 - -
Phe Cα - - 61.3 53.2 56.0 - -
Phe Cβ - - 35.0 39.3 37.9 - -
Phe Cγ 136.7 - 138.4 136.3 137.2 - -
Phe Cδ 129.1 - 127.6 127.6 130.2 - -

13
2D through-bond C-13C DQ/SQ refocused INADEQUATE NMR correlation
spectrum of degummed silk collected from B. mori silkworms that were fed with [U-
2
H/13C/15N]-phenylalanine was shown in Figure 6.4. It is found that three resonances
associated with the Tyr Cβ were observed: 36.3 ppm, 37.4 ppm, and 40.5 ppm, indicating
the Tyr residues have three different conformations in the silk II. The resonance at 40.5
ppm is attributed to the Tyr Cβ in the β-sheet structures while and the resonances at 36.3
ppm and 37.4 ppm correspond is assigned to the Tyr Cβ in disordered structures
including helical, turn, and random coil structures [47, 50]. NMR studies on model
168
peptides have indicated that Tyr Cβ has chemical shifts of 36.1 ppm and 37.1 ppm for α–
helix and random coil structures, respectively. However, it has been reported that the Tyr
Cβ in β-turn structures has a chemical shift around 36 ppm that is similar to the Tyr Cβ in
α–helical structures [10]. To be safe, the resonances at 36.3 ppm and 37.4 ppm are
assigned to the Tyr Cβ present in helix/β-turn and random coil structures, respectively. If
13
C labeling efficiencies for different Tyr Cβ sites are assumed to be equal in the silk
fibroin, the composition of Tyr residues in each individual conformation could be
obtained from signal deconvolution according to the following procedure [69]: first, the
peak positions and peak widths for the Gly Cα and three Tyr Cβ resonances were
extracted from the refocused INADEQUATE NMR spectra. Gly Cα was also considered
in order to get a better deconvolution since it overlaps with the Tyr Cβ resonances;
second, a 13C direct spectrum of the labeled silk with a relaxation delay time of 10 s was
collected (Figure 6.5a). The use of the long relaxation delay time (10 s) ensures the full
relaxation of 13C nuclear, which is critical in obtaining a correct quantitative result; third,
the peak parameters obtained from INADEQUATE spectra were used in deconvolution,
in which only the intensities of peaks were varied. The deconvolution results were shown
in Figure 6.5b.
The ratios of different conformations for the Tyr residues were calculated from
the deconvolution results of the Tyr Cβ region and it indicates that the ratios of the Tyr
residues in the helix, β-sheets, and random coil structures are 36.2%, 40.4%, and 23.4%,
respectively. From the statistical analysis of protein sequence of B. mori silk fibroin
(Supplementary), the total number of the Tyr residues in silk fibroin is 288, and 221 Tyr
residues are found in the segments with repetitive sequences in the H-chain. The ratio can
169
be then estimated to be 76.7%. This is close to the sum of the ratios of the Tyr residues in
the helix (36.2%) and β-sheet structures (40.4%). In addition, the ratio of the Tyr residues
in random coil structures estimated from deconvolution (23.4%) is close to the ratio of
the Tyr residues in the linker and light chain of the protein, which is hypothesized to be in
relaxed conformations in previous studies [18, 47].

spectrum of degummed silk collected from B. mori silkworms that were fed with [U-2H/13C/15N]-
phenylalanine. The inset shows the zoom-in spectrum of Tyr Cβ-Cγ, correlation peak. Three
resonances can be clearly resolved and they correspond to different conformations.

170
Figure 6.5. (a) 13C direct spectrum of the degummed silkworm silk collected from B. mori
silkworms that were fed with [U-2H/13C/15N]-phenylalanine. (b) Deconvolution results of the Tyr
Cβ regions in 13C NMR spectrum.
Previous solid-state NMR study on model peptides has indicated that the Tyr
residues flanking the repetitive GAGAGS and (GA)n sequences are involved in β-sheet
structures {Asakura:2005hs}. Based on this finding, the ratio of this type of Tyr residues
was estimated to be ~ 40% in the sequence of silk fibroin, primarily in the GAGAGY
motif (labeled blue in silk fibroin sequence, supplementary). This ratio agrees well with
the deconvolution result for Tyr Cβ in β-sheet structures (~40.4%), illustrating that the
Tyr residues in the GAGAGY motif flanking the repetitive GAGAGS and the (GA)n
motifs are in β-sheet structures. Additionally, ~36% Tyr residues are found in GAGYGA
motif (labeled red in silk fibroin sequence, supplementary), which is primarily located in
the linking region between repetitive GAGAGS sequences. This ratio is consistent with
171
the deconvolution result for helix/β-turn structures (~36.2%), indicating the Tyr residues
in GAGYGA motif are very likely in helix/β-turn structures.
The conformation of the Tyr residues in silk I was also investigated in this study.
13
The results of 2D C-13C correlation NMR spectroscopy on the 13
C-Tyr labeled
lyophilized silk have indicated that Tyr CO, Cα, and Cβ have 13C chemical shifts of 173.3
ppm, 56.4 ppm, and 36.3 ppm respectively (Figure 6.6). This result is in good agreement
with the chemical shifts reported for the Tyr residues in GAGYGA motif in the liquid
silk [10] where the Tyr residues are indicated to be present in disordered type II β-turn
structures in the silk I.

spectrum of lyophilized silkworm silk made from B. mori silkworms that were fed with [U-
2
H/13C/15N]-phenylalanine. The inset shows the zoom-in spectrum of Tyr Cβ-Cγ, correlation
peak.
172
6.4 Conclusions
In this work, the conformations of the Ala, Ser, and Tyr residues in silk fibroin
were investigated primarily by 2D 13C-13C correlation NMR spectroscopies, including 2D
through-space 13C-13C DARR NMR spectroscopy and 2D through-bond 13C-13C DQ/SQ
refocused INADEQUATE NMR spectroscopy. It is found that the Ala, Ser, and Tyr
residues are all present in the disordered structures in the silk I, while show different
conformations in the silk II. Specifically, the Ala residues are present in both amorphous
domains composed of disordered structures and β-sheet crystalline domains with the
primary sequence (GAGAGS)n and (GA)n, and the Ser residues are present primarily in
β-sheet crystalline domains with the primary sequence (GAGAGS)n in the silk II.
Furthermore, it is presumed that in the silk II the Tyr residues in the GAGAGY motif
flanking the repetitive GAGAGS and (GA)n motifs are very likely in β-sheet structures,
while the Tyr residues in the GAGYGA motif present in the linking regions between the
repetitive GAGAGS sequences are in helix/β-turn structures. Additionally, It is illustrated
that the Tyr residues are not present in β-sheet structures, otherwise, it is very likely
present in disordered structures such as helical and β-turn structures in the silk I.
6.5 References

173
Spectrosc., 69 (2013) 23-68.
1612-1631.
Polymer, 49 (2008) 4309-4327.

741.

119-122.

[14] K. Tanaka, N. Kajiyama, K. Ishikura, S. Waga, A. Kikuchi, K. Ohtomo, T. Takagi,

S. Mizuno, Determination of the site of disulide linkage between heavy and light
chains of silk fbroin produced by Bombyx mori, Biochim. Biophys. Acta, 1432
(1999) 92-103.
[16] R.E. Marsh, R.B. Corey, L. Pauling, An invesitgation of the structure of silk fibroin,
Biochim. Biophys. Acta, 16 (1955) 1-34.
3908-3913.
174
Am. Chem. Soc., 124 (2002) 8794-8795.
2713.


[23] L.F. Drummy, B.L. Farmer, R.R. Naik, Correlation of the β-sheet crystal size in silk
fibers with the protein amino acid sequence, Soft Matter, 3 (2007) 877-882.

(2001) 521-525.

Mol. Biol., 61 (1971) 201-215.
Biol., 32 (1967) 289-295.

(1966) 580-582.
[30] B. Lotz, F.C. Cesari, The chemical structure and the crystalline structures of
Bombyx mori silk fibroin, Biochimie, 61 (1979) 205-214.
175

3344-3349.


Vib. Spectrosc, 46 (2008) 63-68.

78 (2005) 249-258.

324.

[39] E. Callone, S. Dirè, X. Hu, A. Motta, Processing influence on molecular assembling

and structural conformations in silk fibroin: elucidation by solid-state NMR, ACS
Biomater. Sci. Eng., 2 (2016) 758-767.

13
7397-7403.

176
2654-2657.

680-688.
266.


2

(2001) 291-305.

33 (2000) 9682-9687.

8495.

(1999) 7166-7171.
177
Biol. Macromol., 24 (1999) 187-195.
[53] T. Asakura, M. Minami, R. Shimada, M. Demura, M. Osanai, T. Fujito, M. Imanari,

A.S. Ulrich, 2H-labeling of silk fibroin fibers and their structural characterization by
solid-state 2H NMR, Macromolecules, 30 (1997) 2429-2435.

study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C- NMR



Macromolecules, 18 (1985) 1841-1845.

[59] T. Asakura, Y. Sato, A. Aoki, Stretching-induced conformational transition of the

crystalline and noncrystalline domains of 13C-labeled Bombyx mori silk fibroin
monitored by solid state NMR, Macromolecules, 48 (2015) 5761-5769.
[60] T. Asakura, Y. Watanabe, A. Uchida, H. Minagawa, NMR of silk fibroin. 2. 13C

NMR study of the chain dynamics and solution structure of Bombyx mori silk
fibroin, Macromolecules, 17 (1984) 1075-1081.
[61] H. Saito, M. Ishida, M. Yokoi, T. Asakura, Dynamic features of side chains in

tyrosine and serine residues of some polypeptides and fibroins in the solid as
studied by high-resolution solid-state 13C NMR spectroscopy, Macromolecules, 23
(1990) 83-88.
[62] A. Lesage, M. Bardet, L. Emsley, Through-bond carbon-carbon connectivities in

178
Phys., 118 (2003) 2325-2341.

Macromolecules, 40 (2007) 1995-2001.

6.6.1 Protein sequence of silk fibroin
Heavy chain (5263 residues, 391,593 Da)
MRVKTFVILCCALQYVAYTNANINDFDEDYFGSDVTVQSSNTTDEIIRDASGAVI
EEQITTKKMQRKNKNHGILGKNEKMIKTFVITTDSDGNESIVEEDVLMKTLSDGT
VAQSYVAADAGAYSQSGPYVSNSGYSTHQGYTSDFSTSAAV (N-terminal)
GAGAGAGAAAGSGAGAGAGYGAASGAGAGAGAGAGAGYGTGAGAGAGAGY
GAGAGAGAGAGYGAGAGAGAGAGYGAGAGAGAGAGYGAGAGAGAGAGYG
AGAGAGAGAGYGAASGAGAGAGYGQGVGSGAASGAGAGAGAGSAAGSGAG
AGAGTGAGAGYGAGAGAGAGAGYGAASGTGAGYGAGAGAGYGGASGAGAG
AGAGAGAGAGAGYGTGAGYGAGAGAGAGAGAGAGYGAGAGAGYGAGYGVG
AGAGYGAGYGAGAGSGAASGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGA
GSGAGAGSGAGAGSGAGAGSGTGAGSGAGAGYGAGAGAGYGAGAGSGAASG
AGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGAGAGYGAGAGAG
YGAGAGVGYGAGAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGA
179
GAGSGAGAGSGAGAGSGAGAGSGAGVGYGAGVGAGYGAGYGAGAGAGYGA
GAGSGAAS
GAGAGAGAGAGTGSSGFGPYVANGGYSRSDGYEYAWSSDFGTGS (Linker 1)
GAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGVGVGYGAGYGA
GAGAGYGAGAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAG
SGAGAGSGAGAGSGAGAGSGAGAGSGAGVGSGAGAGSGAGAGVGYGAGAGV
GYGAGAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAG
AGSGAGAGSGAGAGSGAGVGYGAGVGAGYGAGYGAGAGAGYGAGAGSGAA
SGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGA
GAGSGAGAGSGAGAGSGAGAGYGAGAGAGYGAGYGAGAGAGYGAGAGSGA
ASGAGSGAGAGSGAGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSG
AGAGYGAGVGAGYGAGYGAGAGAGYGAGAGSGAASGAGAGSGAGAGSGAG
AGSGA
GAGSGAGAGSGAGAGSGAGAGSGAGVGYGAGYGAGAGAGYGAGAGSGAASG
AGAGAGAGAGTGSSGFGPYVAHGGYSGYEYAWSSESDFGTGS (Linker 2)
GAGAGSGAGAGSGAGAGSGAGAGSGAGYGAGVGAGYGAGYGAGAGAGYGA
GAGSGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSG
AGAGSGAGAGYGAGYGAGAGAGYGAGAGSGAGSGAGAGSGAGAGSGAGAGS
GAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGVGAGYGAGYGAGAGAGY
GAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGVGSGAGAGSGAGAGSGAGAG
SGAGAGYGAGYGAGAGAGYGAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGA
GSGAGAGSGAGAGSGAGAGSGAGVGYGAGVGAGYGAGYGAGAGAGYGAGA
GSGAAS
GAGAGAGAGAGTGSSGFGPYVANGGYSGYEYAWSSESDFGTGS (Linker 3)
GAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGYGAGAGAGYGAGAGSGA
GSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGSGSGA
GAGSGAGAGSGAGAGYGAGVGAGYGVGYGAGAGAGYGAGAGSGAAS
GAGAGAGAGAGTGSSGFGPYVAHGGYSGYEYAWSSESDFGTGS (Linker 4)
GAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGVGAGYG
AAYGAGAGAGYGAGAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGAGAGS
GAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGAGAGYGAGAGSG
AGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGSGSGAGAGSGAGAGSG
AGAGYGAGVGAGYGAGYGAGAGAGYGAGAGSGAGSGAGAGSGAGAGYGAG
AGAGYGAGYGAGAGAGYGAGAGTGAGSGAGAGSGAGAGSGAGAGSGAGAGS
GAGAGSGAGAGSGAGSGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAG
AGYGAGAGAGYGAGYGAGAGAGYGAGAGSGAGSGAGAGSGAGAGSGAGAG
SGAGAGYGAGYGAGAGSGAAS
180
GAGAGAGAGAGTGSSGFGPYVAHGGYSGYEYAWSSESDFGTGS (Linker 5)
GAGAGSGAGAGAGAGAGSGAGAGYGAGVGAGYGAGYGAGAGAGYGAGAGS
GTGSGAGAGSGAGAGYGAGVGAGYGAGAGSGAAFGAGAGAGAGSGAGAGSG
AGAGSGAGAGSGAGAGSGAGAGYGAGYGAGVGAGYGAGAGSGAASGAGAGS
GAGAGSGAASGAGAGSGAGAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAG
SGAGAGSGAGAGYGAGAGSGAAS
GAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAG
YGAGAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGA
GYGAGVGAGYGVGYGAGAGAGYGAGAGSGAGSGAGAGSGAGAGSGAGAGS
GAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGYGVGYGAG
AGAGYGAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSGA
GAGSGAGAGYGAGVGAGYGVGYGAGAGAGYGAGAGSGAGSGAGAGSGAGA
GSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSGAG
AGSGAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGVGAGYGVGYGA
GVGAGYGAGAGSGAASGAGAGSGAGAGAGSGAGAGSGAGAGSGAGAGSGAG
AGSGAGAGSGAGAGYGAGYGAGVGAGYGAGAGVGYGAGAGAGYGAGAGSG
AASGAGAGAGSGAGAGTGAGAGSGAGAGYGAGAGSGAAS
GAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGVGAGYGAGAGSGAGSGAG
AGSGAGAGSGAGAGSGAGAGSGAGAGYGAGAGSGTGSGAGAGSGAGAGSGA
GAGSGAGAGSGAGAGSGAGAGSGVGAGYGVGYGAGAGAGYGVGYGAGAGA
GYGAGAGSGTGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAG
AGYGAGVGAGYGVGYGAGAGAGYGAGAGSGAGSGAGAGSGAGAGSGAGAG
SGAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGYGVGYGA
GAGAGYGAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSG
AGAGSGAGAGYGAGVGAGYGVGYGAGAGAGYGAGAGSGAGSGAGAGSGAG
AGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSGA
GAGSGAGAGYGAGVGAGYGVGYGAGAGAGYGAGAGSGAAS
GAGAGSGAGAGSGAGAGYGAGYGAGVGAGYGAGAGVGYGAGAGAGYGAGA
GSGAASGAGAGAGAGAGSGAGAGSGAGAGAGSGAGAGYGAGYGIGVGAGYG
AGAGVGYGAGAGAGYGAGAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGA
GAGSGAGAGSGAGAGYGAGYGAGVGAGYGAGAGVGYGAGAGAGYGAGAGS
GAASGAGAGAGAGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAG
181
AGSGAGAGSGAGAGSGAGAGYGAGVGAGYGAGYGGAGAGYGAGAGSGAAS
GAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGAGSGAAS
GAGAGSGAGAGSGAGAGYGAGVGAGYGAGYGAGAGAGYGAGAGSGAASGA
GAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGSGAGAGSGAGAGSG
AGAGSGAGAGYGAGVGAGYGAGYGAGAGAGYGAGAGSGAASGAGAGSGAG
AGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGSGAGAGY
GAGYGAGVGAGYGAGAGVGYGAGAGAGYGAGAGSGAASGAGAGSGSGAGS
GAGAGSGAGAGSGAGAGAGSGAGAGSGAGAGSGAGAGYGAGYGAGAGSGAA
S
GAGAGSGAGAGSGAGAGYGAGVGAGYGAGYGAGAGAGYGAGAGSGAGSGA
GAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGYG
AGAGAGYGAGAGVGYGAGAGAGYGAGAGSGAGSGAGAGSGSGAGAGSGSGA
GSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGYGIG
VGAGYGAGAGVGYGAGAGAGYGAGAGSGAASGAGAGSGAGAGSGAGAGSG
AGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGYGAGAGVGYGA
GAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSG
AGSGAGAGSGAGAGYGAGYGAGVGAGYGAGAGYGAGYGVGAGAGYGAGAG
SGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGSGAGAGYGAGAGA
GYGAGAGAGYGAGAGSGAASGAGAGAGAGSGAGAGSGAGAGSGAGSGAGAG
SGAGAGYGAGAGSGAASGAGAGSGAGAGAGAGAGAGSGAGAGSGAGAGYGA
GAGSGAAS
GAGAGAGAGTGSSGFGPYVANGGYSRREGYEYAWSSKSDFETGS (Linker 11)
GAASGAGAGAGSGAGAGSGAGAGSGAGAGSGAGAGS
VSYGAGRGYGQGAGSAASSVSSASSRSYDYSRRNVRKNCGIPRRQLVVKFRALP
CVNC (C-terminal)
Light Chain (262 residues, 27669 Da)

MKPIFLVLLVATSAYAAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDK
SIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYT
DGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRI
YDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVF
HQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHV
182
6.6.2 Supporting figures and tables
Figure 6.7. 1Hà13C CP-MAS spectra of (a) degummed silk collected from B. mori silkworms
that were fed with [U-2H/13C/15N]-serine, (b) lyophilized silk made from [U-2H/13C/15N]-serine
labeled degummed B. mori silk, (c) degummed silk collected from B. mori silkworms that were
fed with [U-2H/13C/15N]-phenylalanine, and (d) lyophilized silk made from [U-2H/13C/15N]-
phenylalanine labeled degummed B. mori silk. The spectra were collected at 20 kHz MAS and a 1
ms CP contact time. The spectra were collected with the same number of scans (4096 scans) and
processed with the same exponential line broadening (lb = 100 Hz).
183
Table 6.2. 13C Chemical Shifts (in ppm from TMS) of Amino Acid Residues in liquid silk in B.
mori gland, regenerated B. mori silk solution, degummed B. mori silk and lyophilized B. mori
silk.
Liquid Silk in Gland Regenerated Silk

Residue Degummed Silk Lyophilized Silk
[10] Solution
Gly Cα 43.0 43.0 43.3 43.2
Gly CO 171.9 - 169.5 171.0
Gly NH 107.5/107.9/110.8 107.5/107.9/110.8 - -
Ala Cα 50.5 50.5 49.6 50.8
Ala Cβ 16.9 16.9 17.3/20.8 16.6
Ala CO 175.9 - 172.8 173.9
Ala NH 123.5 123.5 - -
Ser Cα 56.3 56.4 55.3 56.2
Ser Cβ 61.7 61.7 63.8 61.6
Ser CO 173.0 - 170.9 171.8
Ser NH 114.7/115.3 114.7/115.3 - -
Tyr Cα 56.1 56.0 54.7 56.4
Tyr Cβ 36.6 36.5 36.3/40.5 36.3
Tyr Cγ 128.4 - 127.4 127.3
Tyr Cδ 130.9 131.0 130.3 130.6
Tyr Cε 115.8 116.0 115.3 115.4
Tyr Cζ 155.0 - 155.8 156.0
Tyr CO 173.0 - 171.2 173.3
Tyr NH 119.8 119.8 - -

184
REFERENCES
CHAPTER 1
[1] F. Bloch, Nuclear Induction, Phys. Rev., 70 (1946) 460-474.
[2] F. Bloch, W.W. Hansen, M. Packard, Nuclear Induction, Phys. Rev., 69 (1946) 127.
[3] E.M. Purcell, H.C. Torrey, R.V. Pound, Resonance absorption by nuclear magnetic
moments in a solid, Phys. Rev., 69 (1946) 37-38.
[4] R.R. Ernst, W.A. Anderson, Application of fourier transform spectroscopy to

magnetic resonance, Rev. Sci. Instrum., 37 (1966) 93-102.
[5] W.P. Aue, E. Bartholdi, R.R. Ernst, Two-dimensional spectroscopy. Application to

nuclear magnetic resonance, J. Chem. Phys., 64 (1976) 2229-2246.
[6] A.W. Overhauser, Polarization of nuclei in metals, Phys. Rev., 92 (1953) 411-415.
[7] K. Wuthrich, The way to NMR structures of proteins, Nature Struct. Biol., 8 (2001)
923-925.
[8] E.D. Becker, A brief history of nuclear magnetic resonance., Anal. Chem., 65
(1993) 295A-302A.
[9] E.R. Andrew, A. Bradbury, R.G. Eades, Nuclear magnetic resonance spectra from a
crystal rotated at high speed, Nature, 182 (1958) 1659-1659.
[10] A. Pines, M.G. Gibby, J.S. Waugh, Proton-enhanced NMR of dilute spins in solids,
J. Chem. Phys., 59 (1973) 569-590.
Rev., 128 (1962) 2042-2053.
[12] O.N. Antzutkin, J.J. Balbach, R.D. Leapman, N.W. Rizzo, J. Reed, R. Tycko,
Multiple quantum solid-state NMR indicates a parallel, not antiparallel,
organization of beta-sheets in Alzheimer's β-amyloid fibrils, Proc. Natl. Acad. Sci.,
97 (2000) 13045-13050.
[13] R. Tycko, Solid state NMR studies of amyloid fibril structure, Annu. Rev. Phys.
Chem., 62 (2011) 279-299.
[14] F. Creuzet, A. McDermott, R. Gebhard, K. Van Der Hoef, M.B. Spijker-Assink, J.

Herzfeld, J. Lugtenburg, M.H. Levitt, R.G. Griffin, Determination of membrane
protein structure by rotational resonance NMR: bacteriorhodopsin., Science, 251
(1991) 783-786.
185
[15] O.C. Andronesi, S. Becker, K. Seidel, H. Heise, a. Howard S Young, M. Baldus,
Determination of membrane protein structure and dynamics by magic-angle-
spinning solid-state NMR spectroscopy, J. Am. Chem. Soc., 127 (2005) 12965-
12974.
Spectrosc., 69 (2013) 23-68.
[18] D.A. Torchia, Solid state NMR studies of protein internal dynamics, Ann. Rev.
Biophys. Bioeng., 13 (1984) 125-144.
[19] D.D. Laws, H.-M.L. Bitter, A. Jerschow, Solid-state NMR spectroscopic methods
in chemistry Angew. Chem. Int. Ed., 41 (2002) 3096-3129.
4300.
[21] S. Paasch, E. Brunner, Trends in solid-state NMR spectroscopy and their relevance
for bioanalytics., Anal. Bioanal. Chem., 398 (2010) 2351-2362.
[22] A. Roehrich, G. Drobny, Solid-state NMR studies of biomineralization peptides and

proteins., Acc. Chem. Res., 46 (2013) 2136-2144.
[23] P. Schanda, M. Ernst, Studying dynamics by magic-angle spinning solid-state NMR

spectroscopy: Principles and applications to biomolecules, Prog. Nucl. Magn.
Reson. Spectrosc., 96 (2016) 1-46.
[24] M.J. Duer, Solid state NMR spectroscopy: Principles and applications, John Wiley
& Sons, 2008.
[27] J. Cavanagh, W.J. Fairbrother, A.G. Palmer, N.J. Skelton, Protein NMR
spectroscopy: Principles and practice, Elsevier Academic Press, 2007.
[28] R.W. Schurko, Ultra-wideline solid-state NMR spectroscopy, Acc. Chem. Res., 46
(2013) 1985-1995.
186
[29] D.A. Giljohann, D.S. Seferos, W.L. Daniel, M.D. Massich, P.C. Patel, C.A. Mirkin,
Gold nanoparticles for biology and medicine, Angew. Chem. Int. Ed., 49 (2010)
3280-3294.
[30] O.S. Wolfbeis, An overview of nanoparticles commonly used in fluorescent

bioimaging., Chem Soc Rev, 44 (2015) 4743-4768.
[31] T. Vo-Dinh, Y. Liu, A.M. Fales, H. Ngo, H.-N. Wang, J.K. Register, H. Yuan, S.J.
Norton, G.D. Griffin, SERS nanosensors and nanoreporters: golden opportunities in
biomedical applications., WIREs Nanomed. Nanobiotechnol., 7 (2015) 17-33.
[32] K. Saha, S.S. Agasti, C. Kim, X. Li, V.M. Rotello, Gold nanoparticles in chemical
and biological sensing, Chem. Rev., 112 (2012) 2739-2779.
[33] M.A. Hayat, Colloidal gold: Principles, methods, and applications, Elsevier, 2012.
Rev, 37 (2008) 2028-2019.

[37] S. Eustis, M.A. El-Sayed, Why gold nanoparticles are more precious than pretty
gold: Noble metal surface plasmon resonance and its enhancement of the radiative
and nonradiative properties of nanocrystals of different shapes, Chem Soc Rev, 35
(2006) 209-217.
[38] M.-C. Daniel, D. Astruc, Gold nanoparticles: Assembly, supramolecular chemistry,

quantum-size-related properties, and applications toward biology, catalysis, and
nanotechnology, Chem. Rev., 104 (2004) 293-346.
[39] J. Turkevich, P.C. Stevenson, J. Hillier, A study of the nucleation and growth
processes in the synthesis of colloidal gold, Discuss. Faraday Soc., 11 (1951) 55-75.

Science, 298 (2002) 2176-2179.

Chem. Commun., (1994) 801-802.
[42] C.A. Mirkin, R.L. Letsinger, R.C. Mucic, J.J. Storhoff, A DNA-based method for
rationally assembling nanoparticles into macroscopic materials., Nature, 382 (1996)
607-609.
187
[43] X. Ji, X. Song, J. Li, Y. Bai, W. Yang, X. Peng, Size control of gold nanocrystals in
citrate reduction: The third role of citrate, J. Am. Chem. Soc., 129 (2007) 13939-
13948.
(2008) 3754-3755.

[46] Y. Shichibu, Y. Negishi, H. Tsunoyama, M. Kanehara, T. Teranishi, T. Tsukuda,

Extremely high stability of glutathionate-protected Au25 clusters against core
etching, Small, 3 (2007) 835-839.

nanoparticles by inductively coupled plasma-mass spectrometry, ACS Nano, 7
(2013) 1129-1136.

8608-8610.
Chem. Soc., 131 (2009) 6535-6542.
10413.

188
135 (2013) 10011-10013.
430.
[56] D.W. Schaefer, K.D. Keefer, Structure of random porous materials: Silica aerogel,
Phys. Rev. Lett., 56 (1986) 2199-2202.
Res., 46 (2013) 792-801.


303-305.
397.
Nano Today, 4 (2009) 165-179.

Int. Ed., 47 (2008) 8582-8594.
[64] C. Bharti, U. Nagaich, A.K. Pal, N. Gulati, Mesoporous silica nanoparticles in

target drug delivery system: A review., Int J Pharm Investig, 5 (2015) 124-133.
[65] S. Kwon, R.K. Singh, R.A. Perez, E.A. Abou Neel, H.-W. Kim, W. Chrzanowski,
Silica-based mesoporous nanoparticles for controlled drug delivery, J Tissue Eng, 4
(2013) 2041731413503357.
189
[66] I.I. Slowing, J.L. Vivero-Escoto, C.-W. Wu, V.S.-Y. Lin, Mesoporous silica
nanoparticles as controlled release drug delivery and gene transfection carriers.,
Adv. Drug Deliv. Rev., 60 (2008) 1278-1288.

[69] R.I. Nooney, D. Thirunavukkarasu, Y. Chen, a. Robert Josephs, A.E. Ostafin,

Synthesis of nanoscale mesoporous silica spheres with controlled particle size,
Chem. Mater., 14 (2002) 4721-4728.
Soc., 118 (1996) 5103-5119.

[75] W.A. Shear, J.M. Palmer, J.A. Coddington, P.M. Bonamo, A devonian spinneret:
Early evidence of spiders and silk use, Science, 246 (1989) 479-481.
[76] R.V. Lewis, Spider silk: Ancient ideas for new biomaterials, Chem. Rev., 106
(2006) 3762-3774.
(2006) 377-385.
[78] C.Y. Hayashi, R.V. Lewis, Evidence from flagelliform silk cDNA for the structural
basis of elasticity and modular nature of spider silks., J. Mol. Biol., 275 (1998) 773-
784.
190
Natl. Acad. Sci., 87 (1990) 7120-7124.
[80] O. Emile, A.L. Floch, F. Vollrath, Biopolymers: Shape memory in spider draglines,
Nature, 440 (2006) 621-621.
[81] P.M. Cunniff, S.A. Fossey, M.A. Auerbach, J.W. Song, D.L. Kaplan, W.W. Adams,
R.K. Eby, D. Mahoney, D.L. Vezie, Mechanical and thermal properties of dragline
silk from the spider Nephila clavipes, Polymer. Adv. Tech., 5 (1994) 401-410.
[82] J.M. Gosline, P.A. Guerette, C.S. Ortlepp, K.N. Savage, The mechanical design of
spider silks: From fibroin sequence to mechanical function., J. Exp. Biol., 202
(1999) 3295-3303.

1480-1481.
392-398.
741.
[90] C. Vepari, D.L. Kaplan, Silk as a biomaterial, Prog. Polym. Sci., 32 (2007) 991-
1007.
[91] L. Yang, K.O. van der Werf, C.F.C. Fitié, M.L. Bennink, P.J. Dijkstra, J. Feijen,
Mechanical properties of native and cross-linked type I collagen fibrils, Biophys. J.,
94 (2008) 2204-2211.
Chem. Commun., 46 (2010) 6714-6713.
191
367.
Polymer, 49 (2008) 4309-4327.

1612-1631.

119-122.

[105] R.E. Marsh, R.B. Corey, L. Pauling, An invesitgation of the structure of silk
fibroin, Biochim. Biophys. Acta, 16 (1955) 1-34.
3908-3913.
192
Am. Chem. Soc., 124 (2002) 8794-8795.
[108] T. Asakura, J. Yao, 13C CP/MAS NMR study on structural heterogeneity in

Bombyx mori silk fiber and their generation by stretching, Protein Sci., 11 (2002)
2706-2713.


[111] L.F. Drummy, B.L. Farmer, R.R. Naik, Correlation of the β-sheet crystal size in
silk fibers with the protein amino acid sequence, Soft Matter, 3 (2007) 877-882.

(2001) 521-525.

Mol. Biol., 61 (1971) 201-215.
Biol., 32 (1967) 289-295.

(1966) 580-582.
[118] E. Callone, S. Dirè, X. Hu, A. Motta, Processing influence on molecular

assembling and structural conformations in silk fibroin: elucidation by solid-state
NMR, ACS Biomater. Sci. Eng., 2 (2016) 758-767.
193

13
7397-7403.


2654-2657.

680-688.
266.


2

(2001) 291-305.
194
33 (2000) 9682-9687.

8495.

(1999) 7166-7171.
Biol. Macromol., 24 (1999) 187-195.
[133] T. Asakura, M. Minami, R. Shimada, M. Demura, M. Osanai, T. Fujito, M.

Imanari, A.S. Ulrich, 2H-labeling of silk fibroin fibers and their structural
characterization by solid-state 2H NMR, Macromolecules, 30 (1997) 2429-2435.

study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR



Macromolecules, 18 (1985) 1841-1845.

resolution 13C NMR study of silk fibroin in the solid state by the cross-
polarization-magic angle spinning method. Conformational characterization of silk I
and silk II type forms of Bombyx mori fibroin by the conformation-dependent 13C
chemical shifts, Macromolecules, 17 (1984) 1405-1412.

195
3344-3349.


Vib. Spectrosc, 46 (2008) 63-68.
[143] T. Lefèvre, M.-E. Rousseau, M. Pézolet, Protein secondary structure and

orientation in silk as revealed by Raman spectromicroscopy, Biophys. J., 92 (2007)
2885-2895.

78 (2005) 249-258.

324.


CHAPTER 2

Rev, 37 (2008) 2028-2019.
196
10413.
[6] M. Stratakis, H. Garcia, Catalysis by supported gold nanoparticles: Beyond aerobic

oxidative processes, Chem. Rev., 112 (2012) 4469-4506.
[7] B. Sharma, R.R. Frontiera, A.-I. Henry, E. Ringe, R.P. Van Duyne, SERS:
Materials, applications, and the future, Materials Today, 15 (2012) 16-25.
[8] J.N. Anker, W.P. Hall, O. Lyandres, N.C. Shah, J. Zhao, R.P. Van Duyne,
Biosensing with plasmonic nanosensors, Nat. Mater., 7 (2008) 442-453.
[9] D.-K. Lim, K.-S. Jeon, J.-H. Hwang, H. Kim, S. Kwon, Y.D. Suh, J.-M. Nam,
Highly uniform and reproducible surface-enhanced Raman scattering from DNA-
tailorable nanoparticles with 1-nm interior gap, Nat. Nanotechnol., 6 (2011) 452-
460.
[10] J.F. Li, Y.F. Huang, Y. Ding, Z.L. Yang, S.B. Li, X.S. Zhou, F.R. Fan, W. Zhang,
Z.Y. Zhou, D.Y. Wu, B. Ren, Z.L. Wang, Z.Q. Tian, Shell-isolated nanoparticle-
enhanced Raman spectroscopy, Nature, 464 (2010) 392-395.
[11] V. Marjomaki, T. Lahtinen, M. Martikainen, J. Koivisto, S. Malola, K. Salorinne,

M. Pettersson, H. Hakkinen, Site-specific targeting of enterovirus capsid by
functionalized monodisperse gold nanoclusters, Proc. Natl. Acad. Sci., 111 (2014)
1277-1281.
[12] B.V. Enustun, J. Turkevich, Coagulation of colloidal gold, J. Am. Chem. Soc., 85
(1963) 3317-3328.

[14] K. Pyo, V.D. Thanthirige, K. Kwak, P. Pandurangan, G. Ramakrishna, D. Lee,

Ultrabright luminescence from gold nanoclusters: rigidifying the Au(I)–thiolate
shell, J. Am. Chem. Soc., 137 (2015) 8244-8250.
[15] Z. Tang, B. Xu, B. Wu, M.W. Germann, G. Wang, Synthesis and structural
determination of multidentate 2,3-dithiol-stabilized Au clusters, J. Am. Chem. Soc.,
132 (2010) 3367-3374.
[16] J. Xie, Y. Zheng, J.Y. Ying, Protein-directed synthesis of highly fluorescent gold
nanoclusters, J. Am. Chem. Soc., 131 (2009) 888-889.
197
[17] Y. Yu, Z. Luo, D.M. Chevrier, D.T. Leong, P. Zhang, D.-e. Jiang, J. Xie,
Identification of a highly luminescent Au22(SG)18 nanocluster, J. Am. Chem. Soc.,
136 (2014) 1246-1249.
[18] N. Zheng, J. Fan, G.D. Stucky, One-step one-phase synthesis of monodisperse

noble-metallic nanoparticles and their colloidal crystals, J. Am. Chem. Soc., 128
(2006) 6550-6551.
[19] M. Zhu, E. Lanni, N. Garg, M.E. Bier, R. Jin, Kinetically controlled, high-yield
synthesis of Au25 clusters, J. Am. Chem. Soc., 130 (2008) 1138-1139.

Chem. Commun., (1994) 801-802.
[21] Z. Wu, J. Suhan, R. Jin, One-pot synthesis of atomically monodisperse, thiol-

functionalized Au25 nanoclusters, J. Mater. Chem., 19 (2009) 622-626.

Science, 298 (2002) 2176-2179.
[23] C. Yu, L. Zhu, R. Zhang, X. Wang, C. Guo, P. Sun, G. Xue, Investigation on the
mechanism of the synthesis of gold(I) thiolate complexes by NMR, J. Phys. Chem.
C, 118 (2014) 10434-10440.
[24] L. Zhu, C. Zhang, C. Guo, X. Wang, P. Sun, D. Zhou, W. Chen, G. Xue, New
insight into intermediate precursors of Brust–Schiffrin gold nanoparticles synthesis,
J. Phys. Chem. C, 117 (2013) 11399-11404.
[25] H. Häkkinen, The gold–sulfur interface at the nanoscale, Nature Chemistry, 4

(2012) 443-455.
[26] J.C. Azcárate, G. Corthey, E. Pensa, C. Vericat, M.H. Fonticelli, R.C. Salvarezza,
P. Carro, Understanding the surface chemistry of thiolate-protected metallic
nanoparticles, J. Phys. Chem. Lett., 4 (2013) 3127-3138.

nanoparticles by inductively coupled plasma–mass spectrometry, ACS Nano, 7
(2013) 1129-1136.

8608-8610.
198
Chem. Soc., 131 (2009) 6535-6542.

(2008) 3754-3755.
135 (2013) 10011-10013.
[35] C. Zeng, H. Qian, T. Li, G. Li, N.L. Rosi, B. Yoon, R.N. Barnett, R.L. Whetten, U.
Landman, R. Jin, Total structure and electronic properties of the gold nanocrystal
Au36(SR)24, Angew. Chem. Int. Ed., 51 (2012) 13114-13118.
[36] H. Qian, W.T. Eckenhoff, Y. Zhu, T. Pintauer, R. Jin, Total structure determination
of thiolate-protected Au38 nanoparticles, J. Am. Chem. Soc., 132 (2010) 8280-8281.
430.
[38] Z. Hens, J.C. Martins, A solution NMR toolbox for characterizing the surface
chemistry of colloidal nanocrystals, Chem. Mater., 25 (2013) 1211-1221.
[39] L.E. Marbella, J.E. Millstone, NMR techniques for noble metal nanoparticles,
Chem. Mater., 27 (2015) 2721-2739.
[40] B.S. Zelakiewicz, A.C. de Dios, Tong, 13C NMR spectroscopy of 13C1-labeled
octanethiol-protected Au nanoparticles: Shifts, relaxations, and particle-size effect,
J. Am. Chem. Soc., 125 (2003) 18-19.
199
[41] G.C. Lica, B.S. Zelakiewicz, Y.Y. Tong, Electrochemical and NMR
characterization of octanethiol-protected Au nanoparticles, J. Electroanal. Chem.,
554-555 (2003) 127-132.
[42] Z. Wu, R. Jin, Stability of the two Au-S binding modes in Au25(SG)18 nanoclusters
probed by NMR and optical spectroscopy, ACS Nano, 3 (2009) 2036-2042.
[43] A.M. Smith, L.E. Marbella, K.A. Johnston, M.J. Hartmann, S.E. Crawford, L.M.
Kozycz, D.S. Seferos, J.E. Millstone, Quantitative analysis of thiolated ligand
exchange on gold nanoparticles monitored by 1H NMR spectroscopy, Anal. Chem.,
87 (2015) 2771-2778.
[44] S.E. Lehman, Y. Tataurova, P.S. Mueller, S.V.S. Mariappan, S.C. Larsen, Ligand
characterization of covalently functionalized mesoporous silica nanoparticles: an
NMR toolbox approach, J. Phys. Chem. C, 118 (2014) 29943-29951.
[45] R. Sharma, G.P. Holland, V.C. Solomon, H. Zimmermann, S. Schiffenhaus, S.A.

Amin, D.A. Buttry, J.L. Yarger, NMR characterization of ligand binding and
exchange dynamics in triphenylphosphine-capped gold nanoparticles, J. Phys.
Chem. C, 113 (2009) 16387-16393.
[46] O.A. Wong, C.L. Heinecke, A.R. Simone, R.L. Whetten, C.J. Ackerson, Ligand
symmetry-equivalence on thiolate protected gold nanoclusters determined by NMR
spectroscopy, Nanoscale, 4 (2012) 4099-4094.
[47] R.L. Donkers, Y. Song, R.W. Murray, Substituent effects on the exchange dynamics
of ligands on 1.6 nm diameter gold nanoparticles, Langmuir, 20 (2004) 4703-4707.
[48] K.F. Morris, C.S. Johnson, Diffusion-ordered two-dimensional nuclear magnetic

resonance spectroscopy, J. Am. Chem. Soc., 114 (1992) 3139-3141.
[49] K. Salorinne, T. Lahtinen, J. Koivisto, E. Kalenius, M. Nissinen, M. Pettersson, H.

Häkkinen, Nondestructive size determination of thiol-stabilized gold nanoclusters in
solution by diffusion ordered NMR spectroscopy, Anal. Chem., 85 (2013) 3489-
3492.
[50] G. Canzi, A.A. Mrse, C.P. Kubiak, Diffusion-ordered NMR spectroscopy as a

reliable alternative to tem for determining the size of gold nanoparticles in organic
solutions, J. Phys. Chem. C, 115 (2011) 7972-7978.
[51] K. Salorinne, T. Lahtinen, S. Malola, J. Koivisto, H. Häkkinen, Solvation chemistry

of water-soluble thiol-protected gold nanocluster Au102 from DOSY NMR
spectroscopy and DFT calculations, Nanoscale, 6 (2014) 7823-7824.
[52] H. Kato, T. Saito, M. Nabeshima, K. Shimada, S. Kinugasa, Assessment of

diffusion coefficients of general solvents by PFG-NMR: Investigation of the
sources error, J. Magn. Reson., 180 (2006) 266-273.
200
[53] H. Qian, M. Zhu, C. Gayathri, R.R. Gil, R. Jin, Chirality in gold nanoclusters
probed by NMR spectroscopy, ACS Nano, 5 (2011) 8935-8942.
[54] N. Goswami, F. Lin, Y. Liu, D.T. Leong, J. Xie, Highly luminescent thiolated gold
nanoclusters impregnated in nanogel, Chem. Mater., 28 (2016) 4009-4016.
CHAPTER 3
[1] F.S. Emami, V. Puddu, R.J. Berry, V. Varshney, S.V. Patwardhan, C.C. Perry, H.
Heinz, Prediction of specific biomolecule adsorption on silica surfaces as a function
of pH and particle size, Chem. Mater., 26 (2014) 5725-5734.
Res., 46 (2013) 792-801.
[5] A.A. Shemetov, I. Nabiev, A. Sukhanova, Molecular interaction of proteins and

peptides with nanoparticles, ACS Nano, 6 (2012) 4585-4602.
6256.
303-305.
397.
201
[10] G.A. Somorjai, H. Frei, J.Y. Park, Advancing the frontiers in nanocatalysis,
biointerfaces, and renewable energy conversion by innovations of surface
techniques, J. Am. Chem. Soc., 131 (2009) 16589-16605.
Nano Today, 4 (2009) 165-179.

Int. Ed., 47 (2008) 8582-8594.
[13] N.F. Breen, T. Weidner, K. Li, D.G. Castner, G.P. Drobny, A solid-state deuterium
NMR and sum-frequency generation study of the side-chain dynamics of peptides
adsorbed onto surfaces, J. Am. Chem. Soc., 131 (2009) 14148-14149.
[14] O. Mermut, D.C. Phillips, R.L. York, K.R. McCrea, R.S. Ward, G.A. Somorjai, In
situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic
polystyrene and hydrophilic silica surfaces using quartz crystal microbalance,
atomic force microscopy, and sum frequency generation vibrational spectroscopy, J.
Am. Chem. Soc., 128 (2006) 3598-3607.
[15] J.M. Gibson, J.M. Popham, V. Raghunathan, P.S. Stayton, G.P. Drobny, A solid-
state NMR study of the dynamics and interactions of phenylalanine rings in a
statherin fragment bound to hydroxyapatite crystals, J. Am. Chem. Soc., 128 (2006)
5364-5370.

7598-7613.

[18] A. Rimola, M. Sodupe, P. Ugliengo, Amide and peptide bond formation: Interplay
between strained ring defects and silanol groups at amorphous silica surfaces, J.
Phys. Chem. C, 120 (2016) 24817-24826.

15 (2013) 13371-13310.
202
Biosph., 28 (1998) 227-234.

J. Phys. Chem., 62 (1958) 1168-1178.


[28] M. Lelli, D. Gajan, A. Lesage, M.A. Caporini, V. Vitzthum, P. Miéville, F.

Héroguel, F. Rascón, A. Roussey, C. Thieuleux, M. Boualleg, L. Veyre, G.
Bodenhausen, C. Coperet, L. Emsley, Fast characterization of functionalized silica
materials by silicon-29 surface-enhanced NMR spectroscopy using dynamic nuclear
polarization, J. Am. Chem. Soc., 133 (2011) 2104-2107.
Soc., 118 (1996) 5103-5119.
[30] C.C. Perry, X. Li, Structural studies of gel phases. Part 1.-Infrared spectroscopic
study of silica monoliths; the effect of thermal history on structure, J. Chem. Soc.,
Faraday Trans., 87 (1991) 761-766.

[32] D.D. Goller, R.T. Phillips, I.G. Sayce, Structural relaxation of SiO2 at elevated
temperatures monitored by in situ Raman scattering, J. Non-Cryst. Solids, 355
(2009) 1747-1754.
J. Phys. Chem., 96 (1992) 1395-1400.

203
Phys. Chem. C, 113 (2009) 13894-13898.

[37] G. Vaccaro, S. Agnello, G. Buscarino, F.M. Gelardi, Thermally induced structural

modification of silica nanoparticles investigated by Raman and Infrared absorption
spectroscopies, J. Phys. Chem. C, 114 (2010) 13991-13997.


[42] C.T. Kresge, M.E. Leonowicz, W.J. Roth, J.C. Vartuli, J.S. Beck, Ordered
mesoporous molecular sieves synthesized by a liquid-crystal template mechanism,
Nature, 359 (1992) 710-712.
[43] A. Rimola, S. Tosoni, M. Sodupe, P. Ugliengo, Does silica surface catalyse peptide
bond formation? New insights from first-principles calculations, Chem. Phys.
Chem, 7 (2006) 157-163.
19 (2007) 321-331.

204

level characterization of the inorganic-bioorganic interface by solid state NMR:
2267.

C, 116 (2012) 9691-9702.
7901-7909.

21825.
[53] N. Folliet, C. Gervais, D. Costa, G. Laurent, F. Babonneau, L. Stievano, J.-F.

Lambert, F. Tielens, A molecular picture of the adsorption of glycine in
mesoporous silica through NMR experiments combined with DFT-D calculations,
J. Phys. Chem. C, 117 (2013) 4104-4114.
Phys. Chem. C, 113 (2009) 18163-18172.

(2004) 914-923.
[57] J. Bujdák, B.M. Rode, Silica, alumina and clay catalyzed peptide bond formation:
enhanced efficiency of alumina catalyst., Orig. Life Evol. Biosph., 29 (1999) 451-
461.
205
Chem. Phys., 12 (2010) 6763-6711.
[59] J. Bujdák, B.M. Rode, Glycine oligomerization on silica and alumina, React. Kinet.
Catal. Lett., 62 (1997) 281-286.
Technol., 48 (2006) 197-201.

81 (1989) 196-200.
Rev., 128 (1962) 2042-2053.
[66] D.G. Cory, W.M. Ritchey, Suppression of signals from the probe in Bloch decay
spectra, J. Magn. Reson., 80 (1988) 128-132.


[70] R.K. Hester, J.L. Ackerman, B.L. Neff, J.S. Waugh, Separated local field spectra in
NMR: Determination of structure of solids, Phys. Rev. Lett., 36 (1976) 1081-1083.
[71] E. Vinogradov, P.K. Madhu, S. Vega, High-resolution proton solid-state NMR

spectroscopy by phase-modulated Lee–Goldburg experiment, Chem. Phys. Lett.,
314 (1999) 443-450.
206
peptides based on Nephila clavipes dragline silk spidroin (MaSp1) studied by 13C
3226.
[73] S. Fuchida, H. Masuda, K. Shinoda, Peptide formation mechanism on

montmorillonite under thermal conditions., Orig. Life Evol. Biosph., 44 (2014) 13-
28.
[74] M.P. Bhate, J.C. Woodard, M.A. Mehta, Solvation and hydrogen bonding in
alanine- and glycine-containing dipeptides probed using solution- and solid-state
Chem. Commun., 46 (2010) 6714-6713.
[76] A.R. Garcia, R.B. de Barros, J.o.P. Lourenço, L.M. Ilharco, The infrared spectrum
of solid L-alanine: Influence of pH-induced structural changes, J. Phys. Chem. A,
112 (2008) 8280-8287.
[77] N. Lahav, D. White, S. Chang, Peptide formation in the prebiotic era: Thermal
condensation of glycine in fluctuating clay environment, Science, 201 (1978) 67-69.
[78] A. Brack, From interstellar amino acids to prebiotic catalytic peptides: a review.,
Chem. Biodivers., 4 (2007) 665-679.
[80] R. Hemley, H. Mao, P. Bell, Raman spectroscopy of SiO2 glass at high pressure,
Phys. Rev. Lett., 57 (1986) 747-750.
CHAPTER 4

15 (2013) 13371-13310.
207
Res., 46 (2013) 792-801.
6256.

7598-7613.
[10] C.E. Ashley, E.C. Carnes, K.E. Epler, D.P. Padilla, G.K. Phillips, R.E. Castillo,
D.C. Wilkinson, B.S. Wilkinson, C.A. Burgard, R.M. Kalinich, J.L. Townson, B.
Chackerian, C.L. Willman, D.S. Peabody, W. Wharton, C.J. Brinker, Delivery of
small interfering rna by peptide-targeted mesoporous silica nanoparticle-supported
lipid bilayers, ACS Nano, 6 (2012) 2174-2188.
303-305.
397.
[13] J. Liu, A. Stace-Naughton, X. Jiang, C.J. Brinker, Porous nanoparticle supported

lipid bilayers (protocells) as delivery vehicles, J. Am. Chem. Soc., 131 (2009)
1354-1355.

208
[15] M. Vallet-Regi, A. Rámila, R.P. del Real, J. Pérez-Pariente, A new property of
MCM-41: Drug delivery system, Chem. Mater., 13 (2001) 308-311.
Biosph., 28 (1998) 227-234.
[19] F. Tielens, C. Gervais, J.F.o. Lambert, F. Mauri, D. Costa, Ab initio study of the
hydroxylated surface of amorphous silica: A representative model, Chem. Mater.,
20 (2008) 3336-3344.
Soc., 118 (1996) 5103-5119.

J. Phys. Chem., 62 (1958) 1168-1178.
J. Phys. Chem., 96 (1992) 1395-1400.


Phys. Chem. C, 113 (2009) 13894-13898.
209

[31] K. Saalwächter, M. Krause, W. Gronski, Study of molecular interactions and

dynamics in thin silica surface layers by proton solid-state NMR spectroscopy,
Chem. Mater., 16 (2004) 4071-4079.

[33] T. Yokoi, J. Wakabayashi, Y. Otsuka, W. Fan, M. Iwama, R. Watanabe, K.

Aramaki, A. Shimojima, T. Tatsumi, T. Okubo, Mechanism of formation of
uniform-sized silica nanospheres catalyzed by basic amino acids, Chem. Mater., 21
(2009) 3719-3729.
[34] M.M. Tomczak, D.D. Glawe, L.F. Drummy, C.G. Lawrence, M.O. Stone, C.C.
Perry, D.J. Pochan, T.J. Deming, R.R. Naik, Polypeptide-templated synthesis of
hexagonal silica platelets, J. Am. Chem. Soc., 127 (2005) 12577-12582.
[35] T. Yokoi, Y. Sakamoto, O. Terasaki, Y. Kubota, T. Okubo, T. Tatsumi, Periodic

arrangement of silica nanospheres assisted by amino acids, J. Am. Chem. Soc., 128
(2006) 13664-13665.
[36] S.H. Behrens, D.G. Grier, The charge of glass and silica surfaces, J. Chem. Phys.,
115 (2001) 6716-6721.
[37] N. Kitadai, T. Yokoyama, S. Nakashima, In situ ATR-IR investigation of L-lysine

adsorption on montmorillonite, J. Colloid Interface Sci., 338 (2009) 395-401.

[39] A.D. Roddick-Lanzilotta, P.A. Connor, A.J. McQuillan, An in situ infrared

spectroscopic study of the adsorption of lysine to TiO2 from an aqueous solution,
Langmuir, 14 (1998) 6479-6484.
210
[40] T.F. Koetzle, M.S. Lehmann, J.J. Verbist, W.C. Hamilton, Precision neutron
diffraction structure determination of protein and nucleic acid components. VII. The
crystal and molecular structure of the amino acid L-lysine monohydrochloride
dihydrate, Acta Cryst., B28 (1972) 3207-3214.
[41] D.A. Wright, R.E. Marsh, The crystal structure of L-lysine monohydrochloride
dihydrate, Acta Cryst., 15 (1962) 54-64.
19 (2007) 321-331.


level characterization of the inorganic−bioorganic interface by solid state NMR:
2267.

C, 116 (2012) 9691-9702.
7901-7909.
Phys. Chem. C, 113 (2009) 18163-18172.

(2004) 914-923.
Chem. Phys., 12 (2010) 6763-6711.
211
Technol., 48 (2006) 197-201.

21825.
4300.
[56] P.A. Mirau, J.L. Serres, M. Lyons, The structure and dynamics of poly(L-lysine) in
templated silica nanocomposites, Chem. Mater., 20 (2008) 2218-2223.
[57] R. Manríquez, F.A. López-Dellamary, J. Frydel, T. Emmler, H. Breitzke, G.

Buntkowsky, H.-H. Limbach, I.G. Shenderovich, Solid-state NMR studies of
aminocarboxylic salt bridges in L-lysine modified cellulose, J. Phys. Chem. B, 113
(2009) 934-940.

81 (1989) 196-200.
[60] S.P. Brown, Probing proton–proton proximities in the solid state, Prog. Nucl. Magn.
Reson. Spectrosc., 50 (2007) 199-251.
[61] P. Hohenberg, W. Kohn, Inhomogeneous electron gas, Phys. Rev., 136 (1964) 864-
871.
[62] W. Kohn, L.J. Sham, Self-consistent equations including exchange and correlation
effects, Phys. Rev., 140 (1965) 1133-1138.
Rev., 128 (1962) 2042-2053.
[65] K. Takegoshi, S. Nakamura, T. Terao, 13C-1H dipolar-assisted rotational resonance

212
[66] K. Takegoshi, S. Nakamura, T. Terao, 13C–1H dipolar-driven 13C–13C recoupling
Phys., 118 (2003) 2325-2341.
[67] A. Lesage, C. Auger, S. Caldarelli, L. Emsley, Determination of through-bond

carbon-carbon connectivities in solid-state NMR using the INADEQUATE
experiment, J. Am. Chem. Soc., 119 (1997) 7867-7868.
[68] A. Lesage, M. Bardet, L. Emsley, Through-bond carbon−carbon connectivities in

[70] M.e.a. Feike, Broadband multiple-quantum NMR spectroscopy, J. Magn. Reson.,

122 (1996) 214-221.
[71] I. Schnell, H.W. Spiess, High-resolution 1H NMR spectroscopy in the solid state:
Very fast sample rotation and multiple-quantum coherences, J. Magn. Reson., 151
(2001) 153-227.


[74] A.D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J.
Chem. Phys., 98 (1993) 5648-5652.
[75] K. Wolinski, J.F. Hinton, P. Pulay, Efficient implementation of the gauge-

independent atomic orbital method for NMR chemical shift calculations, J. Chem.
Soc., Chem. Commun., 112 (1990) 8251-8260.
[76] A. Dos, V. Schimming, M.C. Huot, H.-H. Limbach, Acid-induced amino side-chain
interactions and secondary structure of solid poly-L-lysine probed by 15N and 13C
solid state NMR and ab initio model calculations, J. Am. Chem. Soc., 131 (2009)
7641-7653.
[77] A. Dos, V. Schimming, S. Tosoni, H.-H. Limbach, Acid−base interactions and

secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR and
ab initio model calculations, J. Phys. Chem. B, 112 (2008) 15604-15615.
[78] G. Schreckenbach, T. Ziegler, Calculation of NMR shielding tensors using gauge-

including atomic orbitals and modern density functional theory, J. Phys. Chem., 99
(1995) 606-611.
213
[79] V. Schimming, C.-G. Hoelger, G. Buntkowsky, I. Sack, J.-H. Fuhrhop, S.
Rocchetti, H.-H. Limbach, Evidence by 15N CPMAS and 15N−13C REDOR NMR
for fixation of atmospheric CO2 by amino groups of biopolymers in the solid state,
J. Am. Chem. Soc., 121 (1999) 4892-4893.
[80] S. Maeda, S. Oumae, S. Kaneko, K.-K. Kunimoto, Formation of carbamates and

cross-linking of microbial poly(ε-L-lysine) studied by 13C and 15N solid-state NMR,
Polym. Bull., 68 (2011) 745-754.
[81] J. Schmidt, A. Hoffmann, H.W. Spiess, D. Sebastiani, Bulk chemical shifts in

hydrogen-bonded systems from first-principles calculations and solid-state-NMR, J.
Phys. Chem. B, 110 (2006) 23204-23210.
CHAPTER 5
[1] M. Heim, D. Keerl, T. Scheibel, Spider silk: From soluble protein to extraordinary
fiber, Angew. Chem. Int. Ed., 48 (2009) 3584-3596.
[2] R.W. Work, Force-elongation behavior of web fibers and silks forcibly obtained
from orb-web-spinning spiders., Text. Res. J., 46 (1976) 485-492.
[3] F. Vollrath, D.P. Knight, Liquid crystalline spinning of spider silk., Nature, 410
(2001) 541-548.
[4] L.W. Jelinski, A. Blye, O. Liivak, C.A. Michal, G. LaVerde, A. Seidel, N. Shah, Z.
Yang, Orientation, structure, wet-spinning, and molecular basis for supercontraction
of spider dragline silk, Int. J. Biol. Macromol., 24 (1999) 197-201.
[5] Z. Shao, F. Vollrath, J. Sirichaisit, R.J. Young, Analysis of spider silk in native and
supercontracted states using Raman spectroscopy, Polymer, 40 (1999) 2493-2500.
[6] J.D. van Beek, J. Kummerlen, F. Vollrath, B.H. Meier, Supercontracted spider
dragline silk: a solid-state NMR study of the local structure, Int. J. Biol. Macromol.,
24 (1999) 173-178.
[7] Z. Yang, O. Liivak, A. Seidel, G. LaVerde, D.B. Zax, L.W. Jelinski,

Supercontraction and backbone dynamics in spider silk: 13C and 2H NMR studies,
J. Am. Chem. Soc., 122 (2000) 9019-9025.
(2006) 377-385.
Natl. Acad. Sci., 87 (1990) 7120-7124.
392-398.
214

[14] C. Riekel, B. Madsen, D. Knight, F. Vollrath, X-ray diffraction on spider silk

during controlled extrusion under a synchrotron radiation X-ray beam,
[15] C. Riekel, F. Vollrath, Spider silk fibre extrusion: combined wide- and small-angle
X-ray microdiffraction experiments, Int. J. Biol. Macromol., 29 (2001) 203-210.
[17] G.R. Plaza, J. Pérez-Rigueiro, C. Riekel, G.B. Perea, F. Agulló-Rueda, M.

Burghammer, G.V. Guinea, M. Elices, Relationship between microstructure and
mechanical properties in spider silk fibers: identification of two regimes in the
microstructural changes, Soft Matter, 8 (2012) 6015-6012.
[18] S. Sampath, T. Isdebski, J.E. Jenkins, J.V. Ayon, R.W. Henning, J.P.R.O. Orgel, O.
Antipoa, J.L. Yarger, X-ray diffraction study of nanocrystalline and amorphous
structure within major and minor ampullate dragline spider silks, Soft Matter, 8
(2012) 6713-6711.
[19] J.E. Jenkins, S. Sampath, E. Butler, J. Kim, R.W. Henning, G.P. Holland, J.L.
Yarger, Characterizing the secondary protein structure of black widow dragline silk
using solid-state NMR and X-ray diffraction, Biomacromolecules, 14 (2013) 3472-
3483.
[20] S. Sampath, J.L. Yarger, Structural hysteresis in dragline spider silks induced by
supercontraction: an X-ray fiber micro-diffraction study, RSC Adv., 5 (2014) 1462-
1473.
[21] R. Madurga, T.A. Blackledge, B. Perea, G.R. Plaza, C. Riekel, M. Burghammer, M.

Elices, G. Guinea, J. Pérez-Rigueiro, Persistence and variation in microstructural
design during the evolution of spider silk, Sci. Rep., 5 (2015) 1-11.
[22] C. Riekel, M. Burghammer, T.G. Dane, C. Ferrero, M. Rosenthal, Nanoscale

structural features in major ampullate spider silk, Biomacromolecules, 18 (2017)
231-241.
215
[23] A. Simmons, E. Ray, L.W. Jelinski, Solid-state 13C NMR of Nephila clavipes
dragline silk establishes structure and identity of crystalline regions,
Macromolecules, 27 (1994) 5235-5237.
[24] J. Kummerien, J.D. van Beek, F. Vollrath, B.H. Meier, Local structure in spider
dragline silk investigated by two-dimensional spin-diffusion nuclear magnetic
resonance Macromolecules, 29 (1996) 2920-2928.
[25] A. Simmons, C.A. Michal, L.W. Jelinski, Molecular orientation and two-component
nature of the crystalline fraction of spider dragline silk, Science, 271 (1996) 84-87.
1480-1481.
[27] O. Liivak, A. Flores, R.V. Lewis, L.W. Jelinski, Conformation of the polyalanine
repeats in minor ampullate gland silk of the spider Nephila clavipes ,
Macromolecules, 30 (1997) 7127-7130.
[28] A.D. Parkhe, S.K. Seeley, K. Gardner, L. Thompson, R.V. Lewis, Structural studies
of spider silk proteins in the fiber, J. Mol. Recogn., 10 (1997) 1-6.
[29] C.A. Michal, L.W. Jelinski, Rotational-echo double-resonance in complex

biopolymers: a study of Nephila clavipes dragline silk, J. Biomol. NMR, 12 (1998)
231-241.
[30] J.D. van Beek, S. Hess, F. Vollrath, B.H. Meier, The molecular structure of spider
dragline silk: Folding and orientation of the protein backbone, Proc. Natl. Acad.
Sci., 99 (2002) 10266-10271.
[31] T. Asakura, M. Yang, T. Kawase, Structure of characteristic sequences in Nephila

clavipes dragline silk (Masp1) studied with 13C solid state NMR, Polym. J., 36
(2004) 999-1003.
[32] P.T. Eles, C.A. Michal, Strain dependent local phase transitions observed during
controlled supercontraction reveal mechanisms in spider silk, Macromolecules, 37
(2004) 1342-1345.
[33] G.P. Holland, R.V. Lewis, J.L. Yarger, WISE NMR characterization of nanoscale
heterogeneity and mobility in supercontracted Nephila clavipes spider dragline silk,
J. Am. Chem. Soc., 126 (2004) 5867-5872.

peptides based on Nephila clavipes dragline silk spidroin (Masp1) studied by 13C
3226.
216
[35] B. Bonev, S. Grieve, M.E. Herberstein, A.I. Kishore, A. Watts, F. Separovic,
Orientational order of Australian spider silks as determined by solid-state NMR,
Biopolymers, 82 (2006) 134-143.
[36] J.D. van Beek, B.H. Meier, A DOQSY approach for the elucidation of torsion angle
distributions in biopolymers: Application to silk, J. Magn. Reson., 178 (2006) 106-
120.
Macromolecules, 40 (2007) 1995-2001.
[39] G.P. Holland, J.E. Jenkins, M.S. Creager, R.V. Lewis, J.L. Yarger, Quantifying the
fraction of glycine and alanine in β-sheet and helical conformations in spider
dragline silk using solid-state NMR, Chem. Commun., (2008) 5568-5563.
[40] G.P. Holland, J.E. Jenkins, M.S. Creager, R.V. Lewis, J.L. Yarger, Solid-state
NMR investigation of major and minor ampullate spider silk in the native and
hydrated states, Biomacromolecules, 9 (2008) 651-657.
[41] M.S. Creager, J.E. Jenkins, L.A. Thagard-Yeaman, A.E. Brooks, J.A. Jones, R.V.
Lewis, G.P. Holland, J.L. Yarger, Solid-state NMR comparison of various spiders’
dragline silk fiber, Biomacromolecules, 11 (2010) 2039-2043.
Chem. Commun., 46 (2010) 6714-6713.
[45] J.E. Jenkins, G.P. Holland, J.L. Yarger, High resolution magic angle spinning NMR
investigation of silk protein structure within major ampullate glands of orb weaving
spiders, Soft Matter, 8 (2012) 1947-1954.
[46] G.P. Holland, Q. Mou, J.L. Yarger, Determining hydrogen-bond interactions in

spider silk with 1H-13C HETCOR fast MAS solid-state NMR and DFT proton
chemical shift calculations, Chem. Commun., 49 (2013) 6680-6683.
217
[47] X. Shi, J.L. Yarger, G.P. Holland, 2H-13C HETCOR MAS NMR for indirect
detection of 2H quadrupole patterns and spin-lattice relaxation rates, J. Magn.
Reson., 226 (2013) 1-12.
[48] X. Shi, J.L. Yarger, G.P. Holland, Elucidating proline dynamics in spider dragline
silk fibre using 2H-13C HETCOR MAS NMR, Chem. Commun., 50 (2014) 4856-
4854.
[49] D. Xu, J.L. Yarger, G.P. Holland, Exploring the backbone dynamics of native
spider silk proteins in Black Widow silk glands with solution-state NMR
spectroscopy, Polymer, 55 (2014) 3879-3885.
[50] X. Shi, G.P. Holland, J.L. Yarger, Molecular dynamics of spider dragline silk fiber
investigated by 2H MAS NMR, Biomacromolecules, 16 (2015) 852-859.
[51] D. Xu, X. Shi, F. Thompson, W.S. Weber, Q. Mou, J.L. Yarger, Protein secondary
structure of Green Lynx spider dragline silk investigated by solid-state NMR and
X-ray diffraction, Int. J. Biol. Macromol., 81 (2015) 171-179.
[52] G. Gray, A. van der Vaart, C. Guo, J. Jones, D. Onofrei, B. Cherry, R. Lewis, J.
Yarger, G. Holland, Secondary structure adopted by the Gly-Gly-X repetitive
regions of dragline spider silk, International Journal of Molecular Sciences, 17
(2016) 2023-2013.

Vib. Spectrosc., 46 (2008) 63-68.
[54] R. Ene, P. Papadopoulos, F. Kremer, Combined structural model of spider dragline

silk, Soft Matter, 5 (2009) 4568-4567.
[55] P. Papadopoulos, R. Ene, I. Weidner, F. Kremer, Similarities in the structural

organization of major and minor ampullate spider silk, Macromol. Rapid Commun.,
30 (2009) 851-857.
[56] P. Papadopoulos, J. Sölter, F. Kremer, Hierarchies in the structural organization of

spider silk-a quantitative model, Colloid Polym Sci, 287 (2009) 231-236.
[57] R. Ene, P. Papadopoulos, F. Kremer, Partial deuteration probing structural changes

in supercontracted spider silk, Polymer, 51 (2010) 4784-4789.
[58] R. Ene, P. Papadopoulos, F. Kremer, Quantitative analysis of infrared absorption

coefficient of spider silk fibers, Vib. Spectrosc., 57 (2011) 207-212.
[59] S. Ling, Z. Qi, D.P. Knight, Z. Shao, X. Chen, Synchrotron FTIR microspectroscopy
of single natural silk fibers, Biomacromolecules, 12 (2011) 3344-3349.
218
[61] Z. Shao, R.J. Young, F. Vollrath, The effect of solvents on spider silk studied by
mechanical testing and single-fibre Raman spectroscopy, Int. J. Biol. Macromol., 24
(1999) 295-300.
[62] J. Sirichaisit, R.J. Young, F. Vollrath, Molecular deformation in spider dragline silk
subjected to stress, Polymer, 41 (2000) 1223-1227.

[65] P. Colomban, H.M. Dinh, J. Riand, L.C. Prinsloo, B. Mauchamp, Nanomechanics

of single silkworm and spider fibres: a Raman and micro-mechanical in situ study
of the conformation change with stress, J. Raman Spectrosc., 39 (2008) 1749-1764.
[66] M.-E. Rousseau, T. Lefèvre, M. Pézolet, Conformation and orientation of proteins

in various types of silk fibers produced by Nephila clavipes spiders,
[67] P. Colomban, H.M. Dinh, A. Bunsell, B. Mauchamp, Origin of the variability of the
mechanical properties of silk fibres: 1-The relationship between disorder, hydration
and stress/strain behaviour, J. Raman Spectrosc., 43 (2011) 425-432.
[68] T. Lefèvre, F. Paquet-Mercier, J.-F. Rioux-Dubé, M. Pézolet, Structure of silk by

raman spectromicroscopy: From the spinning glands to the fibers, Biopolymers, 97
(2011) 322-336.
[69] T. Lefèvre, M. Pézolet, Unexpected β-sheets and molecular orientation in

flagelliform spider silk as revealed by Raman spectromicroscopy, Soft Matter, 8
(2012) 6350-6358.
[70] S.L. Adrianos, F. Teulé, M.B. Hinman, J.A. Jones, W.S. Weber, J.L. Yarger, R.V.
Lewis, Nephila clavipes flagelliform silk-like GGX motifs contribute to
extensibility and spacer motifs contribute to strength in synthetic spider silk fibers,

219
Phys., 118 (2003) 2325-2341.
[73] Y. Shen, A. Bax, Identification of helix capping and β-turn motifs from NMR
chemical shifts, J. Biomol. NMR, 52 (2012) 211-232.

2654-2657.
680-688.
266.

Am. Chem. Soc., 124 (2002) 8794-8795.
2713.

2

(2001) 291-305.
[82] H. Saito, Conformation‐dependent 13C chemical shifts: A new means of

conformational characterization as obtained by high‐resolution solid‐state 13C NMR,
Magn. Reson. Chem., 24 (1986) 835-852.
220

367.
[86] A. Nova, S. Keten, N.M. Pugno, A. Redaelli, M.J. Buehler, Molecular and
nanostructural mechanisms of deformation, strength and toughness of spider silk
fibrils., Nano Lett., 10 (2010) 2626-2634.
[87] S.W. Cranford, A. Tarakanova, N.M. Pugno, M.J. Buehler, Nonlinear material
behaviour of spider silk yields robust webs, Nature, 482 (2012) 72-76.
CHAPTER 6

Spectrosc., 69 (2013) 23-68.
221
1612-1631.
Polymer, 49 (2008) 4309-4327.

741.

119-122.

[14] K. Tanaka, N. Kajiyama, K. Ishikura, S. Waga, A. Kikuchi, K. Ohtomo, T. Takagi,

S. Mizuno, Determination of the site of disulide linkage between heavy and light
chains of silk fbroin produced by Bombyx mori, Biochim. Biophys. Acta, 1432
(1999) 92-103.
[16] R.E. Marsh, R.B. Corey, L. Pauling, An invesitgation of the structure of silk fibroin,
Biochim. Biophys. Acta, 16 (1955) 1-34.
3908-3913.

Am. Chem. Soc., 124 (2002) 8794-8795.
2713.
222

[23] L.F. Drummy, B.L. Farmer, R.R. Naik, Correlation of the β-sheet crystal size in silk
fibers with the protein amino acid sequence, Soft Matter, 3 (2007) 877-882.

(2001) 521-525.

Mol. Biol., 61 (1971) 201-215.
Biol., 32 (1967) 289-295.

(1966) 580-582.
[30] B. Lotz, F.C. Cesari, The chemical structure and the crystalline structures of
Bombyx mori silk fibroin, Biochimie, 61 (1979) 205-214.


3344-3349.
223

Vib. Spectrosc, 46 (2008) 63-68.

78 (2005) 249-258.

324.

[39] E. Callone, S. Dirè, X. Hu, A. Motta, Processing influence on molecular assembling

and structural conformations in silk fibroin: elucidation by solid-state NMR, ACS
Biomater. Sci. Eng., 2 (2016) 758-767.

13
7397-7403.


2654-2657.

224
680-688.
266.


2

(2001) 291-305.

33 (2000) 9682-9687.

8495.

(1999) 7166-7171.
Biol. Macromol., 24 (1999) 187-195.
[53] T. Asakura, M. Minami, R. Shimada, M. Demura, M. Osanai, T. Fujito, M. Imanari,

A.S. Ulrich, 2H-labeling of silk fibroin fibers and their structural characterization by
solid-state 2H NMR, Macromolecules, 30 (1997) 2429-2435.
225
study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C- NMR



Macromolecules, 18 (1985) 1841-1845.

[59] T. Asakura, Y. Sato, A. Aoki, Stretching-induced conformational transition of the

crystalline and noncrystalline domains of 13C-labeled Bombyx mori silk fibroin
monitored by solid state NMR, Macromolecules, 48 (2015) 5761-5769.
[60] T. Asakura, Y. Watanabe, A. Uchida, H. Minagawa, NMR of silk fibroin. 2. 13C

NMR study of the chain dynamics and solution structure of Bombyx mori silk
fibroin, Macromolecules, 17 (1984) 1075-1081.
[61] H. Saito, M. Ishida, M. Yokoi, T. Asakura, Dynamic features of side chains in

tyrosine and serine residues of some polypeptides and fibroins in the solid as
studied by high-resolution solid-state 13C NMR spectroscopy, Macromolecules, 23
(1990) 83-88.
[62] A. Lesage, M. Bardet, L. Emsley, Through-bond carbon-carbon connectivities in


Phys., 118 (2003) 2325-2341.

226
Macromolecules, 40 (2007) 1995-2001.


227

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Nuclear Magnetic Resonance (NMR) Spectroscopic Characterization of Nanomaterials

A Dissertation Presented in Partial Fulfillment

Approved February 2017 by the

Jeffery L. Yarger, Co-Chair

ARIZONA STATE UNIVERSITY

Nanomaterials have attracted considerable attention in recent research due to their

applications. However, in many cases, a molecular level understanding of nanoparticles

chemistry of nanomaterials is of great significance for obtaining a better understanding of

spectroscopy can provide a familiar means of looking at the molecular structure of

molecules bound to surfaces of nanomaterials as well as a method to determine the size

of nanoparticles in solution. Here, a combination of NMR spectroscopic techniques

example, thiol-protected gold nanostructures and biomolecule-capped silica

mechanical properties, biocompatibility, and non-linear optical properties. These

is very likely to be present in a disordered structure with evidence for 31-helix

present primarily in β-sheet structures.

better advisor and mentor for my Ph.D. study.

Their encyclopedic knowledge of NMR spectroscopy and instrumentation is awe-

inspiring and they have helped me on many NMR experiments.

track of being a good, motivated and independent scientist.

encouragement. I thank my parents for raising me up and supporting me in all my

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES .............................................................................................................x

1 GENERAL INTRODUCTION .................................................................................1

1.1 Nuclear Magnetic Resonance (NMR) Spectroscopy........................................1

1.2 Gold Nanoparticles ...........................................................................................8

1.3 Silica Nanoparticles ........................................................................................11

1.4 Silk Materials..................................................................................................13

1.5 References ......................................................................................................21

2 CHARACTERIZING GOLD NANOPARTICLES BY NMR SPECTROSCOPY ....

2.1 Introduction ....................................................................................................34

2.2 Materials and Methods ...................................................................................36

2.3 Results and Discussion ...................................................................................39

2.4 Conclusions ....................................................................................................48

2.5 References ......................................................................................................49

2.6 Supplementary Materials ................................................................................54

3 ALANINE ADSORPTION AND THERMAL CONDENSATION AT THE

INTERFACE OF FUMED SILICA NANOPARTICLES ......................................56

3.1 Introduction ....................................................................................................56

3.2 Materials and Methods ...................................................................................59

3.3 Results and Discussion ...................................................................................64

3.4 Conclusions ....................................................................................................91

3.5 References ......................................................................................................92

3.6 Supplementary Materials ................................................................................99

4 INVESTIGATING LYSINE ADSORPTION ON FUMED SILICA

4.1 Introduction ..................................................................................................103

4.2 Materials and Methods .................................................................................106

4.3 Results and Discussion .................................................................................110

4.4 Conclusions ..................................................................................................124

4.5 References ....................................................................................................124

4.6 Supplementary Materials ..............................................................................131

5 SECONDARY STRUCTURE OF GLY-GLY-X (X=LEU, TYR, GLN) MOTIF IN

SPIDER DRAGLINE SILK INVESTIGATED BY SOLID-STATE NMR

5.1 Introduction ..................................................................................................136

5.2 Materials and Methods .................................................................................139

5.3 Results and Discussion .................................................................................141

5.4 Conclusions ..................................................................................................148

5.5 References ....................................................................................................148

6 PROTEIN SECONDARY STRUCTURE IN BOMBYX MORI SILK

INVESTIGATED BY SOLID-STATE NMR SPECTROSCOPY .......................157

6.1 Introduction ..................................................................................................157

6.2 Materials and Methods .................................................................................160

6.3 Results and Discussion .................................................................................162