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NMR characterization of peptide bonds in linear and cyclic peptides

Darrien James and Markus Germann

Georgia State University, Atlanta, GA 30303

Abstract

NMR spectroscopy is used to study the peptide bond conformations in the linear and

cyclic forms of the tetrapeptide IGGN. Five minor peptide bond conformers were

discovered in the linear peptide. However, only a G3 peptide bond minor conformer was

observed for the cyclic tetrapeptide. At 293K, 0.41 % cis G-3 peptide bond isomer

population was detected for the linear tetrapeptide. The trans to cis isomerization of the

G-3 peptide bond is exothermic. The enthalpy of the process is – 35.5 kJ/mol. It was also

found that the rate of the isomerization increases with increasing temperature.

Introduction

Peptide bonds form the backbone of protein chemistry. The amino acid sequence of a

protein contains important folding information. These unique folding patterns define the

function of the protein. 1 A peptide bond has partial double bond character rendering the

bond planar. The π electrons of the carbonyl bond are delocalized via resonance (figure

1), consequently, the bond is rigid but some rotation is possible. 2 This characteristic

allows us to study the cis (Z) /trans (E) isomerization at peptide bonds. 3 NMR

spectroscopy can be employed in the study of the conformational change of peptide

bonds. 4

2

2 Figure 1. Illustration of the peptide bond resonance. Currently, most academic and pharmaceutical labs study

Figure 1. Illustration of the peptide bond resonance.

Currently, most academic and pharmaceutical labs study peptides in their trans form. Our

research in cis peptide bond analysis could provide the foundation for a more holistic

study of peptides. Moreover, most small peptide drugs have trans peptide bonds.

Therefore, investigating the cis isomer will further diversify peptide drug chemistry.

Proteases naturally metabolize trans peptide bonds. Therefore, peptide drugs with their

peptide bonds locked in the cis conformation could be less susceptible to enzymatic

degradation. This innovation could potentially lead to a substantial improvement in the

bioavailability of peptide drugs.

Prolyl peptide bonds have been studied extensively. Proline residues are common at the

edge of beta strands and as the 1 st residue in alpha helices. In a typical peptide bond the

trans isomer is preferred greatly over the cis isomer. However, the trans peptide bond is

only slightly preferred in a prolyl peptide bonds. This is due to the fact that the nitrogen

of proline is bonded to two tetrahedral carbon atoms. 5

The proline cis isomer population of an unstructured peptide is 5%-50%. 6 The amount is

dependent on the amino acid that is adjacent to the proline. Prolines flanked by aromatic

3

residues have higher cis content when compared to prolines flanked by residues of other

amino acids. This is due the steric restrictions generated by the large aromatic side chains

of tyrosine, tryptophan, and phenylalanine. Reimer found that the unstructured

oligopeptide acetyl-Ala-X aa -Pro-Ala-Lys-NH 2 showed proline cis populations were

highest for Trp-Pro peptide bonds, 38% and lowest for Pro-Pro, 6%. 7

While, researchers studying non-prolyl peptide bonds report a cis secondary amide

peptide bond population ranging from 0.1% -1.00% for the following peptides: Ala-Tyr,

Tyr-Ala, Phe-Ala, Ala-Phe, Ala-Ala-Tyr, Ala-Ala-Tyr-Ala and Ala-Ala-Tyr-Ala-Ala.

The cis-trans isomerization was quantified by NMR line shape analysis and

magnetization- transfer experiments.

The 1 H NMR analysis of Ala-Tyr was carried out at different temperatures. The chemical

shifts for the cis and trans methyl protons of alanine in Ala-Tyr were recorded at 369 and

295K. The first run was performed by gradually raising the temperature to 369K. The line

broadening and signal shifts increased gradually. At 295K, the line shapes and signal

ratios of the alanine methyl protons were identical to those observed at 369K. New

signals were observed as time progressed at higher temperatures. However, unlike the cis

and trans methyl protons of alanine, these new minor conformers, due to partial

decomposition products, showed no reversibility. Consequently, Scherer et al. were able

to infer that the conformational isomerism of the dipeptide Ala-Tyr rendered the alanine

methyl group chemically different in both conformers. Exchange spectroscopy provided

further evidence of two inter-converting Ala-Tyr species. Thereafter, Scherer uses this

evidence and data as a model to study the secondary peptide bond isomerization of the

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Ala-X aa moiety in all the peptides mentioned above. It was found that the cis populations

were dependent on chain length and ionization state of the peptides. 8 Similarly, our

research involves the study of peptide bond conformations. However, our efforts are

focused on the cis (Z) / trans (E) isomerization at the primary peptide and not the

secondary peptide bond. 1 H and 13 C NMR spectroscopy are used to determine the

population of the cis isomer present in a solution of the linear tetrapeptide with sequence

IGGN. NMR is also used to explore if conditions can be found that increase the

population of the cis isomer. NMR along with molecular modeling software is used to

determine the structure of the cyclic peptide IGGN. It is believed that all the peptide

bonds in the cyclic peptide are in the trans form. This is due to the conformational

restriction produced by the ring.

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5 STRUCTURES OF THE LINEAR AND CYCLIC TETRAPEPTIDES I 1 -G 2 -G 3 -N 4
5 STRUCTURES OF THE LINEAR AND CYCLIC TETRAPEPTIDES I 1 -G 2 -G 3 -N 4

STRUCTURES OF THE LINEAR AND CYCLIC TETRAPEPTIDES I 1 -G 2 -G 3 -N 4

6

Procedure

Instrumentation

All NMR experiments were performed on a Bruker Avance 500 MHz spectrometer operating at a

500.18 MHz proton frequency. A 5 mm TBI probe head was used throughout the study.

Assignment

The assignments for both peptides were determined with 1-dimensional and 2-

dimensional NMR experiments. These techniques include 1D proton NMR, correlation

spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear single

quantum coherence spectroscopy (HSQC), nuclear overhauser effect spectroscopy

(NOESY) and heteronuclear multiple bond correlation spectroscopy (HMBC).

COSY experiments provided connectivity information for neighboring protons separated

by 3 bonds. The TOCSY experiment provided further connectivity information going

down the amino acid chain. In all TOCSY experiments the mixing time was set to 80 ms.

The NOESY technique was used to assign resonances that could not be assigned by other

experiments. The experiment requires that the atoms are close in space. The atoms must

be within 5-6 Å. 9 The technique was also used to support other chemical shift

assignments obtained using correlation experiments. The NOESY mixing times that were

used in the cyclic and linear chemical shift assignments were 50, 75, 150 and 300 ms. A

50 ms experiment was performed on the linear peptide while a 75 ms experiment was

performed on the cyclic peptide.

The following heteronuclear experiments were performed to obtain data on C-H

connectivity, long-range carbon to hydrogen coherence and nitrogen chemical shifts. The

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13 C HSQC provided data on C-H connectivity. While, the 15 N HSQC provided the

nitrogen chemical shifts for the cyclic peptide. The HSQC, was indirectly referenced

using the method outlined by Wishart et. al. 10 The HMBC technique facilitates the

determination of long range (two and three bond) H- heteronucleus connectivity. It was

employed in the determination of long-range carbon to hydrogen coherence in the cyclic

peptide. The experiment was used to assign the carbonyl chemical shifts for the cyclic

peptide.

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Linear peptide

A

6.5 mM solution of the linear peptide was prepared by dissolving 2.6 mg of the peptide

in

1 mL of DMSO. Initially, 1D and NOE spectra were used to detect minor conformers

of

all four peptide bonds. In the NOE spectra, each peptide bond had 1 or more exchange

peaks with smaller diagonal peaks within the amide region (figure 3). These peaks were

identified as the minor conformers of the peptide bonds. The signal intensities of the

peaks were measured and tabulated. However, the G-3 peptide bond was chosen for

further 1D peptide bond characterization studies because it had the strongest NOE

exchange peaks at a temperature of 298K and a mixing time of 300 ms. Four 1 H NMR

experiments were performed at the following temperatures 293, 303, 308, and 313K. The

peak areas for the major and minor conformers of the G-3 peptide bond were then

calculated using the integration function. The signal of the major conformer was used as

the calibration peak. It was assigned an area of 1 then the minor conformer peak was

integrated. These areas were used to determine the equilibrium constant for the

isomerization at each temperature. Thereafter, the free energy, enthalpy and entropy

changes involved in the isomerization were calculated from the equilibrium constants.

8

Cyclic Peptide

A 6.3 mM solution of the cyclic peptide was prepared by dissolving 1.8 mg of the

peptide in 0.6 mL of DMSO. Thereafter, a more concentrated 30mM solution was made

to obtain better signal to noise in NMR experiments. The Sparky program was used to

assign and integrate NOE cross peaks. 12 Cross peak intensities obtained from the distance

map were then used to calculate the distances between all hydrogens that produced NOE

signals. Several models of the cyclic tetrapeptide were constructed using PCModel. The

first had all peptide bonds in the cis conformation, while the other two models each had 1

of the glycine peptide bonds in the cis conformation. There after a series of models were

constructed in which the carbonyl orientations were varied.

Results

The chemical shift assignments for the linear and cyclic peptides were mostly routine.

However, differentiating the 2 glycines was more difficult. NOE experiments allowed for

the differentiation between the glycines. It was also interesting that the alpha hydrogens

of the glycines were degenerate in the linear but not in the cyclic peptide. This provided

evidence of the cyclic peptides conformational restriction. Also, for resonances with 2

protons such as alpha hydrogens of the glycines, the hydrogen with the lower chemical

shift was named alpha-1. Similarly, for the beta and gamma hydrogens of isoleucine, the

hydrogens with the lower chemical shift were named beta-1 and gamma-1. The same

convention was used for naming the beta hydrogens of asparagine. The chemical shift

assignments are in the tables below.

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THE LINEAR TETRAPEPTIDE I 1 -G 2 -G 3 -N 4

   

Ile (I1)

   

Gly (G2)

   

Gly (G3)

   

Asp (N4)

 
 

1

H

13

C

1

H

13

C

1

H

13

C

1

H

13

C

N-H

7.92

 

8.25

 

8.08

 

8.03

 

H

α 1

4.12

57.0

3.71

42.0

3.68

42.0

4.46

50.0

H

α 2

   

3.71

 

3.68

     

H

β 1

1.40

24.3

       

2.50

37.0

H

β 2

           

2.39

 

H

γ 1

1.08

24.3

           

H

γ 2

1.08

             

H

γ '

0.81

15.0

           

H

δ

0.80

11.0

           

γ NH 2 -1

           

6.85

 

γ NH 2 -2

           

7.27

 

Term. NH 2 -1

           

7.04

 

Term. NH 2 -2

           

7.10

 

Acetyl

1.87

22.2

           

Table 1. NMR chemical shifts of the linear peptide hydrogens and carbons in DMSO at 298K.

THE CYCLIC TETRAPEPTIDE I 1 -G 2 -G 3 -N 4

   

Ile (I1)

   

Gly (G2)

   

Gly (G3)

   

Asp (N4)

 
 

1

H

13

C

15

N

1

H

13

C

15

N

1

H

13

C

15

N

1

H

13

C

15

N

N-H

7.68

   

7.75

   

8.67

   

8.32

   

H

α 1

3.92

58.1

 

3.51

42.0

 

3.58

43.0

 

4.46

50.0

 

H

α 2

     

3.92

   

3.72

         

H

β 1

1.81

36.0

             

2.44

36.0

 

H

β 2

                 

2.39

   

H

γ 1

1.14

25.0

                   

H

γ 2

1.47

                     

H

γ '

0.81

15.0

                   

H

δ

0.78

10.0

                   

γ NH 2 -1

                 

6.93

   

γ NH 2 -2

                 

7.33

   

C=O

 

171.7

   

170.9

   

169.0

   

171.1

 

Amide N

   

110.0

   

99.6

   

104.2

   

113.7

γ

N

                     

104.3

Table 2. NMR chemical shifts of the cyclic peptide hydrogens and carbons in DMSO at 298K.

10

10 Figure 2 . 1 H NMR spectra of the linear tetrapeptide within the amide proton

Figure 2. 1 H NMR spectra of the linear tetrapeptide within the amide proton region at 298K in DMSO. The intensity of the G-3 minor peak was far lower than that of the major N-H signals as a result the two spectra with different peak height scales were superimposed.

G - 3 minor ↓ ↑ ↑ N4 I 1 ↑ ↑ G3 G2
G - 3 minor
N4
I 1
G3
G2

Figure 3. NOESY spectra of the amide protons of the linear tetrapeptide at 308 K with a mixing time of 150 ms in DMSO.

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Minor conformers were detected for all four peptide bonds. Interestingly, two exchange

peaks were observed for the G3 peptide bond. However, only 1 was identified as being

due to a minor conformer. This G3 minor conformer is labeled in the spectrum above.

I-1 G-2 exchange peak N-4 exchange peak G-3
I-1
G-2
exchange peak
N-4
exchange peak
G-3

Figure 4. NOESY spectra of the amide protons of the cyclic tetrapeptide at 308 K with a mixing time of 150 ms in DMSO.

In the exchange spectrum above the minor conformer of the G3 N-H proton of the cyclic

tetrapeptide is shown. Though the diagonal peak of the minor conformer was not

completely resolved, the cross peaks in both dimensions provide evidence that there is a

minor conformer of the G3 N-H proton with a chemical shift of 7.95 ppm. It is

hypothesized that this minor conformer is the cis G-3 peptide bond. Since, the

tetrapeptide has very little conformational mobility.

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STRUCTURE OF N-METHYLACETAMIDE

TRANS

12 STRUCTURE OF N-METHYLACETAMIDE TRANS E M M = -7.43 kcal/mol CIS E M M =

E MM = -7.43 kcal/mol

CIS

OF N-METHYLACETAMIDE TRANS E M M = -7.43 kcal/mol CIS E M M = -0.035 kcal/mol

E MM = -0.035 kcal/mol

QM Chemical Shift Calculations (ppm)

QM Chemical Shift Calculations (ppm)

Trans

4.89

Cis

4.66

Table 3. Spartan QM Chemical shift calculations for the cis and trans conformations of a peptide bond amide hydrogen in n-methylacetamide using the B3LYP6-31G** density function.

The quantum mechanical calculations above supported the 1D chemical shift order of the

G-3 N-H major and minor conformers (figure 2). Both procedures showed that the

chemical shift of the major conformer was higher. The minimization energies were

calculated in Hyperchem. The MM+ force field and the steepest decent minimization

algorithm were employed.

13

13 Graph showing the temperature dependence of the cis G-3 peptide bond abundance. The above curve
13 Graph showing the temperature dependence of the cis G-3 peptide bond abundance. The above curve
13 Graph showing the temperature dependence of the cis G-3 peptide bond abundance. The above curve

Graph showing the temperature dependence of the cis G-3 peptide bond abundance. The above

curve illustrates that the amount of the cis isomer decreases with increasing temperature. The R 2 value for the curve is 0.9391.

Figure 5.

T (K)

% Cis

Fraction Cis

Fraction Trans

 

K

ln (K)

1/T

Δ G a

Δ

H

Δ S b

           

(1/K)

(J/mol)

(J/mol)

J/mol-K

293

0.41

0.0041

0.99996

4.1

x10 -3

-5.50

3.41

x10 -3

13399

-35528

-205.2

298

0.39

0.0039

0.99996

3.9

x10 -3

-5.55

3.36

x10 -3

13751

-35528

-203.6

303

0.27

0.0027

0.99997

2.7

x10 -3

-5.91

3.30

x10 -3

14889

-35528

-204.7

308

0.21

0.0021

0.99998

2.1

x10 -3

-6.17

3.25

x10 -3

15801

-35528

-204.9

a From the free energy equation G = -RTlnK b From the free energy equation G = H-T S

Table 4. Thermodynamic data for the G-3 peptide bond isomerization in DMSO.

The calculated Gibbs free energies were all positive therefore the isomerization is

nonspontaneous between 293 and 308K. It was found that the percentage cis G-3 NH

increased as the temperature was lowered. Therefore, the G3 trans cis isomerization

for the linear tetrapeptide IGGN is an exothermic process. The calculated enthalpy for the

G3 trans cis isomerization was –35.5 kJ/mol.

14

14 Figure 6. G-3 peptide bond Isomerization: Van Hoff Plot: ln (K) as a function of
14 Figure 6. G-3 peptide bond Isomerization: Van Hoff Plot: ln (K) as a function of

Figure 6. G-3 peptide bond Isomerization: Van Hoff Plot: ln (K) as a function of reciprocal temperature.

The following equation, lnK =

- Δ H˚/RT + Δ S˚/R, was used to construct a Van Hoff plot

for the isomerization. The lnK was the y term while 1/T was the x term. The slope =

Δ H˚/R= 4273.6 K. This value was then was then multiplied by the gas constant to give

the enthalpy of the isomerization, Δ H= -35.5 kJ/mol. Van Hoff Curve Equation: y =

4286.4x - 20.047

R 2 = 0.9297.

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Temperature

Mixing Time

Amide

Major

Exchange

Exchange

(K)

(ms)

Proton

Peak

Peak A

Peak B

   

G-2

8.8x10 6

7500

 

293

150

G-3

9.2x10 6

8400

7700

   

N-4

7.8x10 6

6400

 
   

G-2

10.0x10 6

9592

 

298

50

G-3

11.0x10 6

8800

7012

   

N-4

9.0x10 6

6212

 
   

G-2

8.6x10 6

12000

 

298

150

G-3

8.6x10 6

12000

10000

   

N-4

7.6x10 6

11400

 
   

G-2

12.5x10 6

32000

 

298

300

G-3

12.6x10 6

33000

26000

   

N-4

12.0x10 6

24000

 
   

G-2

7.5x10 6

20540

 

308

150

G-3

8.2x10 6

21000

21000

   

N-4

7.2x10 6

18000

 

Table 5. Intensity of NOESY spectra exchange peaks for the major and minor conformations of the peptide amide bond protons. All of the above NOESY experiments were done in DMSO.

16

This NOESY analysis was done to get the rate of the trans-cis isomerization. The peak

intensities were measured using both the NOESY and 1D experiments. The exchange

peaks labeled A are for the trans-cis isomerization of I1, G2, G3 and N4 amide hydrogens

with the corresponding diagonal peaks. The exchange peak labeled B was only detected

for G-3 peptide bond. Currently, we are not sure of the identity of the diagonal peak that

corresponds to the exchange peak labeled B. It could be 1 minor form inducing another

minor form.

Isomerization Rates

mt (s)

Xa

Xb

Iaa

Ibb

Iab

Iba

r

k c t (s -1 )

k t c (s -1 )

0.01

0.998

0.0021

6.70x10 7

1.41

x10 5

33000

14000

10.9

120.1

0.252

0.02

0.998

0.0021

6.00

x10 7

1.26

x10 5

39000

38000

5.54

72.02

0.151

0.03

0.998

0.0021

6.10

x10 7

1.28

x10 5

73000

41000

3.49

60.06

0.126

0.05

0.998

0.0021

6.00

x10 7

1.26

x10 5

70000

70000

2.60

44.98

0.095

Table 6. G3 peptide bond isomerization data.

A

model developed by Perrin et .al was employed to calculate the rates of isomerization

of

the G3 peptide bond at 308K. The formula below shows how the data in the table

above was computed. 13

the G3 peptide bond at 308K. The formula below shows how the data in the table
the G3 peptide bond at 308K. The formula below shows how the data in the table

Eqn. 1

Eqn. 2

17

Perrin’s method proved to be ineffective for this system because the relative population

difference was too vast. However, other appropriate models will be tested and

implemented.

Rates analysis

Seven NOE experiments with different mixing times were carried out at 308 K to

examine the intensity change of the cross peaks between the major N-H diagonal peaks

and the smaller cis conformer peaks (figure 3 labeled NOE) The mixing times were 50

ms, 100 ms, 250 ms, 500 ms, 750 ms, 1000 ms and 1500 ms.

The diagonal peak intensities all decreased as the mixing time was increased. While, the

cross peak intensities increased to a maximum at a specific mixing time, thereafter, their

intensities decreased. The following curves summarize the diagonal and cross peak

development as a function of mixing time.

18 Figure 7. NOE Intensity of the I1 N-H diagonal peak as a function of

18

Figure 7. NOE Intensity of the I1 N-H diagonal peak as a function of mixing time.

of the I1 N-H diagonal peak as a function of mixing time. Figure 8. NOE Intensity

Figure 8. NOE Intensity of the G2 N-H diagonal peak as a function of mixing time.

19

19 Figure 9. NOE Intensity of the G-3 N-H diagonal peak as a function of mixing

Figure 9. NOE Intensity of the G-3 N-H diagonal peak as a function of mixing time.

of the G-3 N-H diagonal peak as a function of mixing time. Figure 10. NOE Intensity

Figure 10. NOE Intensity of the N-4 N-H diagonal peak as a function of mixing time.

20

20 Figure 11. NOE intensity of I1 N-H exchange peak A as a function of mixing

Figure 11. NOE intensity of I1 N-H exchange peak A as a function of mixing time.

of I1 N-H exchange peak A as a function of mixing time. Figure 12. NOE intensity

Figure 12. NOE intensity of I1 N-H exchange peak B as a function of mixing time.

21

21 Figure 13. NOE intensity of G2 N-H exchange peak A as a function of mixing

Figure 13. NOE intensity of G2 N-H exchange peak A as a function of mixing time.

of G2 N-H exchange peak A as a function of mixing time. Figure 14. NOE intensity

Figure 14. NOE intensity of G2 N-H exchange peak B as a function of mixing time.

22

22 Figure 15. NOE intensity of G-3 N-H exchange peak 1A as a function of mixing

Figure 15. NOE intensity of G-3 N-H exchange peak 1A as a function of mixing time.

of G-3 N-H exchange peak 1A as a function of mixing time. Figure 16. NOE intensity

Figure 16. NOE intensity of G-3 N-H exchange peak 1B as a function of mixing time.

23

23 Figure 17. NOE intensity of G-3 N-H exchange peak 2A as a function of mixing

Figure 17. NOE intensity of G-3 N-H exchange peak 2A as a function of mixing time.

of G-3 N-H exchange peak 2A as a function of mixing time. Figure 18. NOE intensity

Figure 18. NOE intensity of G-3 N-H exchange peak 2B as a function of mixing time.

24

24 Figure 19. NOE Intensity of the N4 N-H exchange peak A as a function of

Figure 19. NOE Intensity of the N4 N-H exchange peak A as a function of mixing time.

of the N4 N-H exchange peak A as a function of mixing time. Figure 20. NOE

Figure 20. NOE Intensity of the N4 N-H exchange peak B as a function of mixing time.

25

25 Figure 21. T 1 Inversion Recovery plot of the G-3 N-H minor form diagonal peak

Figure 21. T 1 Inversion Recovery plot of the G-3 N-H minor form diagonal peak as a function of delay ( τ ).

The 1D 1 H NMR intensity of the minor form of the G-3 NH resonance obtained from a

T1 IR experiment was plotted as a function of delay to determine the spin-spin lattice

relaxation time (T 1 ). The T 1 value was obtained from a curve fitting to the general

equation y= m1*(1-2*e -x/m2 ). The T 1 value for the minor form of G3 N-H was 583 ms.

This value is close to the average T 1 obtained for the G3 N-H major conformer, 623 ms.

The table below summarizes the T 1 values obtained for the N-H protons at 293, 298, 303,

and 308K.

26

Temperature

   

(K)

N

- H

T1 (ms)

   

I1

600

 

G2

538

293

G3

627

 

N4

600

   

I1

597

 

G2

528

298

G3

615

 

N4

582

   

I1

607

 

G2

534

303

G3

618

 

N4

584

   

I1

624

 

G2

537

308

G3

632

 

N4

597

Table 7. Spin-spin lattice relaxation times for the N-H protons of the linear tetrapeptide at various temperatures.

Structural Elucidation of the Cyclic tetrapeptide IGGN

NOESY spectra were used to determine the distance between the hydrogen atoms in the

cyclic tetrapeptide I 1 -G 2 -G 3 -N 4 . The distance between γNH 2 -1 - γNH 2 -2 amide hydrogens

of the asparagine was used as the reference distance. The table below shows the γNH 2 -1 -

γNH 2 -2 distance measured from a model of the cyclic peptide constructed in PC Model,

alongwith, the NOE cross peak intensities for γNH 2 -1 - γNH 2 -2 at three mixing times.

27

The γNH 2 -1- γNH 2 -2 amide reference distance, the NOE cross peaks intensities for γNH 2

-1- γNH 2 -2 and the individual resonances were substituted into the equation below to

calculate all the tabulated distances. All reported distances are in angstroms (Å).

I a

/

I b

=

r b 6

/ r a 6

Eqn. 3

   

Mixing Time (ms)

 

NOE Intensity

Distance (Å)

 

75

5.64

x 10 6

 

1.65

 

150

7.47

x 10 6

 

1.65

 

300

 

6.38

x 10 6

 

1.65

Table 8. NOE reference data collected at three different mixing times.

 

NH-NH

 

N-H

 

N-H

I1

 

G2

G3

N4

 
 

75

   

1.9

ne

2.6

 

150

I1

2.0

5.2

2.6

300

2.1

4.5

2.5

 

75

 

3.2

3.9

 

150

 

G2

3.1

3.7

300

 

3.0

3.7

   

75

 

3.1

150

G3

3.1

300

3.2

 

75

 

150

N4

300

Table 9. NH-NH distances calculated at 3 different mixing times. The abbreviation ne means there was no

peak.

28

α H

N-H

α H- NH

 

α H δ ’s

I1

 

G2

G3

N4

75

 

2.7

 

+

   
 

ne

ne

ne

150

I1

2.5

 

+

 

ne

ne

ne

300

3.92

2.7

 

+

3.9

 

ne

ne

75

 

1-n 2-n

1- n 2-2.4 +

1-n, vc *2-2.3 1-n *2-2.3 1-n *2-2.2

1-ne

2-ne

150

G2

1-n 2-n

1-2.4 2-2.3 +

1-ne

2-ne

300

3.51, 3.92

1-n 2-n

1-2.3 2-2.2 +

1-ne

2-ne

75

 

1-ne

2- n

1-n

2-n

1- n, vc

2- 2.6

1-3.0

2-2.3

150

G3

1-ne

2-3.5

1-n

2-n

1-2.2

2- 2.5

1-2.8

2-2.3

300

3.59,3.72

1-ne

2-3.5

1-n

2-n

1-2.2

2- 2.7

1-3.1

2-2.2

75

 

2.7

 

3.8

ne

c

150

N4

2.6

4.4

ne

2.4

300

4.46

2.6

4.2

ne

2.4

Table 10. α H-NH distances calculated at 3 different mixing times. The following abbreviations are used

in the above tabulation: ne- none: there were no cross peaks, n-low S/N, c-coupling: spin coupling peak

distortion, and

vc- very close to the diagonal: the NOE cross peak is too close to the diagonal to obtain

accurate volumes. *+ I1 α H and G2 α H2 are isochronous; both hydrogens have the same chemical shift, as a

result, molecular modeling applications were employed to differentiate the alpha hydrogens. The volumes

obtained for these resonances with identical chemical shifts were compared with model distances to

decipher the cross peaks.

29

α H

α H- All other Resonances

All other Resonances

   

I1

G2

G3

N4

75

 

β

-2.8

γ

'-2.7

γ 1- 3.2

γ

2-3.7

ne

ne

ne

150

I1

β

-2.8

γ

'-2.5

γ 1- 3.1

γ

2-3.7

ne

ne

ne

300

β - 2.8

γ '-2.5

γ 1- 3.0

γ

2-3.6

ne

ne

ne

75

   

ne

α H- 1.8 α H- 1.8 α H- 1.8

ne

ne

150

G2

ne

ne

ne

300

ne

ne

ne

75

   

ne

ne

α H- 1.6 α H- 1.7 α H- 1.7

ne

150

G3

ne

ne

ne

300

ne

ne

ne

75

   

ne

ne

ne

ne

150

N4

ne

ne

ne

ne

300

ne

ne

ne

ne

Table 11. α H -All other resonance distances calculated at 3 different mixing times. * I1 α H and G2 α H2 are

isochronous. However, model evidence supports these distances are G2 α H1- G2 α H2. G3 α 1, G3 α 2 and

G2 α 1 all have cross peaks with I1 γ ' however these very weak peaks were in the noise.

β H – All other Resonances

β H

All other Resonances

   

I1

G2

G3

 

N4

75

 

α

H-

2.8

γ

1-ps

γ 2- 2.5

γ '- 2.5

 

ne

 

ne

150

I1 β

α H- 2.8

γ

1-2.9

γ 2- 2.6

γ

'-2.4

ne

ne

300

α

H-

2.8

γ 1- 2.9 γ 2- 2.4

γ '-2.4

ne

ne

75

   

ne

ne

α H- 2.7 α H- 2.8 α H- 2.5

γ NH2-1- 4.2 γ NH2-1- 4.3 γ NH2-1- 3.6

γ NH2-2- 2.9 γ NH2-2- 2.9 γ NH2-2- 2.8

150

N4 β 1

ne

ne

300

ne

ne

75

   

ne

ne

α H- 2.4 α H- 2.2

γ NH2-1- n γ NH2-1- 4.2

γ NH2-2- n γ NH2-2- 4.2

150

N4 β 2

ne

ne

300

ne

ne

α H- 2.6

γ NH2-1- 4.2

γ NH2-2- 4.2

Table 12. All β H distances calculated at 3 different mixing times. The I1 δ and I1 γ ' resonances had similar

chemical shifts; they are 0.78 and 0.81 respectively. The N4 β 1 - N4 β 2 cross peaks were very close to the

diagonal and the peaks volumes were inconsistent.

30

N-H

NH – All other Resonances

All other Resonances

 

N-H

 

I1

G2

G3

 

N4

75

 

β

-2.5

γ 1- 3.1

γ 2-3.3

y'-3.4

   

β 1- 3.8 β 1- 3.6 β 1- 3.4

β 2- 3.8 β 2- 3.6 β 2- 3.3

150

I1 N-H

β - 2.5

γ

1-3.1

y2-3.2

y'-3.3

300

β

-2.4

γ 1- 2.9

y2-3.1

y'-3.1

75

   

β - 3.0

γ '- 3.3 γ '- 3.3 γ '- 4.1

       

150

G2 N-H

β - 2.9

300

β - 2.7

75

         

150

G3 N-H

300

75

       

β 1- 2.6 β 1- 2.7 β 1- 2.6

β 2- 3.1 β 2- 3.1 β 2- 3.0

150

N4 N-H

300

Table 13. N-H- all other resonances distances calculated at 3 different mixing times.

The tables below summarize the N-H to N-H distance data obtained by varying the

backbone carbonyl orientation of each amino acid in various models of the cyclic peptide.

The models that were closest to the experimental distances obtained from the NOE

experiment were G2 carbonyl down others up (table 18) and the G3 carbonyl down others

up (table 17). This data suggests that the actual structure of the peptide could be similar

to 1 of these models.

N-H to N-H

Model (Å)

NOE (mt=150ms) (Å)

Absolute Difference

I1-G2

2.2

2.0

0.2

G2-G3

2.5

3.1

0.6

G3-N4

2.6

3.1

0.5

I1-G3

3.6

5.2

1.6

I1-N4

2.8

2.6

0.2

G2-N4

3.6

3.7

0.1

Table 14. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with all carbonyls pointing downward.

31

N-H to N-H

Model (Å)

NOE (mt=150ms) (Å)

Absolute Difference

I1-G2

4.1

2.0

2.1

G2-G3

4.1

3.1

1.0

G3-N4

4.1

3.1

1.0

I1-G3

4.3

5.2

0.9

I1-N4

4.1

2.6

1.5

G2-N4

4.3

3.7

0.6

Table 15. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with alternating carbonyls.

N-H to N-H

Model (Å)

NOE (mt =150ms) (Å)

Absolute Difference

I1-G2

3.5

2.0

1.5

G2-G3

2.7

3.1

0.4

G3-N4

2.6

3.1

0.5

I1-G3

4.6

5.2

0.6

I1-N4

4.0

2.6

1.4

G2-N4

3.8

3.7

0.1

Table 16. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with N4 carbonyl pointing down and all other carbonyls pointing up.

N-H to N-H

Model (Å)

NOE (mt=150ms) (Å)

Absolute Difference

I1-G2

2.4

2.0

0.4

G2-G3

2.4

3.1

0.7

G3-N4

4.1

3.1

1.0

I1-G3

4.4

5.2

0.8

I1-N4

4.0

2.6

1.4

G2-N4

3.7

3.7

0.0

Table 17. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G3 carbonyl down and all other carbonyls pointing up.

32

N-H to N-H

Model (Å)

NOE (mt=150ms) (Å)

Absolute Difference

I1-G2

2.1

2.0

0.1

G2-G3

4.0

3.1

0.9

G3-N4

3.9

3.1

0.8

I1-G3

4.3

5.2

0.9

I1-N4

2.8

2.6

0.2

G2-N4

4.0

3.7

0.3

Table 18. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G2 carbonyl down and all other carbonyls pointing up.

N-H to N-H

Model (Å)

NOE (mt=150ms) (Å)

Absolute Difference

I1-G2

4.2

2.0

2.2

G2-G3

3.8

3.1

0.7

G3-N4

3.0

3.1

0.1

I1-G3

3.7

5.2

1.5

I1-N4

2.8

2.6

0.2

G2-N4

5.5

3.7

1.8

Table 19. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with I1 carbonyl down and all other carbonyls pointing up.

N-H to N-H

Model (Å)

NOE (mt=150ms) (Å)

Absolute Difference

I1-G2

3.6

2.0

1.6

G2-G3

3.2

3.1

0.1

G3-N4

3.9

3.1

0.8

I1-G3

5.5

5.2

0.3

I1-N4

3.0

2.6

0.4

G2-N4

4.1

3.7

0.4

Table 20. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with G3 and N4 carbonyls down and all other carbonyls pointing up.

33

N-H to N-H

Model (Å)

NOE (mt=150ms) (Å)

Absolute Difference

I1-G2

2.4

2.0

0.4

G2-G3

3.1

3.1

0.0

G3-N4

2.9

3.1

0.2

I1-G3

4.6

5.2

0.6

I1-N4

4.2

2.6

1.6

G2-N4

4.2

3.7

0.5

Table 21. Comparison of NOE and model distance data for the cyclic tetrapeptide IGGN with N4 and I1 carbonyls down and all other carbonyls pointing up.

and I1 carbonyls down and all other carbonyls pointing up . Figure 22. A model of

Figure 22. A model of the cyclic tetrapeptide IGGN with G2 carbonyl down and other carbonyls up. This model has the best agreement with experimental N-H to N-H distances.

34

Discussion

In the linear peptide, the concentration of the cis peptide bond isomers increased with

increasing temperature. This result is consistent with earlier work conducted, which

showed that the concentration of secondary cis peptide bonds increased with increasing

temperatures for small peptides with the Ala-X aa moiety.

Remarkably, thermodynamic calculations revealed that the enthalpy of the G-3 peptide

bond trans to cis isomerization is exothermic. It was hypothesized that the G3 peptide

bond trans-cis isomerization would be an endothermic process since the cis conformer

produces steric clashes between the oxygen and the hydrogen of the amide group.

Furthermore, from the free energy equation, G = H-TS, the negative enthalpy and the

positive free energy shows that the reaction is entropy driven.

While, the NOE rates data plots produced the expected trend; an exponential decay in N-

H diagonal peak intensity as a function of mixing time and an increase in the

cross peak intensities until a maximum intensity was reached, thereafter, the cross peak

intensities decreased. The T1 inversion recovery showed that the spin-spin lattice

relaxation of all four N-H protons were similar. They ranged from 527- 632 ms.

The distance data obtained from the NOE experiments were reliable and made spatial

sense. There was also very good agreement in distances among the 3 mixing times.

Therefore, spin diffusion effects were minimized. The group of cyclic models with varied

backbone carbonyl orientation provided further insight on the structure of the cyclic

tetrapeptide IGGN.

Our research has shown that it is possible to directly study peptide

35

bond isomerization complementing earlier work which placed more emphasis on the

secondary amide peptide bond orientation.

36

References

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2. Pauling, L. The Nature of the Chemical Bond. Cornell. NY. 1948, 10-14.

3. Berg, J. Biochemistry. W. H. Freeman. NY. 2006, 37-38.

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6. Li, P.S., Chen, E., Shulin, S., Asher, A. J. Am. Chem. Soc. 1997, 119, 1116.

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37