Supramolecular Chemistry

ISSN: 1061-0278 (Print) 1029-0478 (Online) Journal homepage: http://www.tandfonline.com/loi/gsch20

Triazolecontaining zinc(II)dipicolylaminefunctionalised peptides as highly selective

pyrophosphate sensors in physiological media
Vincent E. Zwicker, Xuejian Liu, Karen K.Y. Yuen & Katrina A. Jolliffe
To cite this article: Vincent E. Zwicker, Xuejian Liu, Karen K.Y. Yuen & Katrina A. Jolliffe
(2016) Triazolecontaining zinc(II)dipicolylamine-functionalised peptides as highly selective
pyrophosphate sensors in physiological media, Supramolecular Chemistry, 28:1-2, 192-200
To link to this article: http://dx.doi.org/10.1080/10610278.2015.1122789

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Date: 31 January 2016, At: 14:41

Supramolecular Chemistry, 2016

Vol. 28, Nos. 12, 192200, http://dx.doi.org/10.1080/10610278.2015.1122789

Triazolecontaining zinc(II)dipicolylamine-functionalised peptides as highly selective

pyrophosphate sensors in physiological media
Vincent E. Zwicker,, Xuejian Liu,, Karen K.Y. Yuen and Katrina A. Jolliffe*
School of Chemistry, The University of Sydney, Sydney, Australia

Downloaded by [University of Sydney Library] at 14:41 31 January 2016

(Received 21 October 2015; accepted 13 November 2015)

A small family of linear bis[zinc(II)dipicolylamine] (bis[Zn(II)-DPA])-functionalised peptidic anion receptors has been
prepared where the Zn(II)-DPA binding sites have been installed via either a reductive amination reaction or a copper(I)catalysed azide-alkyne cycloaddition reaction. The latter reaction connects the Zn(II)-DPA binding site and the peptide
backbone through a 1,2,3-triazole linkage. Subsequent anion binding studies using indicator displacement assays were
conducted to elucidate the effect of the triazole linker on the anion-binding properties of these novel receptors and it
was found that the triazole-containing receptors exhibited stronger afnity and slightly improved selectivity for
pyrophosphate over adenosine triphosphate and adenosine diphosphate compared to the analogous receptors which did
not bear the triazole linker.
Keywords: anion recognition; pyrophosphate

Introduction
The development of novel receptors for anionic species
has been an area of active research within the eld of
supramolecular chemistry in the last decade (1). Anion
receptors have been applied in a wide range of areas
such as anion sensing, anion extraction, anion transport
through lipid bilayers and organocatalysis (2). Particular
effort has been devoted to the development of receptors
for biologically relevant anions such as chloride, uoride, acetate or phosphates (3).
In recent years, we have focused on the development
of receptors for the pyrophosphate anion (P2O74, PPi)
which is formed by the hydrolysis of adenosine triphosphate (ATP) in cells and as such, plays an important role
in a range of metabolic processes (4). We have reported
a number of cyclic peptide-based scaffolds with two pendant zinc(II)dipicolylamine (Zn(II)-DPA) arms which
were installed via the reductive amination (RA) of free
amine side chain functionalities, with subsequent complexation of the DPA unit to zinc(II) (5). Utilising indicator displacement assays (IDAs) (6), we have shown
that these cyclic receptors were able to selectively recognise PPi in the presence of other inorganic and organic
phosphates such as hydrogen phosphate and ATP in
aqueous and physiological media (HEPES and Krebs
buffer) (5). Subsequently, we created a small library of
synthetically facile linear peptide-based Zn(II)-DPA-functionalised receptors for the recognition of PPi (7). The
pendant Zn(II)-DPA binding sites were again incorpo*Corresponding author. Email: kate.jolliffe@sydney.edu.au
These authors contributed equally to this work.
2016 Taylor & Francis

rated through the RA reaction. Despite their lack of preorganisation compared to the cyclic peptide derived
analogues, these novel linear peptide-based scaffolds displayed high afnity and selectivity for PPi (7).
The copper(I)-catalysed azide-alkyne [3 + 2] cycloaddition (CuAAC) is a well-known, high yielding, reliable
and widely used reaction (click reaction) for the covalent connection of an alkyne and an azide building block
(8); moreover, the CuAAC has been found to be fully
compatible with SPPS (9). We wished to utilise the
CuAAC for the attachment of the DPA unit to our linear
peptide-based scaffolds on solid phase, as this would
connect the DPA moiety to the scaffold through a
[1,2,3]-triazole linker (as compared to an alkyl chain
which is the resulting linker when we install the DPA
units via RA, Figure 1) and it has previously been established that a ligand containing a triazole connected to a
DPA unit via a short spacer coordinates zinc(II) ions in a
tetradentate manner (10). We hypothesised that the incorporation of an additional metal binding site might inuence the anion-binding properties of the receptor.
Herein, we report the novel synthesis of triazole-containing linear peptide-based receptors 1.Zn2 and 2.Zn2
(Figure 2). We prepared both of these diastereomers as it
has previously been found that the relative stereochemistry of the amino acid side chains has an effect on anion
binding afnity and selectivity (7). The synthetic
approach to receptor precursors 1 and 2 was designed
such that it could be entirely performed on solid phase.
Furthermore, we test our hypothesis that the triazole lin-

Supramolecular Chemistry

N
Zn 2+

Zn

2+

N
N

N
Zn 2+

Zn 2+

vs.

Peptide scaffold

(Colour online) Installment of the DPA moiety through (left) the CuAAC reaction and (right) the RA reaction.

4 NO 3-

Zn 2+

Zn 2+

N
N1

N2

N
N

N3

4 NO 3-

Zn 2+

Zn2+

NH

N
O

HN
O

N
N

N
N
O

HN

NH

HN
O

HN
H 2N

H2N

O
1Zn 2: L, L
2Zn 2: L, D

Figure 2.

N
N

Peptide scaffold

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N
N

Figure 1.

193

3 Zn 2: L, L
4 Zn 2: L, D

(Colour online) Structures of receptors 14.Zn2.

ker affects the binding properties of our receptors to PPi

by conducting anion-binding studies using colorimetric
IDAs in Krebs buffer (a physiological medium that is
commonly used for cell and tissue culture and is approximately isotonic with blood serum) (5, 6). Analogous
receptors 3.Zn2 and 4.Zn2 which lack the triazole moiety
were synthesised and then tested under identical conditions as receptors 1.Zn2 and 2.Zn2 to allow a direct comparison of anion-binding abilities.
Results and discussion
Synthesis of receptors
The synthesis of receptors 1.Zn2 and 2.Zn2 was carried
out using a 9-uorenylmethoxycarbonyl solid-phase peptide synthesis (Fmoc-SPPS) strategy on Rink amide resin
using (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexauorophosphate (PyBOP) as the coupling reagent
(Scheme 1). We incorporated a C-terminal Gly residue
as we have previously found that a Gly spacer between

the peptide and the bulky resin facilitates the on-resin

RA reaction to install the DPA units (7) and we anticipated that this would also be the case for the planned
on-resin CuAAC reaction.
Initial Fmoc deprotection of Rink amide resin was
achieved using 20% piperidine in DMF and loading was
performed by treatment with Fmoc-Gly-OH in the presence of PyBOP and N,N-diisopropylethylamine (DIPEA).
Iterative Fmoc deprotection and coupling of Fmocpropargylglycine (Fmoc-L-Pra-OH 5, Fmoc-D-Pra-OH 6)
afforded the desired resin bound peptides with alkyne
functionalities at the side chains. The N-terminal amino
acid of each peptide was then capped upon treatment
with 20% acetic anhydride in pyridine. With the resin
bound tripeptide bearing two propargylglycine side
chains in hand, we conducted the on-resin CuAAC using
the known N,N-bis(2-picolyl)(2-azidoethyl)amine 7 (11),
copper(I) iodide and DIPEA to conjugate the two DPA
moieties to the peptide backbone. For subsequent
removal of the copper catalyst, the functionalised resin-

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V.E. Zwicker et al.

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bound peptide was thoroughly washed with a solution of

the copper chelator sodium diethyldithiocarbamate trihydrate in DMF (12). Cleavage of the peptide from the
resin by treatment with a solution of triuoroacetic acid/
triisopropylsilane/water (95:2.5:2.5, v/v/v), followed by
purication on reversed-phase silica gel and complexation to stoichiometric amounts of zinc nitrate gave the
triazole-containing receptors 1.Zn2 and 2.Zn2 in 25 and
22% yield, respectively, based on an initial resin loading
of 0.55 mmol g1. The synthesis of aliphatic receptors
3.Zn2 and 4.Zn2 was carried out following our previously established method (7) which afforded receptors
3.Zn2 and 4.Zn2 in 32 and 29% yield, respectively, again
based on an initial resin loading of 0.55 mmol g1.
Anion binding studies
The anion-binding abilities of receptors 14.Zn2 were
assessed utilising IDAs[6] with the previously used colorimetric indicator pyrocatechol violet 8 in Krebs buffer
(pH 7.4, 25 C) (57, 13). The association constants for
the formation of the receptorindicator complexes were
determined by UVvis titrations of solutions of indicator
8 (20 M) with aliquots of solutions of receptors 1
4.Zn2 (1000 M) in Krebs buffer. In all cases, the addition of the receptor solution caused a decrease of the
absorbance maximum at 446 nm, which was accompanied by an increase of the absorbance maximum at
646 nm (Figure 3) and a naked-eye observable colour
change of the indicator solution from yellow to blue
upon the addition of one equiv. of receptor. Non-linear
least square tting of the titration data to a 1:1 binding
model using the program HypSpec (Hyperquad package) (14) gave the apparent association constants

Figure 3. (Colour online) Representative UVvis spectra showing the changes in molar absorbance of a solution of 8 (20 M)
in Krebs buffer upon the addition of receptor 2.Zn2, [2.Zn2] = 0,
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 30, 40 M; inset: tting
curve for the titration data at 646 nm to a 1:1 binding model.

Table 1. Apparent association constants (log Ka) for receptors

14.Zn2 and indicator 8, PPi, ATP and ADP.a
Receptor
1.Zn2
2.Zn2
3.Zn2
4.Zn2

Indicator 8

PPi

ATP

ADP

7.2
7.0
6.5
6.3

7.7
7.6
6.9
6.9

4.5
4.4
4.3
3.8

4.2
4.3
4.0
3.5

Titrations were performed at 25 C in Krebs buffer, pH 7.4; estimated

errors in log Ka 15%.

(log Ka) for the receptorindicator complexes (Table 1).

The 1:1 binding stoichiometry between receptors 1
4.Zn2 and indicator 8 was corroborated by Jobs plot
analysis (ESI). Aliphatic receptors 3.Zn2 and 4.Zn2
which differed in the stereochemistry of the functionalised side chains displayed similar afnities for indicator
8 with log Ka values of 6.5 and 6.3, respectively. Interestingly, the triazole-containing receptors 1.Zn2 and
2.Zn2 bound indicator 8 more strongly with log Ka values of 7.2 and 7.0, respectively (Table 1).
We next performed anion screenings in Krebs buffer
by preparing 1:1 receptorindicator mixtures (20 M
each) and adding 10 equiv. of different anions (sodium
salts) to assess which anions are able to displace indicator 8 from the receptorindicator complex. The addition
of PPi resulted in a distinct naked-eye observable colour
change from blue to yellow indicating the successful displacement of 8. Minor colour changes were observed
upon the addition of ATP and ADP suggesting partial
indicator displacement, whereas no colour change was
observed for other anions such as nitrate, sulfate, carbonate, chloride, bromide and iodide, or any of the other
phosphoanions tested (Figure 4). To quantify the interaction of receptors 14.Zn2 with PPi, ATP and ADP, we
prepared a range of 1:1 receptorindicator mixtures
(20 M) in Krebs buffer and titrated them with aliquots
of solutions of the sodium salts of PPi, ATP and ADP
(2000 M) following the course of the titration by UV
vis. In accordance with the previously observed colour
changes, the addition of PPi induced an increase of the
absorbance maximum at 446 nm which was accompanied by a signicant decrease of the absorbance maximum at 646 nm (Figure 5), whereas the addition of ATP
and ADP induced considerably smaller changes in the
absorbance spectra of the respective solutions (Figure 6).
From the titration data, the apparent association constants
(log Ka) for the receptoranion complexes were determined by a non-linear least squares tting procedure
based on the equilibria described for indicator displacement (15) using the HypSpec (Hyperquad package)
software (14) and are summarised in Table 1.
All receptors 14.Zn2 exhibited strong binding to PPi
(log Ka ranging from 6.9 to 7.7) and clearly weaker

Supramolecular Chemistry

195

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Figure 4. (Colour online) Colour changes of 1:12.Zn2:indicator 8 mixtures (20 M each) in Krebs buffer upon the addition of 10
equiv. of a range of phosphoanions. From left to right: PPi, ATP, ADP, AMP, TMP, p-Ser, p-Thr, p-Tyr, blank.

Figure 5. (Colour online) Representative UVvis spectrum

showing the changes in molar absorbance of a 1:12.Zn2:indicator 8 mixture (20 M each) in Krebs buffer upon the addition
of PPi, [PPi] = 0, 2, 3, 5, 6, 8, 10, 12, 14, 16, 18, 25, 35,
51 M; inset: tting curve for the titration data at 646 nm to a
1:1 binding model.

Figure 6. Changes in the normalised absorbance at 646 nm of

a solution of a 1:12.Zn2:indicator 8 mixture (20 M each) in
Krebs buffer upon the addition of PPi, ATP, and ADP (09
equiv.).

binding to ATP and ADP (log Ka ranging from 3.5 to

4.5) in Krebs buffer. The variation of the side chain
stereochemistry had only minor effects on the

Figure 7. (Colour online) 1H NMR spectra (aromatic region,

500 MHz, MeOD) of (top) 2 and (bottom) 2.Zn2 showing the
downeld shift of the aromatic signals; benzylic protons of the
DPA ligands are denoted by black lled circles, aromatic protons
of the two 1,2,3-triazole units are denoted by black lled squares.

anion-binding abilities of both the triazole-containing

receptors and the aliphatic receptors. However, triazolecontaining receptors 1.Zn2 and 2.Zn2 bound PPi more
strongly than aliphatic receptors 3.Zn2 and 4.Zn2 (log Ka
7.7 and 7.6 vs. log Ka 6.9 and 6.9) and ATP and ADP
slightly stronger than the aliphatic receptors. The observation that the triazole-containing receptors did not only
bind indicator 8, but also the tested phosphoanions with
higher afnities than the aliphatic receptors enforced our
initial hypothesis that the triazole linker alters the nature
of the Zn(II)-DPA-binding pocket.
We presumed that, in addition to the DPA nitrogen
atoms, the triazole unit coordinates to the zinc centre,
acting as a 1-ligand via the N2 nitrogen atom, as was
observed in a recent X-ray crystallographic study of a
structurally similar Zn(II)-DPA-functionalised [1,2,3]-triazole compound conducted by Kuang et al. which shows
that the DPA-functionalised triazole moiety can act as a
tetradentate ligand for a Zn(II) ion (10). For receptors
1.Zn2 and 2.Zn2, evidence that the triazole units

196

V.E. Zwicker et al.

N
O
(i)- (iii)

FmocHN

N
H

FmocHN

H
N

(iv)-(vi)

N
H

N
N

NH

HN
O

HN

N
OH

FmocHN

N3

HN

SO3H

HO

Downloaded by [University of Sydney Library] at 14:41 31 January 2016

HO
5: Fmoc- L-Pra-OH
6: Fmoc- D-Pra-OH

1 Zn 2: L, L
2 Zn 2: L, D

(vii), (viii)

Scheme 1. (Colour online) Synthetic route for the preparation of receptors 1.Zn2 and 2.Zn2. Conditions: (i) Fmoc deprotection of
Rink Amide resin: 20 vol% piperidine in DMF; (ii) Loading: Fmoc-Gly-OH (3 equiv.), PyBOP (3 equiv.), DIPEA (6 equiv.), DMF;
(iii) Iterative Fmoc-SPPS: (a) Fmoc deprotection: 20 vol% piperidine in DMF; (b) Coupling: amino acid 5/6 (2.5 equiv.), PyBOP
(2.5 equiv.), DIPEA (5 equiv.), DMF; (iv) N-terminal Fmoc deprotection: 20 vol% piperidine in DMF; (v) Acetylation: 20 vol%
acetic anhydride in pyridine; (vi) CuAAC: azide 7 (4.0 equiv.), CuI (3.0 equiv.), DIPEA (3.0 equiv.), DMF; (vii) Cleavage: TFA/TIS/
H2O (95:2.5:2.5, v/v/v); (viii) Complexation: Zn(NO3)2 (2.0 equiv.), MeOH/H2O (1:1, v/v).

coordinate to the Zn(II) centres was provided by a comparison of the 1H NMR spectra of 1 and 2 in the absence
of and upon complexation with 2.0 equiv. of zinc nitrate
(Figure 7). The aromatic protons of the DPA ligands (denoted by black circles) exhibited downeld shifts of 0.3
0.5 ppm indicative of the successful complexation to Zn
(II) (7). Additionally, the aromatic proton of each of the
two triazole linkers (denoted by black squares) is shifted
downeld by 0.10.2 ppm, suggesting the interaction of
the triazole units with the zinc centres. Gratifyingly, this
change in the coordination environment of the zinc(II)
centres lead to enhancements in binding afnities for
anionic guests with the most signicant enhancements
observed for binding to PPi with the triazole containing
receptors 1.Zn2 and 2.Zn2 exhibiting a six times stronger
binding afnity to PPi than aliphatic receptors 3.Zn2 and
4.Zn2.

the Zn(II)-DPA binding sites. Subsequent anion binding

studies conducted in Krebs buffer showed that triazolecontaining receptors 1.Zn2 and 2.Zn2 exhibited stronger
binding to PPi than aliphatic receptors 3.Zn2 and 4.Zn2
which was attributed to the presence of the [1,2,3]-triazole units linking the peptide backbone and the Zn(II)DPA binding motif. Evidence was provided that the triazole moieties coordinate to the zinc centres of the Zn(II)DPA ligands and thus, presumably induce a higher
degree of receptor preorganisation which is responsible
for the enhanced PPi binding afnity. Future work will
focus on the development of rigid cyclic peptide based
analogues equipped with the triazole-altered Zn(II)-DPA
motifs as we envisage the combination of the rigid peptide scaffold and the preorganised triazole Zn(II)-DPA
binding motif will afford highly complementary PPi
receptors.

Conclusions
In summary, we have synthesised and evaluated four linear peptide-based PPi receptors which were furnished
with the bis-Zn(II)-DPA recognition motif. The installment of the Zn(II)-DPA binding sites was performed
through the previously reported synthetic route using the
RA reaction as well as through a novel synthetic route in
which the attachment of the Zn(II)-DPA binding sites
occurs via on-resin CuAAC. This approach introduces a
[1,2,3]-triazole linker between the peptidic scaffold and

Experimental
General information
All chemicals and solvents were of reagent grade and
used as received unless otherwise noted. Fmoc-Pra-OH
was synthesised by Boc deprotection of Boc-Pra-OH and
subsequent Fmoc protection using Fmoc-OSu. Melting
points were manually observed using a Stanford
Research Systems Optimelt melting point apparatus and
are uncorrected. Optical rotations were obtained using a
Perkin Elmer Model 341 polarimeter at 589 nm and

Downloaded by [University of Sydney Library] at 14:41 31 January 2016

Supramolecular Chemistry
20 C, using the indicated spectroscopic grade solvents.
1
H NMR spectra were recorded using a Bruker Avance
III 500 at a frequency of 500.13 MHz or a Bruker
Avance DPX 400 at a frequency of 400.13 MHz and are
reported as parts per million (ppm) with CD3OD
(H 3.31 ppm) as an internal reference. The data are
reported as chemical shift (), multiplicity (br = broad,
s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (J Hz) and relative integral. 13C NMR
spectra were recorded using a Bruker Avance III 500 at
a frequency of 125.76 MHz or a Bruker Avance DPX
400 at a frequency of 100.61 MHz and are reported as
parts per million (ppm) with CD3OD (C 49.00 ppm) as
an internal reference. Low-resolution mass spectra were
recorded on a Thermo Finnigan LCQ Deca Ion Trap
mass spectrometer using electrospray ionisation (ESI,
positive mode) and are reported as m/z (relative intensity). High-resolution ESI spectra were recorded on a
Bruker BioApex Fourier transform ion cyclotron resonance mass spectrometer (FTICR) with an Analytica ESI
source, operating at 4.7 T or a Bruker Daltonics Apex
Ultra FTICR with an Apollo Dual source, operating at
7 T and are reported as m/z (relative intensity). Infrared
absorption spectra were recorded on a Bruker Alpha-E
FT-IR spectrometer using attenuated total reection
(ATR) of either a solid or a thin lm. Notable vibrational
wavenumbers are recorded in cm1. UVvis data were
recorded using a Varian Cary 4000 UVvis spectrophotometer at 25 C. Temperature control was provided by a
Varian Cary PCB 150 Water Peltier System. pH values
were obtained using an Activon Model 209 pH per mV
meter. Reversed-phase column chromatography was performed on a Biotage Isolera Prime automated ash
purication system equipped with a dual-wavelength
UVvis detector. The detection wavelengths were 254
and 280 nm, respectively. Molecular structures were
modelled employing molecular mechanic force eld
(MMFF) calculations and MMFF structures were fully
optimised using DFT calculations at the B3LYP/631G*
level of theory (Spartan10).
General procedures for Fmoc-SPPS of 14.Zn2
Resin loading
Rink amide resin (loading 0.55 mmol g1) was swollen
in DMF for 1 h. The resin was drained and washed with
DMF (5), CH2Cl2 (5) and DMF (5). The resin was
then treated with 20 vol% piperidine in DMF
(3 5 min) for Fmoc deprotection and washed with
DMF (5), CH2Cl2 (5) and DMF (5). A solution of
Fmoc-Gly-OH (3 equiv.), PyBOP (3 equiv.) and DIPEA
(6 equiv.) in DMF was added to the resin. The suspension was agitated at rt for 2 h and then washed with
DMF (5), CH2Cl2 (5) and DMF (5).

197

N-terminal Fmoc deprotection

The resin-bound peptide was treated with 20 vol% piperidine in DMF (3 10 min) for Fmoc deprotection and
washed with DMF (5), CH2Cl2 (5) and DMF (5).
The resulting free amine was immediately subjected to
the following coupling step.
Estimation of resin loading
The drained Fmoc deprotection solution was diluted with
a solution of 20 vol% piperidine in DMF so that the
maximum concentration of the fulvenepiperidine adduct
was in the range of 2.57.5 105 M. A sample of this
solution (2 3 mL) was transferred to two matched 1cm quartz glass cuvettes and the UVvis absorbance at
301 nm was measured, using the solution of 20 vol%
piperidine in DMF as a reference. An average of the two
absorbance values was used to calculate the loading,
using = 7800 M1 cm1.
Amino acid coupling
A solution of the respective amino acid (2.53 equiv.),
PyBOP (2.53 equiv.) and DIPEA (56 equiv.) in DMF
was added to the resin. The suspension was agitated at
RT for 2 h and then washed with DMF (5), CH2Cl2
(5) and DMF (5).
Acetylation
After coupling of the nal amino acid, the Fmoc protecting group was removed. The free amine functionality
was acetylated by treatment with 20 vol% acetic anhydride in pyridine (3 5 min) and washed with DMF
(5), CH2Cl2 (5) and DMF (5).
Copper(I)-catalysed azide-alkyne [3 + 2] cycloaddition
The resin was swollen in DMF at RT for 1 h before a
solution of DIPEA (3 equiv.), copper(I) iodide (3 equiv.)
and N,N-bis(2-picolyl)(2-azidoethyl)amine (4 equiv.) in
DMF was added to the resin. The resulting suspension
was agitated at RT for 2 days and the reaction progress
was monitored by LCMS. The resin was rinsed and
washed with CH2Cl2 (5), a solution of sodium
diethyldithiocarbamate trihydrate (20 mg mL1) with
1 vol% triethylamine in DMF (10), DMF with 1 vol%
triethylamine (5), DMF (5) and CH2Cl2 (5) and dried
in vacuo overnight.
(Allyloxy)carbonyl (Alloc) deprotection
The resin was swollen in CH2Cl2 at RT for 1 h prior to
the deprotection step. The resin was agitated in a

198

V.E. Zwicker et al.

solution of Pd(PPh3)4 (0.8 equiv.) and phenylsilane (25

equiv.) in CH2Cl2 (2 1 h). The resin was rinsed and
washed with CH2Cl2 (5), a solution of sodium
diethyldithiocarbamate trihydrate (20 mg mL1) with
1 vol% triethylamine in DMF (10), DMF with 1 vol%
triethylamine (5), DMF (5) and CH2Cl2 (5) and dried
in vacuo overnight.

Downloaded by [University of Sydney Library] at 14:41 31 January 2016

Reductive amination
The resin was swollen in DMF at RT for 1 h before
being treated with 2-pyridinecarboxaldehyde (20 equiv.)
with 1 vol% AcOH in DMF (5 mL) and agitated at RT
overnight. The resin was rinsed and washed with DMF
(3). A suspension of sodium triacetoxyborohydride (25
equiv.) and 2-pyridinecarboxaldehyde (20 equiv.) with
1 vol% AcOH in DMF (5 mL) was added to the resin
and the resulting suspension was agitated at RT overnight. The resin was rinsed and washed with MeOH
(3 5 mL), DMF (5), CH2Cl2 (5) and DMF (5) and
dried in vacuo overnight.
Cleavage of peptides from resin
The resin was treated with a solution of triuoroacetic
acid/H2O/triisopropylsilane (95:2.5:2.5, v/v/v) for 1 h.
The resin was drained and then washed with triuoroacetic acid (3). The cleavage solution and acid
washes were combined and the solvent evaporated.

Purication
Peptides were puried by reversed-phase column chromatography (25 g C8-reversed phase silica gel; water/
CH3CN with 1 vol% ammonium hydroxide solution
(25%), 050% CH3CN, 75 mL min1). Appropriate fractions were lyophilised, affording the pure peptides as
colourless solids.

Complexation to zinc
A solution of peptide in MeOH (10 mg mL1) was
added to an aqueous solution of Zn(NO3)24H2O (2
equiv. relative to peptide, 10 mg mL1) and the mixture
was stirred at RT for 1 h. The mixture was concentrated
and the residue lyophilised, affording the bis[Zn(II)]
complex as a nitrate salt.

[]D20 = 20.4 (c 0.70 MeOH); 1H NMR (500 MHz,

MeOD) 8.41 (m, 4H), 7.707.77 (m, 6H), 7.26 (m,
8H), 4.61 (m, 2H), 4.49 (m, 4H), 3.803.90 (m, 10H),
3.283.32 (m, 1H), 3.17 (m, 2H), 3.01 (m, 5H), 1.90 (s,
3H),NH signals not observed; 13C NMR (75 MHz,
MeOD) 174.5, 173.5, 173.3, 173.2, 160.1, 160.0,
149.4, 149.3, 144.2, 143.9, 138.8, 138.7, 125.2, 125.1,
124.8, 123.9, 60.6, 54.9, 54.8, 54.6, 43.3, 28.6, 28.3,
22.5; IR (ATR) max 3275, 3061, 2931, 2830, 1654,
1591, 1534, 1475, 1433, 1370, 1224, 1148, 1130 cm1;
LRMS (ESI) m/z 843 [M+H]+; HRMS (ESI), m/
z = 843.4284
[M+H]+,
843.4274
calcd.
for
+
C42H51N16O4 . Tripeptide 1 (18.0 mg, 21.4 mol) was
complexed to zinc according to the above general procedure, affording 1.Zn2 (tetranitrate salt) as a colourless
solid. Yield: 25.2 mg (quant.); 1H NMR (500 MHz,
MeOD) 8.70 (m, 4H), 8.10 (m, 4H), 7.887.92 (d,
J = 18 Hz, 2H), 7.64 (m, 8H), 4.65 (1H), 4.49 (m, 9H),
4.314.35 (m, 4H), 3.92 (s, 2H), 3.37 (m, 4H), 3.25 (m,
1H), 3.13 (m, 3H), 1.89 (m, 3H), NH signals not
observed.
Receptor 2.Zn2
Tripeptide 2 was synthesised on Rink amide resin
(0.281 g, resin capacity 0.55 mmol g1), utilising the
above general methods. Yield: 28.7 mg (22%);
[]D20 = 7.2 (c 0.70 MeOH); 1H NMR (500 MHz,
MeOD) 8.41 (m, 4H), 7.707.74 (m, 6H), 7.247.30
(m, 8H), 4.61 (m, 1H), 4.57 (m, 1H), 4.49 (m, 4H),
3.813.90 (m, 10H), 3.28 (m, 1H), 3.20 (m, 3H), 3.03
(m, 4H), 1.95 (s, 3H), NH signals not observed; 13C
NMR (75 MHz, MeOD) 174.4, 173.8, 173.7, 173.2,
160.0, 159.9, 149.5, 149.4, 144.3, 143.9, 138.7, 138.6,
125.1, 125.0, 124.8, 124.7, 123.9, 123.8, 60.7, 55.0,
54.9, 54.8, 43.4, 28.4, 28.1, 22.6; IR (ATR) max 3290,
3055, 2921, 2831, 1651, 1588, 1528, 1478, 1433, 1363,
1210, 1122 cm1; LRMS (ESI) m/z 843 [M+H]+; HRMS
(ESI), m/z = 843.4280 [M+H]+, 843.4274 calcd. for
C42H51N16O4+. Tripeptide 2 (17.4 mg, 20.6 mol) was
complexed to zinc according to the above general procedure, affording 2.Zn2 (tetranitrate salt) as a colourless
solid. Yield: 25.2 mg (quant.); 1H NMR (500 MHz,
MeOD) 8.75 (m, 4H), 8.11 (t, J = 7.6 Hz, 4H), 7.86
7.90 (m, 2H), 7.66 (m, 8H), 4.64 (m, 1H), 4.46 (m, 9H),
4.284.32 (m, 4H), 3.87 (m, 2H), 3.37 (m, 4H), 3.20 (m,
1H), 3.10 (m, 3H), 1.92 (m, 3H), NH signals not
observed.

Synthesis

Receptor 3.Zn2

Receptor 1.Zn2

Tripeptide 3 was synthesised on Rink amide resin

(0.328 g, resin capacity 0.55 mmol g1), utilising the
above general methods. Yield: 42.5 mg (32%);
[]D20 = 16.5 (c 0.70 MeOH); 1H NMR (500 MHz,

Tripeptide 1 was synthesised on Rink amide resin

(0.311 g, resin capacity 0.55 mmol g1), utilising the
above general methods. Yield: 36.0 mg (25%);

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Supramolecular Chemistry
MeOD) 8.42 (m, 4H), 7.78 (m, 4H), 7.61 (m, 4H),
7.27 (m, 4H), 4.184.24 (m, 2H), 3.90 (d, J = 17 Hz,
1H), 3.78 (s, 8H), 3.70 (d, J = 17 Hz, 1H), 2.52 (m, 4H),
1.95 (s, 3H), 1.521.78 (m, 8H), 1.351.52 (m, 4H), NH
signals not observed; 13C NMR (125 MHz, MeOD)
174.9, 174.6, 174.2, 173.6, 160.7, 149.4, 138.7, 138.6,
124.9, 123.8, 61.1, 61.0, 55.4, 55.3, 55.2, 55.0, 32.8,
32.1, 27.7, 27.6, 24.7, 22.5; IR (ATR) max 3269, 3077,
2942, 2868, 1650, 1607, 1537, 1442, 1367, 1306, 1199,
1157, 1103 cm1; LRMS (ESI) m/z 737 [M+H]+; HRMS
(ESI), m/z = 737.4244 [M+H]+, 737.4246 calcd. for
C40H53N10O4+. Tripeptide 3 (24.9 mg, 33.8 mol) was
complexed to zinc according to the above general procedure, affording 3.Zn2 (tetranitrate salt) as a colourless
solid. Yield: 37.7 mg (quant.); 1H NMR (500 MHz,
MeOD) 8.71 (s, 4H), 8.16 (t, J = 8.0 Hz, 4H), 7.69 (m,
8H), 4.40 (m, 4H), 4.13 (m, 6H), 3.85 (d, J = 17.0 Hz,
1H), 3.74 (d, J = 17.0 Hz, 1H), 2.67 (m, 4H), 1.92 (s,
3H), 1.70 (m, 2H), 1.50 (m, 6H), 1.21 (m, 4H), NH signals not observed.
Receptor 4.Zn2
Tripeptide 4 was synthesised on Rink amide resin
(0.274 g, resin capacity 0.55 mmol g1), utilising the
above general methods. Yield: 32.2 mg (29%);
[]D20 = 21.1 (c 0.70 MeOH); 1H NMR (500 MHz,
MeOD) 8.42 (m, 4H), 7.78 (m, 4H), 7.60 (m, 4H),
7.27 (m, 4H), 4.16 (m, 2H), 3.733.88 (m, 10H), 2.50
(m, 4H), 1.95 (s, 3H), 1.541.95 (m, 8H), 1.33 (m, 4H),
NH signals not observed; 13C NMR (125 MHz, MeOD)
175.7, 174.6, 174.4, 173.8, 160.5, 160.4, 149.4, 149.3,
138.7, 138.6, 124.8, 123.8, 61.00, 60.9, 55.6, 55.4, 55.3,
43.2, 32.2, 31.9, 27.5, 27.4, 24.8, 24.6, 22.3; IR (ATR)
max 3272, 3056, 2933, 2861, 1650, 1591, 1540, 1433,
1371, 1300, 1200, 1148, 1093 cm1; LRMS (ESI) m/z
737 [M+H]+; HRMS (ESI), m/z = 737.4246 [M+H]+,
737.4246 calcd. for C40H53N10O4+. Tripeptide 4
(16.8 mg, 22.8 mol) was complexed to zinc according
to the above general procedure, affording 4.Zn2 (tetranitrate salt) as a colourless solid. Yield: 25.5 mg (quant.);
1
H NMR (500 MHz, MeOD) 8.73 (s, 4H), 8.17 (t,
J = 8.0 Hz, 4H), 7.71 (m, 8H), 4.31 (m, 4H), 3.98 (m,
6H), 3.75 (d, J = 17.0 Hz, 1H), 3.63 (d, J = 17.0 Hz,
1H), 2.68 (m, 4H), 1.92 (s, 3H), 1.72 (m, 2H), 1.43 (m,
6H), 1.25 (m, 4H), NH signals not observed.

199

buffer solution as the blank (2.5 mL). The absorbance

spectrum was recorded from 250 to 750 nm. Aliquots of
a solution containing the respective receptor (1000 M)
were then added to both the sample and the blank cuvettes. After each addition, the resulting solution was stirred for at least 30 s before the absorbance spectrum was
recorded. Typically, up to 10 equiv. of the receptor were
added to the solution. All titrations were repeated three
times. To determine association constants for the receptorindicator complexes, global analysis of the absorbance data over the range of 300720 nm was carried
out using a non-linear least squares curve tting procedure, based on the equilibria described for 1:1 binding,
using the commercially available software program HypSpec (Hyperquad package).
General procedure for titrating receptorindicator
ensembles with anions: stock solutions of the respective
1:1receptor:indicator 8 ensemble (20 M both) and of
the tested phosphoanions as sodium salts (2000 M)
were prepared. To a 1-cm quartz glass cuvette was
added a solution of the receptorindicator ensemble
(2.5 mL) and to another matched quartz glass cuvette
was added a solution of the same concentration of the
receptor as the blank (2.5 mL). The absorbance spectrum was recorded from 250 to 750 nm. Aliquots of
the respective anion solution were then added to both
the sample and the blank cuvettes. After each addition,
the resulting solution was stirred for at least 30 s before
the absorbance spectrum was recorded. Typically, up to
10 equiv. of the anion were added to the solution. All
titrations were repeated three times. To determine association constants for the receptoranion complexes, global analysis of the absorbance data over the range of
300720 nm was carried out using a non-linear least
square curve tting procedure, based on the equilibria
described for indicator displacement, using the commercially
available
software
program
HypSpec
(Hyperquad package).

Supplementary material
Copies of 1H and 13C NMR spectra for all new compounds
and anion titration data are available online here: http://dx.
doi.org/10.1080/10610278.2015.1122789

Acknowledgements
Anion binding studies
All measurements were performed in Krebs buffer or
articial urine 25 at pH 7.4 and at 25 C.
General procedure for titrating indicator 8 with receptors 14.Zn2: To a 1-cm quartz glass cuvette was added
a solution of the indicator PV (2.5 mL, 20 M) and to
another matched quartz glass cuvette was added the

This project was supported by the Australian Research Council

(DP140100227). VEZ thanks the DAAD for the award of a
scholarship. KKYY thanks the University of Sydney for the
award of a Gritton Postgraduate Scholarship.

Disclosure statement
No potential conict of interest was reported by the authors.

200

V.E. Zwicker et al.

Funding
Australian Research Council10.13039/501100000923 [Grant
Number DP140100227].

ORCID
Katrina A. Jolliffe

http://orcid.org/0000-0003-1100-4544

Downloaded by [University of Sydney Library] at 14:41 31 January 2016

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