Bis[zinc(ii)dipicolylamino]-functionalised peptides as high affinity receptors for pyrophosphate ions in water

Article abstract of DOI:10.1039/c3cc40937f

A library of bis[zinc(ii)dipicolylamine]-functionalised linear peptides was prepared using an efficient solid phase peptide synthesis approach and their use as chemosensors for anions in water was investigated using indicator displacement assays. High affinity and selectivity for pyrophosphate (PPi) over adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were observed and additional aromatic side chains provided enhanced discrimination between PPi and ATP.

Full text of DOI:10.1039/c3cc40937f

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Bis[zinc(II)dipicolylamino]-functionalised peptides as
high affinity receptors for pyrophosphate ions in
water†
Cite this: Chem. Commun., 2013,
49, 4824
Received 4th February 2013,
Accepted 5th April 2013
Karen K. Y. Yuen and Katrina A. Jolliffe*
DOI: 10.1039/c3cc40937f
www.rsc.org/chemcomm
A library of bis[zinc(II)dipicolylamine]-functionalised linear peptides was
To enable rapid access to a library of receptors, we developed
prepared using an efficient solid phase peptide synthesis approach and a synthetic route that would enable the entire synthesis, prior to
their use as chemosensors for anions in water was investigated addition of the zinc(II) ions, to be performed on the solid phase.
using indicator displacement assays. High affinity and selectivity for We initially designed receptors 1–7ÁZn2 (Fig. 1) containing two or
pyrophosphate (PPi) over adenosine triphosphate (ATP) and adenosine three amino acids and attempted their synthesis using Fmoc solid
diphosphate (ADP) were observed and additional aromatic side chains phase peptide synthesis (Fmoc-SPPS) (Scheme 1). Orthogonal
provided enhanced discrimination between PPi and ATP.
allyloxycarbonyl (Alloc) protecting groups were employed for
protection of the side chain amino groups. Following synthesis of
Anions play fundamental roles in numerous important biological the desired linear peptide sequences using standard Fmoc-SPPS
and chemical processes. In particular, phosphate oxoanions such protocols on Rink amide resin and acetyl capping of the N-terminal
as pyrophosphate (PPi) and adenosine triphosphate (ATP) are
involved in a number of bioenergetic and metabolic processes.
Therefore there is intense current interest in developing receptors
that can efficiently and selectively detect phosphate oxoanions
under physiological conditions.1,2
Bis[Zn(II)dipicolylamine] [Zn(II)Dpa] receptors have been
found to exhibit good selectivity for phosphates over other
anions in aqueous solution.2 In recent studies we have observed
that cyclic peptide derived bis[Zn(II)Dpa] receptors can be tuned
to show selectivity for PPi over ATP using indicator displacement
assays through altering of the cyclic peptide structure.3,4 Notably,
both the structure of ‘non-binding’ amino acid side chains
decorating the scaffolds and the size of the cyclic peptide have
a significant impact on receptor selectivity and affinity.4 In order
to further probe the interesting effect of ‘non-binding’ side
chains on receptor selectivity we desired rapid access to libraries
of peptide scaffolds with varying structures. However, the synthesis
and purification of such cyclic peptides is experimentally complex
and time-consuming. We envisaged that receptors with simpler
linear peptides scaffolds, which should be readily accessible by
solid phase synthesis, would provide efficient access to a library
of receptors for screening the effects of different side chains on
receptor selectivity.
School of Chemistry, The University of Sydney, 2006, NSW, Australia.
E-mail: kate.jolliffe@sydney.edu.au; Fax: +61 2 9351 3329; Tel: +61 2 9351 2297
† Electronic supplementary information (ESI) available: Synthetic procedures and
NMR spectra for all new compounds and anion titration data. See DOI: 10.1039/
Fig. 1 Structures of receptors 1–10ÁZn2 and indicator 11.
c3cc40937f
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Fig. 2 Qualitative screening of 9ÁZn2Á11 after addition of 5 equiv. of anions to a
20 mM receptor : indicator solution. UV-vis spectra (top) and visual screening (bottom).
2À
SO4 and HPO42À), it was found that in most cases only PPi, ATP
and ADP resulted in a colour change indicative of indicator dis-
placement (Fig. 2). UV-Vis spectroscopic analysis of the resulting
solutions indicated that in some cases a small change in absorbance
was also observed upon addition of citrate, although the change was
not large enough to be visible to the naked eye.
Scheme 1 General method for synthesis of linear peptides on Rink Amide AM
resin via Fmoc-SPPS strategy.
Association constants for the indicator–receptor complexes
were determined by UV-Vis spectroscopic titration experiments
and non-linear least squares fitting of the titration data to a 1 : 1
binding model using Hypspecs.8 (Table 1 and Fig. 3). The 1 : 1
binding stoichiometry was confirmed by Job plot analysis
(Fig. 3, bottom). Subsequently, the 1 : 1 indicator : receptor mixtures
were titrated with aliquots of the sodium salts of PPi, ATP and ADP
(Fig. 4). Curve fitting of the absorbance data (250–750 nm) based on
the previously described equilibria for competition assays9
using Hypspecs provided apparent association constants
for the receptor : anion complexes. In all cases the receptors
exhibited very strong binding to PPi (log Ka ranging from 7.8 to 9) in
these mimicked physiological conditions and some selectivity for
PPi over ATP and ADP was also observed. Of this library of receptors,
dipeptide receptor 1ÁZn2 was the least effective at discriminating
between polyphosphate species. In contrast, dipeptide receptor
2ÁZn2, which contains alternating L- and D-amino acids exhibited
amino acid, the Alloc groups were removed upon treatment
with Pd(PPh3)4 (1.05 equiv.) in the presence of acetic acid and
morpholine.5 Subsequent reductive aminations with picolyl-
aldehyde6 to install the dipicolylamino groups were only partially
successful and in some cases the reaction could not be forced to
completion despite subjecting the solid phase bound peptides to
repetitive reaction attempts. After analysis of the partially alkylated
products following cleavage from the resin, we established that
reductive amination of the amino acid directly attached to the
resin was not proceeding to completion, presumably as a result of
difficulties of reagent access to this hindered amino group. There-
fore, for the synthesis of receptors 8–10ÁZn2, a C-terminal glycine
residue was incorporated as a spacer between the resin and the
residue to be reacted. In these cases, the reductive aminations
readily proceeded to completion after two reaction cycles, confirm-
ing that steric hindrance by the resin was the cause of the previous
incomplete conversions. Subsequent cleavage of the peptides from
the resin was followed by HPLC purification and addition of two
equivalents of Zn(NO3)2 provided receptors 1–10ÁZn2.
Table 1 Apparent association constants (log Ka) determined for receptors
1–10ÁZn2 and various polyphosphate anions
Apparent association constants (log Ka)
The anion binding capabilities of receptors 1–10ÁZn2 were
investigated using indicator displacement assays (IDAs) with the
colourimetric indicator pyrocatechol violet (11), which has pre-
viously been employed in IDAs with bis[Zn(II)Dpa] complexes.7
This indicator provides the ability to rapidly screen anion binding
to receptor libraries through naked eye visualisation, which can
then be followed by measurement of binding affinities by UV-Vis
spectroscopy for positive hits. IDAs were performed with 1 : 1
receptor–indicator chemosensing ensembles prepared by mixing
equimolar amounts (20 mM) of receptors 1–10ÁZn2 and indicator
11 in buffered saline solution (pH 7.4, 5 mM HEPES, 145 mM
NaCl). Upon addition of five equivalents of a range of anions as
their sodium salts (PPi, ATP, ADP, AMP, cAMP, pThr, pSer, citrate,
‘Spacer’
Receptor amino acid Indicator 11
PPi
ATP
ADP
1ÁZn2
2ÁZn2
3ÁZn2
4ÁZn2
5ÁZn2
6ÁZn2
7ÁZn2
8ÁZn2
9ÁZn2
10ÁZn2
—
—
6.6
6.6
5.4
7.4
6.8
8.3
6.9
8.2
7.0
6.1
8.7
>9
7.8
>9
>9
>9
>9
>9
>9
>9
7.2
6.6
5.4
7.1
6.5
8.2
6.7
7.8
6.4
5.9
5.8
5.3
4.2
5.3
4.9
5.5
5.0
5.3
4.6
4.7
Gly
Leu
Pro
Phe
Tyr
Phe
Trp
Nal
a
Titrations performed at 25 1C in aqueous solutions buffered at pH 7.4
with 5 mM HEPES in the presence of 145 mM NaCl. Estimated error
in log Ka > 0.2.
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a Gly residue at the C-terminus (to facilitate solid phase synthesis)
has little effect on binding affinity or selectivity. While 4–10ÁZn2 all
bound PPi with affinity too high to quantify (log Ka > 9) under the
current competitive conditions, analysis of the difference in their
affinity for ATP provides some indication of receptor ability to
discriminate between ATP and PPi. Incorporation of a Pro residue
to provide a more defined peptide backbone conformation did
not have a significant impact on binding affinity or selectivity, with
similar binding behaviour observed for the Leu (4ÁZn2) and Pro
(5ÁZn2) derivatives. Notably compounds 9ÁZn2 and 10ÁZn2, which
bear large aromatic spacer residues (Trp and Nal, respectively)
had reduced affinity for ATP. While the reason for this requires
further investigation, these results indicate that the incorporation of
hydrophobic and aromatic residues can be used to tune the anion
recognition abilities of such scaffolds to provide better discrimina-
tion between anions of similar charge.
In conclusion, the design, synthesis, and anion binding
capabilities of a library of linear peptide-based receptors
(1–10ÁZn2), bearing two Zn(II)Dpa binding sites are reported. Recep-
tors 2, 9 and 10ÁZn2 show remarkably strong binding to PPi with
significant selectivity over ATP and ADP under mimicked physio-
logical conditions. Studies to elucidate the reasons for the
enhanced binding selectivities exhibited by receptors with hydro-
phobic and aromatic side chains are currently in progress.
The Australian Research Council is acknowledged for financial
support. KKYY thanks the University of Sydney for the award of a
Gritton Postgraduate Scholarship. We thank Dr H. T. Ngo (USyd)
for assistance with data analysis using Hyperquads.
Fig. 3 Representative spectra showing the change in molar absorbance of
indicator 11 (20 mM) upon addition of increasing amounts of 9ÁZn2 (top); fitting
curve for 1 : 1 binding between 9ÁZn2 and indicator 11 at 646 nm (bottom; Job
Plot inset). Measurement conditions: aqueous solution of HEPES buffer (5 mM,
pH 7.4, 145 mM NaCl), labs 250–750 nm, 25 1C.
Fig. 4 Representative spectra showing absorbance changes at 646 nm of 9ÁZn2Á Notes and references
11 ensemble upon addition of various anions (up to 10 equiv.). Measurement
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high affinity and excellent selectivity for PPi over ATP and ADP,
indicating the importance of receptor stereochemistry and sug-
gesting that, despite the inherent flexibility in the Lys derived
Zn(II)Dpa side chains, positioning these on the same face of the
scaffold increases selectivity.
We reasoned that the inclusion of a spacer amino acid between
two L-configured Zn(II)Dpa residues should provide similar geome-
trical positioning of the sidechains,10 with an increased space
between them which might also provide an increase in binding
affinity. As anticipated, most of the tripeptide and tetrapeptide-based
receptors (3–10ÁZn2) displayed enhanced affinity and selectivity for
PPi over ATP and ADP when compared to 1ÁZn2 and similar affinities
and selectivity to 2ÁZn2. The introduction of side chain functionalities
on the spacer amino acid was found to have significant effects on
both binding affinity and selectivity. Notably, the incorporation of a
hydrophobic residue in the peptide sequence (4–10ÁZn2) resulted in a
substantial increase in affinity for both PPi and ATP, when compared
to the affinities obtained for the Gly derivative 3ÁZn2. A comparison
of the binding data for 6ÁZn2 and 8ÁZn2 indicates that the addition of
c
4826 Chem. Commun., 2013, 49, 4824--4826
This journal is The Royal Society of Chemistry 2013