Selective pyrophosphate recognition by cyclic peptide receptors in physiological saline

Article abstract of DOI:10.1002/asia.201200627

The anion binding ability of a family of bis(Zn^II-Dpa) functionalized cyclic peptides has been investigated using displacement assays with a fluorescent coumarin indicator in water, saline solution, and Krebs buffer. Non-binding side-chain steric bulk, the relative position of binding sites, and the scaffold size were all found to affect the ability of these receptors to discriminate between polyphosphate ions. Most receptors showed some selectivity for pyrophosphate over ATP and ADP in water and saline, and this selectivity was significantly enhanced in the biologically relevant Krebs buffer giving chemosensing ensembles capable of selective recognition of pyrophosphate in the presence of excess ATP. Copyright

Full text of DOI:10.1002/asia.201200627

DOI: 10.1002/asia.201200627
Selective Pyrophosphate Recognition by Cyclic Peptide Receptors in
Physiological Saline
Stephen J. Butler and Katrina A. Jolliffe*[a]
Abstract: The anion binding ability of
a family of bis
(ZnII-Dpa) functional-
scaffold size were all found to affect
the ability of these receptors to dis-
criminate between polyphosphate ions.
Most receptors showed some selectivity
for pyrophosphate over ATP and ADP
in water and saline, and this selectivity
was significantly enhanced in the bio-
logically relevant Krebs buffer giving
chemosensing ensembles capable of se-
lective recognition of pyrophosphate in
the presence of excess ATP.
ACHTUNGTRENNUNG
ized cyclic peptides has been investigat-
ed using displacement assays with a flu-
orescent coumarin indicator in water,
saline solution, and Krebs buffer. Non-
binding side-chain steric bulk, the rela-
tive position of binding sites, and the
Keywords: anions
·
molecular
recognition · peptides · receptors ·
supramolecular chemistry
Introduction
ing PPi from the nucleoside triphosphates with a useful level
of discrimination[9,11] and few have been evaluated under
ꢀrealꢁ physiological conditions such as cell growth media.
We have previously reported the use of a backbone-modi-
fied cyclic peptide bearing dipicolylamino (Dpa) arms as
a selective receptor for PPi ions in water as determined
using a fluorescent indicator displacement assay (IDA).[11]
One of the advantages of peptide-derived receptors is their
synthetic versatility, which allows each subunit of the recep-
tor to be modified, enabling ready fine-tuning of the recep-
tor properties.[12,13] We have recently illustrated how this ap-
proach can be used to prepare a small library of cyclic pep-
tide anion receptors (1–6·Zn2, Figure 1)[14,15] and report here
on the ability of these receptors to bind PPi in aqueous solu-
tion and physiological saline (Krebs buffer; commonly used
for cell and tissue culture and approximately isotonic with
blood serum[16]) with high levels of selectivity over ATP and
ADP.
Anions are involved in almost every biological process, with
phosphates amongst the most important anions in the natu-
ral world. They are involved in bioenergetic and metabolic
processes and can be either substrates for, or products of,
enzyme-catalyzed reactions. For example, hydrolysis of
anionic ATP results in the formation of both pyrophosphate
(P2O74ꢀ, PPi) and AMP anions. PPi is also released when nu-
cleoside triphosphates are incorporated into DNA/RNA
during polymerase reactions and in the biosynthesis of sec-
ondary messengers such as cAMP and cGMP.[1] Elevated
levels of PPi in synovial fluid have been found in patients
with pseudogout and osteoarthritis,[2] and the levels of PPi
in urine can be used to monitor clinical disorders in bone
metabolism[3] and urolithiasis.[4] Therefore, there has been
significant recent interest in the development of molecular
receptors capable of discriminating between PPi and other
phosphate derivatives, for use in bioanalytical assays.[5–9] The
major challenges in the development of sensors for PPi for
use in bioanalytical applications are the development of
molecules capable of recognition of these highly hydrated
anions[10] in a complex physiological environment, where nu-
merous competing analytes and proteins are present, and
the development of receptors capable of discriminating be-
tween PPi and nucleoside triphosphates, which have an
identical charge.[6,8] While there has been significant recent
progress in the development of sensors capable of binding
PPi in aqueous solution, very few are capable of distinguish-
Results and Discussion
Synthesis and Characterization of the Cyclic Peptide
Receptor
Based on previous studies,[11,14,17] we designed a series of re-
ceptors 1–6·Zn2 to probe the effects that 1) spacing between
the two ZnIIDpa binding sites, 2) steric bulk of the ꢀnon-
bindingꢁ side chains, and 3) size of the cyclic peptide scaffold
would have on anion binding affinity and selectivity. The
synthesis of C2-symmetric receptors 1·Zn2-3·Zn2 and unsym-
metrical receptors 4·Zn2-5·Zn2 was described previously.[11,14]
The smaller receptor 6·Zn2, based on a hexapeptide scaffold,
was synthesized using solid-phase peptide synthesis (SPPS)
followed by macrocyclization and functionalization in
a manner similar to the other systems. Briefly, the linear
precursor to cyclic peptide 6 was prepared on 2-chlorotrityl
chloride resin, using Fmoc/HATU chemistry to couple the
[a] Dr. S. J. Butler, Prof. Dr. K. A. Jolliffe
School of Chemistry (Building F11)
The University of Sydney
NSW, 2006, Australia
Fax : (+61)2-9351-3329
E-mail: kate.jolliffe@sydney.edu.au
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201200627.
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FULL PAPER
of 10 (10 mm, pH 7.4, 258C) re-
sulted in concentration-depen-
dent quenching of the indicator
fluorescence (a 4.5-fold de-
crease in fluorescence intensity
upon addition of 1 equiv of 1–
6·Zn2 was observed in all cases,
with quenching reaching a maxi-
mum upon addition of 2 equiv
of 1–6·Zn2). A shift in the emis-
sion maximum from 480 nm to
530 nm was also observed, as
Figure 1. Series of cyclic peptide-based receptors 1–6·Zn2.
well as
a
pseudo-isosbestic
ornithine- and leucine-derived building blocks, which have
been previously reported.[14] The linear peptide was cleaved
from the resin using mildly acidic conditions to give 7 in ap-
proximately 75% yield (based on reversed-phase HPLC
analysis). Cyclization of 7 was effected using the coupling
point at 568 nm, thus indicating that the indicator–receptor
complexes were the predominant species in solution
(Figure 2). All receptors bound 10 strongly [log Kin =6.2–7.3
(ꢁ0.1)], as determined by nonlinear least-squares curve fit-
ting of the titration data to a 1:1 binding model[21] (the stoi-
chiometry was confirmed by the mole-ratio method[22] for all
receptors and additionally using Job plots for receptors 1[11]
and 2), with receptor 6·Zn2 having the highest affinity for
10.
Titration of 1:1 solutions of the indicator–receptor com-
plexes (10 mm) with aliquots of the sodium salts of PPi,
ATP, and ADP resulted in significant increases in fluores-
cence intensity indicating displacement of the indicator,
while AMP, cAMP, phosphoserine, phosphotyrosine, hydro-
genphosphate (Figure 3), and the polycarboxylates acetyl-
glutamate and Ac-Glu-Gly-Glu (see the Supporting Infor-
mation) were not able to displace the indicator from any of
the receptors to an appreciable extent (<10% fluorescence
recovery upon addition of 10 equiv hydrogenphosphate),
thus indicating the selectivity of these receptors for di- and
triphosphate anions over monophosphate anions and poly-
carboxylates. Addition of 1 equivalent of PPi resulted in
almost complete restoration of the fluorescence intensity,
while the addition of ATP or ADP resulted in 4-fold and
3.5-fold fluorescence enhancements, respectively.
reagent
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmor-
pholinium tetrafluoroborate (DMTMM)[18] in the presence
of Hꢃnigꢁs base to afford the desired cyclic peptide 8 in
32% yield. The Cbz protecting groups of cyclic peptide 8
were removed using a solution of 45% hydrogen bromide in
acetic acid giving the diamine 9 upon a basic work-up proce-
dure. Afterwards, 9 was subjected to reductive amination
with 2-pyridinecarboxaldehyde using our previously estab-
lished conditions,[11] thus providing the Dpa-functionalized
cyclic peptide 6 in 76% yield over two steps. Subsequent ad-
dition of two equivalents of ZnACHTUNTRGNEUNG(NO3)2 gave the fully-func-
tionalized anion receptor 6·Zn2 (Scheme 1).
Anion Binding Studies
In order to determine the anion binding capabilities of 1–
6·Zn2, we employed indicator displacement assays[19] using
the previously reported fluorescent coumarin derivative
10.[11,14,20] Our initial studies were performed in 5 mm
HEPES buffer at pH 7.4, as all receptors were soluble in
this medium. Titration of solutions of 1–6·Zn2 into a solution
Fluorescence emission intensities (lex =347 nm, lem
=
480 nm) from these titrations
were analyzed using a curve-fit-
ting procedure based on the
equilibria previously described
for competition assays,[23] with
our pre-determined values for
Kin to determine the apparent
stability constants (Table 1).
The assumed 1:1 binding mode
was supported by mass spectro-
metric data of the PPi-receptor
complexes, in which the major
signals observed corresponded
to the singly charged PPi com-
plexes
[receptor+PPi+H]+
Scheme 1. Solid-phase synthesis of receptor 6·Zn2 and structure of indicator 10 (inset). Conditions:
a) DMTMM (3 equiv), Hꢃnigꢁs base (5 equiv), DMF (0.05m); b) 45% HBr in AcOH, followed by basic work-
(see the Supporting Informa-
tion).
up with 0.3m NaOH; c) 2-pyridinecarboxaldehyde (20 equiv), NaHB
ACHTUNGTRENNUNG(NO3)2·4H2O (2 equiv), CD3OD.
ACHTUNGRTEN(NUNG OAc)3 (20 equiv), DMF; d) Zn-
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Selective Pyrophosphate Recognition
Table 1. Apparent association constants (log Ka) determined for recep-
tors 1–6·Zn2 and various polyphosphate anions in aqueous solution (5 mm
HEPES, pH 7.4).
Anion
1·Zn2
2·Zn2
3·Zn2
4·Zn2
5·Zn2
6·Zn2
Indicator 10
PPi
ATP
6.3
7.2
6.5
6.3
6.2
7.4
7.6
7.4
6.9
8.4
8.6
7.5
6.9
8.8
8.3
7.7
6.8
7.9
7.2
7.0
7.3
9.8
9.1
7.9
ADP
Each value represents the average of at least two separate titration ex-
periments. Errors<10%.
Notably, significant differences in binding affinity and se-
lectivity were observed between members of the family of
receptors. Comparing binding data for the series 1–3·Zn2, in
which the steric bulk of the non-binding side chains is al-
tered, indicates that while the hydrophobic cleft provided by
the Leu side chains of 2·Zn2 results in similar affinity for all
three anions [log Ka 7.4–7.6 (ꢁ0.2)], the smaller Ala sub-
stituents of 1·Zn2 provide some selectivity for PPi over ATP
and ADP. In contrast, for 3·Zn2 in which the non-binding
substituents are Phe side chains, binding is in the order
ATP ꢂPPi>ADP. Molecular models (SPARTAN 06,
MMFF94) of the complexes of 3·Zn2 with ATP and ADP
suggest that the reason for the increased affinity of this re-
ceptor for ATP may be a result of a secondary p–p interac-
tion between one of the Phe residues and the adenine
moiety of ATP. In contrast, the backbone of ADP is too
short to allow this interaction to occur (Figure 4).
Figure 2. Representative spectra showing the change in fluorescence
emission of indicator 10 (10 mm) upon the addition of increasing amounts
of receptor 5·Zn2 (top); change in fluorescence intensity of 10 (10 mm) at
480 nm upon the addition of increasing amounts of receptor 5·Zn2
(bottom). Measurement conditions: aqueous solution of HEPES buffer
(5 mm, pH 7.4), lex =347 nm, 258C.
Figure 4. Molecular structures of: (left) the ATP complex of receptor
3·Zn2, and (right) the ADP complex of receptor 3·Zn2, modelled using
SPARTAN 06 (MMFF94).
Moving the ZnIIDpa sites closer together on the scaffold
resulted in increased selectivity for PPi over ATP and ADP
in both the Phe and Leu series. Notably, the affinity of 4·Zn2
(which has proximal ZnIIDpa side chains) for PPi is more
than an order of magnitude higher than that of 2·Zn2, in
which the Dpa side chains are distal, while for 5·Zn2 affinity
for all three anions drops slightly in comparison to that of
3·Zn2. Surprisingly, changing the size of the cyclic peptide
scaffold while maintaining the same distance between the
binding sites had a significant effect on binding affinity with
6·Zn2 binding PPi an order of magnitude more strongly than
4·Zn2 and with increased selectivity over both ATP and
ADP. Since the ZnIIDpa binding sites are expected to be at
almost identical distances apart in 4·Zn2 and 6·Zn2, this dif-
ference in binding affinity might reflect reduced steric inter-
Figure 3. Representative change in fluorescence emission of the complex
10-2·Zn2 (10 mm) upon the addition of: (top) 10 equivalents of various
anions (sodium salts), and (bottom) 10 equivalents of pyrophosphate
(sodium salt) in incremental steps. Measurement conditions: aqueous so-
lution of HEPES buffer (5 mm, pH 7.4), lex =347 nm, 258C.
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Table 2. Apparent association constants (log Ka) determined for recep-
tors 1·Zn2, 2·Zn2, 4·Zn2, and 6·Zn2 and various polyphosphate anions in
aqueous solution (5 mm HEPES, pH 7.4, 145 mm NaCl).
ference from the single Leu side chain in 6·Zn2 compared to
the two bulky side chains present in 4·Zn2.
These initial binding studies revealed that selectivity be-
tween di- and triphosphate anions can be modulated by
changing both the nature and position of the non-binding
side chains in receptors 1–6·Zn2, as well as the size of the
cyclic peptide scaffold. Next, we investigated the binding
properties of the receptors in a medium that closely mimics
physiological conditions (i.e., aqueous solution, pH 7.4,
154 mm NaCl). It should be noted that, in a saline solution,
the high concentration of chloride anions may provide com-
petition for the anion binding sites. In addition, sodium and
chloride ions may interfere with the IDA by quenching the
fluorescence of coumarin indicator 10.[24]
Titrations of each of the receptors 1·Zn2, 2·Zn2, 4·Zn2, and
6·Zn2 into aqueous solutions of indicator 10 (10 mm, 5 mm
HEPES, pH 7.4, 145 mm NaCl, 258C) resulted in quenching
of fluorescence emission of the indicator in a concentration-
dependent manner (Figure 5). The addition of one equiva-
Anion
1·Zn2
2·Zn2
4·Zn2
6·Zn2
Indicator 10
PPi
ATP
5.5
6.8
6.3
6.0
5.4
6.4
6.4
6.4
5.6
7.5
6.1
6.3
5.8
7.9
6.8
6.2
ADP
Each value represents the average of at least two separate titration ex-
periments. Errors<10%.
displacement of the indicator from each of the 1:1 indica-
tor–receptor complexes, as demonstrated by large enhance-
ments in fluorescence emission (Figure 6). By contrast, none
of the monophosphate derivatives were able to displace the
indicator by a significant amount. Apparent association con-
stants between each of the receptors and PPi, ATP, and
ADP are given in Table 2. As expected, the receptors
showed slightly lower affinity for all three anions in a saline
solution as compared to that determined in the absence of
NaCl, with similar patterns of selectivity to those observed
in 5 mm HEPES buffer above.
Figure 5. Representative change in fluorescence emission of indicator 10
(10 mm) upon the addition of increasing amounts of receptor 2·Zn2. Inset:
change in fluorescence intensity of 10 (10 mm) at 480 nm upon increasing
amounts of receptor 2·Zn2. Measurement conditions: aqueous solution of
5 mm HEPES (pH 7.4) containing 145 mm NaCl, lex =347 nm, 258C.
Figure 6. Change in fluorescence intensity (at 480 nm) of the complex 10-
2·Zn2 upon addition of various phosphate oxoanions (sodium salts). Mea-
surement conditions: aqueous solution (5 mm HEPES, pH 7.4, 145 mm
NaCl), lex =347 nm, 258C.
In order to utilize these complexes in a biological assay,
such as for the detection of PPi in extracellular fluid or
urine, they must be functional in a medium that contains
both a large excess of anions (chloride and phosphate) as
well as numerous other species such as metal ions that could
potentially interfere with the recognition process. Therefore,
we assessed the binding capabilities of receptors 1·Zn2,
2·Zn2, 4·Zn2, and 6·Zn2 towards PPi, ATP, and ADP in
a Krebs buffer solution, which is commonly used for cell
and tissue culture in biological research. Krebs buffer (com-
position: 137 mm NaCl, 5.4 mm KCl, 1.2 mm MgSO4, 2.8 mm
CaCl2, 0.4 mm KH2PO4, 0.3 mm NaH2PO4, 10 mm Tris base,
10 mm glucose, buffered to pH 7.4) contains high concentra-
tions of phosphate anions (700 mm) and Ca2+ and Mg2+ ions,
both of which are known to bind strongly to PPi, ATP, and
ADP.[25–27] Using fluorescence titrations, we determined that
all four receptors bind indicator 10 with similar affinities
(log Kas 5.1–5.4) to those determined above in NaCl/
lent of each receptor gave rise to a 2-fold decrease in fluo-
rescence intensity. Receptors 3·Zn2 and 5·Zn2, bearing phe-
nylalanine residues, were insoluble in the saline solution and
were not tested under these conditions. The titration data
were analyzed using a 1:1 binding model (Figure 5, inset) as
described previously, giving association constants in the
range log Kin =5.4–5.8 (ꢁ0.1) (Table 2). As expected, each
receptor displayed a lower affinity for indicator 10 under
these conditions as compared to those determined in the ab-
sence of NaCl. The association constant determined for the
binding of indicator 10 to receptor 1·Zn2 was in agreement
with the previously reported log Kin value of 5.1ꢁ0.1, which
was determined at a slightly lower pH of 7.2.[11]
Subsequent competition assays revealed that each of the
receptors retain their selectivity for di- and triphosphate
anions over monophosphate derivatives in a saline solution.
Pyrophosphate, ATP, and ADP were capable of competitive
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Selective Pyrophosphate Recognition
HEPES buffer solution, thereby indicating that the addition-
al species in Krebs buffer should not interfere with the dis-
placement assay (Figure 7).
Remarkably, competition assays in Krebs buffer indicated
that selectivity of the chemosensing ensembles for PPi over
Figure 7. Representative change in fluorescence emission of indicator 10
(10 mm) in Krebs buffer upon the addition of increasing amounts of re-
ceptor 6·Zn2. Inset: change in fluorescence intensity of 10 (10 mm) at
480 nm upon addition of receptor 6·Zn2. Measurement conditions: aque-
ous solution of Krebs buffer (pH 7.4), lex =347 nm, 258C.
ATP and ADP was enhanced in this more complex medium.
In particular, ensemble 10-6·Zn2 showed a remarkable abili-
ty to discriminate between PPi and ATP/ADP in Krebs
buffer. Only PPi was able to completely displace the indica-
tor from the receptor under these conditions, as demonstrat-
ed by full recovery of the fluorescence signal (Figure 8).
Both ATP and ADP were unable to displace the indicator
by a significant amount, giving minor yet reproducible fluo-
rescence enhancements. Analysis of the titration data using
the curve-fitting procedure described above indicated that
in Krebs buffer 6·Zn2 has a similar affinity for PPi to that
observed in saline, although data did not fit as well to a 1:1
competitive binding model, reflecting the additional equili-
bria present between PPi and species such as Ca2+ and
Mg2+. The other chemosensing ensembles exhibited similar
behavior. The notable improvement in selectivity for PPi
over ATP and ADP in Krebs saline relative to NaCl solu-
tion is readily apparent from a comparison of the titration
plots of ensemble 10–6·Zn2 with these anions (Figure 9),
thus illustrating the lack of fluorescence recovery afforded
by ATP and ADP in Krebs saline. This improved selectivity
enables low micromolar concentrations of PPi to be detect-
ed in the presence of 100 mm ATP (Figure 10), which should
prove useful in monitoring enzymatic reactions in which PPi
is generated.
Figure 8. Representative change in fluorescence emission of the complex
10-6·Zn2 (10 mm) in Krebs buffer upon the addition of 10 equivalents of:
PPi (top), ATP (middle), and ADP (bottom). Measurement conditions:
aqueous solution of Krebs buffer (pH 7.4), lex =347 nm, 258C.
CaADPꢀ =2.84[27]). Therefore, the enhanced selectivity ob-
served for PPi over ATP (and ADP) in Krebs saline cannot
be attributed to the difference in affinity of PPi and ATP
for either Ca2+ or Mg2+ since both of these metal ions bind
with similar affinity to the two phosphate derivatives. This
suggests that the enhanced selectivity observed for PPi in
Krebs solution is predominantly a result of the higher affini-
ty for this ion by receptor 6·Zn2 compared to ATP or ADP,
resulting in both an increased ability of PPi to displace the
indicator 10 from the receptor and increased competition
between the receptor and metal ions for PPi. The numerous
competitive equilibrium processes occurring in this complex
mixture appear to be significantly affected by these differen-
ces in binding affinity, highlighting one of the advantages of
indicator displacement assays for sensing purposes and sug-
gesting that alternative indicators could also be employed to
tune the affinity of the various chemosensing ensembles for
different polyphosphate anions.
Notably, all three phosphate derivatives are known to
bind strongly and with similar affinity (slightly lower for
ADP) to Mg2+ and Ca2+ ions (reported log Ka values at
similar pH and ionic strength to those in the current study
for MgPPi2ꢀ =4.26,[25] Mg2PPi=2.59,[25] MgATP2ꢀ =4.22,[25]
MgADPꢀ =3.6,[26] CaPPi2ꢀ =3.17,[27] CaATP2ꢀ =3.18,[27] and
Conclusions
In summary, we have investigated the anion binding ability
of a family of bis
using indicator displacement assays and found that these flu-
[ZnIIDpa] functionalized cyclic peptides
ACHTUNGTRENNUNG
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Katrina A. Jolliffe et al.
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ability, enabling the tuning of the receptor to optimize selec-
tivity for the analyte of interest (PPi). Current studies are
focused on tuning the selectivity of these receptors for a vari-
ety of anions using a larger library of cyclic peptide scaffolds
and alternative indicators for displacement assays.
Experimental Section
General methods and instrumentation used were identical to those re-
ported previously.[14] Abbreviations used: dimethylformamide (DMF); O-
(7-aza-1H-benzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium hexafluoro-
phosphate (HATU); 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmor-
pholinium tetrafluoroborate (DMTMM); trifluoroacetic acid (TFA); re-
versed-phase high performance liquid chromatography (RP-HPLC).
General Procedures for Solid-Phase Peptide Synthesis
1. Loading of amino acid onto 2-chlorotrityl chloride resin: Under an at-
mosphere of nitrogen, 2-chlorotrityl chloride resin (resin capacity 1.01–
1.4 mmolgꢀ1) was swollen in a sinter-fitted syringe in anhydrous dichloro-
methane for 1 h. The resin was drained and treated with a solution of
Fmoc-protected amino acid (1.5 equiv relative to resin capacity) and
Hꢃnigꢁs base (5 equiv relative to resin capacity) in anhydrous dichloro-
methane/DMF (9:1 v/v, 0.1m) and the resulting suspension was gently
agitated at rt for 24 h under an atmosphere of nitrogen. At this time the
resin was drained and then treated with a mixture of methanol/Hꢃnigꢁs
base/dichloromethane (2:1:17 v/v/v, 2ꢄ5 mLꢄ15 min). The resin was
then washed sequentially with DMF (2ꢄ10 mL), dichloromethane (3ꢄ
10 mL), and diethyl ether (3ꢄ10 mL). The residual solvent was removed
under reduced pressure.
Figure 9. A comparison of the changes in fluorescence emission observed
for the complex 10-6·Zn2 (10 mm) upon the addition of various anions in:
(top) saline solution and (bottom) Krebs buffer solution. Measurement
conditions: pH 7.4, lex =347 nm, 258C.
2. N-terminal Fmoc deprotection: The resin-bound peptide was agitated
in a solution of 10% piperidine in DMF (2ꢄ5 mLꢄ15 min) and then
drained and washed sequentially with DMF (2ꢄ5 mL), dichloromethane
(3ꢄ5 mL), and DMF (3ꢄ5 mL). The resulting resin-bound amine was
used immediately in the next peptide coupling step.
3. Estimation of resin loading: The drained Fmoc deprotection solution
was diluted with a solution of 10% piperidine in DMF so that the maxi-
mum concentration of the fulvene-piperidine adduct was in the range of
2.5–7.5ꢄ10ꢀ5 m. A sample of this solution (2ꢄ3 mL) was transferred to
two matched 1 cm quartz glass cuvettes and the UV/Vis absorbance at
301 nm was measured, using a solution of 10% piperidine in DMF as
a reference. An average of the two absorbance values was used to calcu-
late the loading, using e=7800mꢀ1 cmꢀ1
.
4. SPPS Peptide coupling: Under an atmosphere of nitrogen, a solution
of Fmoc-protected oxazole (1.5 equiv relative to loading), HATU
(2 equiv relative to peptide) and Hꢃnigꢁs base (3 equiv relative to pep-
tide) in anhydrous DMF (0.1m relative to dipeptide) was added to the
resin and the resulting suspension was agitated at rt for 24 h. The resin
was then washed sequentially with DMF (2ꢄ5 mL), dichloromethane
(3ꢄ10 mL), and DMF (3ꢄ5 mL).
Figure 10. Change in fluorescence emission of 10-6·Zn2 (10 mm) in Krebs
buffer containing excess ATP (100 mm) upon the addition of 10 equiva-
lents of PPi (0–100 mm). Measurement conditions: aqueous solution of
Krebs buffer (pH 7.4), ATP (100 mm), lex =347 nm, 258C.
5. Cleavage of peptides from the resin: A mixture of hexafluoroisopropa-
nol/dichloromethane (1:4 v/v, 3ꢄ5 mL) was added to the resin and the re-
sulting suspension was agitated at rt for 15 min. The solutions were
drained and combined, and the solvent was removed under reduced pres-
sure to afford the crude linear peptide.
orescent chemosensing ensembles show excellent selectivity
for PPi over ATP and ADP in Krebs buffer. Notably, selec-
tivity for PPi is significantly enhanced in this biologically
relevant medium compared to aqueous buffer, illustrating
the importance of the composition of the medium and the
presence of competing species in determining selectivity for
anions, and suggesting that other receptors that bind anions
with some selectivity in water should be reinvestigated in
this biorelevant aqueous fluid. Use of the cyclic peptide
scaffold has allowed an investigation of the effects of
a number of features (binding site position, ꢀnon-bindingꢁ
side chain steric bulk, and scaffold size) on anion binding
Synthesis
H2N-Leu-OxzACHTUGNTREN(UGNN Ser)-[OrnACHTUTGNERNNG(U Cbz)-OxzACTHUNTGRENNUGN
The oxazole building block Fmoc-Orn
G
(Ser)-OH[14] was loaded
ACHTUNGTRENNUNG
onto 2-chlorotrityl chloride resin (0.150 g, resin capacity 1.1 mmolgꢀ1) ac-
cording to general procedure 1 (loading 0.88 mmolgꢀ1). Building blocks
Fmoc-Orn
coupled sequentially to the resin-bound amine according to the SPPS
ACHTUNGTRENNU(GN Cbz)-OxzAHCTUNRTGENNUGN CAHTNUGTRENNGUN
(Ser)-OH[14] and Fmoc-Leu-Oxz(Ser)-OH[14] were
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Selective Pyrophosphate Recognition
procedure 4 to give the resin-bound trioxazole. The N-terminal Fmoc
protecting group was removed (general procedure 2), and the resulting
amine was cleaved from the resin (general procedure 5) to afford H2N-
Acknowledgements
We thank the Australian Research Council for financial support and Dr
C. E. Marjo (UNSW), Prof. S. Kable (USyd), and Assoc. Prof. T. Schmidt
(USyd) for helpful discussions.
Leu-OxzACHTUNGTRENNUNG(Ser)-[OrnACHTUNGTRENNUNG(Cbz)-OxzACHTNGURTEN(NUGN Ser)]2-OH (7) (138 mg, quant.) as a color-
less solid, which was used in the next step without further purification.
LRMS (ESI) m/z=829 [M+H]+.
Cyclo-Leu-OxzACHTUNGTRENNUNG(Ser)-[OrnAHCNUTRTGENG(NUN Cbz)-OxzACHTNUGRTEN(NUGN Ser)]2 (8)
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To a solution of the linear trioxazole 7 (138 mg, 0.132 mmol) in anhy-
drous DMF (0.05m) was added DMTMM (3 equiv) and Hꢃnigꢁs base
(5 equiv), and the resulting mixture was stirred at rt for 72 h under an at-
mosphere of nitrogen. The mixture was acidified by the addition of hy-
drochloric acid (0.3m, pH 5) and then partitioned between water (20 mL)
and chloroform/isopropanol (3:1 v/v, 20 mL). The aqueous phase was ex-
tracted with chloroform/isopropanol (3:1 v/v, 3ꢄ20 mL) and the com-
bined organic fractions were dried (MgSO4) and concentrated under re-
duced pressure to give a yellow oil. Subjection of the crude material to
preparative RP-HPLC [gradient: 20–80% acetonitrile (0.05% TFA) in
water (0.05% TFA) over 50 min] gave the desired cyclic trioxazole 8
(tR =50.5 min, 34 mg, 32%) as a colorless solid. ½aꢃ2D0 =ꢀ7.18 (c=0.85,
MeOH); 1H NMR (400 MHz, CD3OD): d=0.95 (d, J=6.4 Hz, 3H), 1.02
(d, J=6.4 Hz, 3H), 1.48 (m, 2H), 1.65 (m, 2H), 1.76 (m, 1H), 1.84 (m,
2H), 1.96 (m, 2H), 2.10 (m, 2H), 3.14 (t, J=6.6 Hz, 4H), 5.02 (s, 4H),
5.22–5.30 (m, 3H), 7.23–7.33 (m, 10H), 8.42 (s, 2H), 8.44 ppm (s, 1H), N-
H signals not observed; 13C NMR (100.6 MHz, CD3OD): d=22.9, 23.1,
25.9, 26.3, 32.6, 32.7, 41.2, 45.4, 48.1, 67.3, 128.7, 128.9, 129.4, 136.2, 136.3,
138.4, 143.4, 143.6, 143.7, 158.9, 161.3, 161.4, 165.0(7), 165.1(3),
165.9 ppm, twelve signals obscured or overlapping; HRMS (ESI) calcd.
for C41H46N8O10Na [M+Na]+ 833.3229, found 833.3203.
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Cyclo-Leu-Oxz
ACHTUNGTRENNUNG(Ser)-[OrnACHTUNTRGEN(GNUN Dpa)-OxzACTHNUGTREN(NUGN Ser)]2 (6)
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4965.
Step 1. A solution of hydrogen bromide in acetic acid (33% v/v, 2.0 mL)
was added to Cyclo-Leu-Oxz(Ser)-[Orn(Cbz)-Oxz(Ser)]2 (8, 32 mg,
G
R
ACHTUNGTRENNUNG
[9] D. H. Lee, J. H. Im, S. U. Son, Y. K. Chung, J.-I. Hong, J. Am. Chem.
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Chemistry: From Molecules to Nanomaterials (Eds.: J. W. Steed,
P. A. Gale), Wiley, Hoboken, NJ, 2012.
0.039 mmol), and the resulting mixture was stirred under nitrogen at rt
for 24 h. Anhydrous ether (10 mL) was added to give a cream colored
precipitate which was condensed by centrifugation. Subsequent tritura-
tion of the precipitate with anhydrous ether (10ꢄ10 mL) and removal of
the final clear ethereal layer under reduced pressure gave the dihydro-
bromide salt as a cream colored solid. The crude material was partitioned
between chloroform/isopropanol (3:1 v/v, 10 mL) and NaOH (0.3m,
10 mL) and the aqueous phase was extracted with chloroform/isopropa-
nol (3:1 v/v, 4ꢄ10 mL). The combined organic fractions were dried
(MgSO4) and concentrated under reduced pressure to give the crude dia-
mine 9 as a colorless solid.
Step 2. To a degassed solution of the diamine 9 (0.039 mmol) in anhy-
drous DMF (2.0 mL) was added 2-pyridinecarboxaldehyde (20 equiv)
and sodium triacetoxyborohydride (25 equiv), and the resulting mixture
was stirred at 358C for 72 h. The mixture was concentrated to almost dry-
ness and then partitioned between chloroform-isopropanol (3:1 v/v,
20 mL) and NaOH (0.3m, pH 8). The aqueous phase was extracted with
chloroform/isopropanol (3:1 v/v, 4ꢄ20 mL) and the combined organic
fractions were washed with half-strength brine solution (40 mL), dried
(MgSO4), and the solvent was removed under reduced pressure to give
a yellow solid. Subjection of the crude material to preparative RP-HPLC
[gradient of 5–50% acetonitrile (0.05% TFA) in water (0.05% TFA)
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´
´
´
[18] Z. J. Kaminski, B. Kolesinska, J. Kolesinska, G. Sabatino, M. Chelli,
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over 50 min] gave the desired Dpa-functionalised trioxazole
6 (tR =
30.3 min, 27 mg, 76%) as a yellow oil. ½aꢃ2D0 =ꢀ49.0 (c=1.0, MeOH);
1H NMR (400 MHz, CD3OD): d=0.97 (d, J=6.4 Hz, 3H), 1.04 (d, J=
6.4 Hz, 3H), 1.73–2.12 (complex m, 11H), 3.11–3.29 (m, 4H), 4.50 (s,
8H), 5.20 (m, 2H), 5.29 (m, 1H), 7.61 (m, 4H), 7.69 (m, 4H), 8.10 (m,
4H), 8.45 (m, 3H), 8.60 (m, 1H), 8.72 ppm (m, 4H), two N-H signals not
observed; 13C NMR (100.6 MHz, CD3OD): d=22.0, 22.1, 22.9, 23.1, 25.9,
33.0, 33.2, 45.4, 45.5, 48.0(9), 48.1(3), 55.8, 58.3, 126.0, 126.3, 136.2(3),
136.2(5), 136.4, 142.0, 143.6, 143.7, 143.9, 148.2, 153.0, 161.2, 161.6, 161.7,
164.8, 164.9, 166.0 ppm, seven signals obscured or overlapping; HRMS
(ESI) calcd. for C49H55N12O6 [M+H]+ 907.4362, found 907.4343.
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[21] All curve fitting was performed using the Equilibria program, C. E.
Marjo, University of New South Wales Analytical Centre, Sydney,
Australia; http://www.sseau.unsw.edu.au/Index.htm, 2009.
[22] P. Thordarson “Binding Constants and Their Measurement” in
Supramolecular Chemistry: From Molecules to Nanomaterials, DOI:
10.1002/9780470661345.smc018, Wiley, 2012.
Chem. Asian J. 2012, 00, 0 – 0
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Katrina A. Jolliffe et al.
FULL PAPER
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plex Stability, Wiley, New York, 1987.
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108, 441–448.
Received: July 12, 2012
Published online: && &&, 0000
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FULL PAPER
Mixing in different circles: Cyclic pep-
tide-based receptors bind preferen-
tially to pyrophosphate (PPi) over
ATP, ADP, and phosphate in physio-
logical saline (Krebs buffer). Selectiv-
ity for PPi over ATP and ADP is sig-
nificantly enhanced in this biologically
relevant fluid in comparison to that
observed in water.
Molecular Recognition
Stephen J. Butler,
Katrina A. Jolliffe*
&&&& — &&&&
Selective Pyrophosphate Recognition
by Cyclic Peptide Receptors in Physio-
logical Saline
Chem. Asian J. 2012, 00, 0 – 0
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