Positive ion pair cooperativity exhibited for the binding of phosphate under physiological conditions

Article abstract of DOI:10.1039/b510262f

The synthesis of a heteroditopic receptor which exhibits positive cooperativity for the binding of phosphate ion pairs under physiological conditions. Optimised complementarity between crown ether host and metal guest leads to increased binding affinity, K_a. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b510262f

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C O M M U N I C A T I O N
Positive ion pair cooperativity exhibited for the binding of phosphate
under physiological conditions†
Patrick T. Gunning*
University of Glasgow, Joseph Black Building, Dept. of Chemistry, Glasgow, UK G12 8QQ.
E-mail: patrick.gunning@yale.edu; Fax: 203 432 5570; Tel: (001)203 432 5218
Received 25th July 2005, Accepted 6th September 2005
First published as an Advance Article on the web 27th September 2005
The synthesis of a heteroditopic receptor which exhibits
positive cooperativity for the binding of phosphate ion
pairs under physiological conditions. Optimised comple-
mentarity between crown ether host and metal guest leads
to increased binding affinity, Ka.
Fig. 1 Schematic structure of ZnL·(OTf)2.
Introduction
Ion pair cooperativity1 is an exciting facet of molecular
recognition which has yet to be fully explored by biomimetic
chemists. The thermodynamics of binding interactions in aque-
ous environments have come to prominence with the use of
techniques such as isothermal titration calorimetry (ITC).2
Through the determination of DH◦ and the affinity constant
Ka it is possible to obtain a full thermodynamic profile of
the binding event. In particular, calorimetric techniques have
been used to great effect in biological research to measure the
affinities of substrates for specific proteins and to elucidate
the stoichiometries of the interactions.3 More recently ITC, in
conjunction with other spectroscopic methods, has been used to
probe the thermodynamic contributions of artificial host–guest
complexation.4
There have been a number of calorimetric investigations into
the binding of ion pairs by artificial guest receptors in non-polar
solvents,5 but there are limited examples of such experiments
in an aqueous environment.6 The focus of this work was to
determine the nature of the binding cooperativity between ions
in established heteroditopic ion pair receptors.7 Previous study
by Anslyn into the origin of negative cooperativity was based
around the findings of Jencks.8
optimisation. The ligand was tested on (X+)(H2PO4−), where
X was either Na+, K+ or the Li+ cation. The different sized
metal cations offered the opportunity to determine the effect
of the cation on binding cooperativity. Optimum dimensions
for metal/crown ether coordination have been well-established9
and the use of a 19-benzocrown-67b specifically favours the
binding of K+ over Na+ and Li+. 19-Benzocrown-6 was synthe-
sised with a pendant aldehyde using 2,4-dihydroxybenzaldehyde
and 1,2-bis(2-chloroethoxy-)ethane with K2CO3 in 40% yield
(Scheme 1). The aldehyde functional group was oxidized to the
corresponding carboxylic acid through the use of NaO2Cl and
sulfamic acid to give 4. Tri-N-Boc protected cyclen was coupled
to Cbz protected glycine using DCC and DMAP in 78% yield,
furnishing 2. Cbz removal was mediated by 1,4-cyclohexadiene
and 10% Pd/C. The deprotected primary amine 3 was then
coupled to the pendant carboxylic acid of the 19-benzocrown-
6 4 in 85% yield. Boc deprotection in TFA–DCM yielded the
ligand precursor 6, which was complexed to zinc triflate in an
equimolar ratio to form the desired ligand complex, ZnL·(OTf)2.
Binding experiments were principally carried out using
isothermal titration calorimetry (ITC), UV-vis titrations and
1H NMR titrations.†
DG◦ = DG◦ + DG◦ − DG◦
As has been stated, experimental calculation of the sign and
S
A
B
AB
value of DG◦ required the values of DG◦AB, DG◦ and DG◦
S
A
B
Jencks formulated an equation which considered the Gibb’s
individually. DG◦ was found from the binding of the ion
AB
free energies of ion pair binding.1b This stated that the Gibb’s
pair (X+)(H2PO4−), and the binding of the individual cation
and anion calculated through the use of a non-coordinating
counter ion. In the case of the cation binding (DG◦A), we used
the corresponding metal perchlorate salt. With all three metal
cations there was no observable metal–ligand interactions in wa-
ter. This was experimentally confirmed using 1H NMR titrations.
Therefore DG◦A can be assumed to be 0 kcal mol−1. Determining
free energy of connection or tether between the two ions DG◦
,
A
S
is equal to the sum of the individual component parts, DG◦
and DG◦ and the overall free energy for the binding of the
B
ion pair DG◦AB. Therefore, if DG◦ is more favourable than the
AB
combined free energies of DG◦A and DG◦B, then a positive DG◦
S
value will result. Positive cooperativity is denoted by a positive
DG◦S and counter-wise a negative DG◦S value represents negative
cooperativity. Utilisation of this mathematical model, allows the
calculation of DG◦ from DG◦AB, DG◦ and DG◦ through ITC.
the individual phosphate anion binding contribution (DG◦
)
B
was problematic. Attempts to measure the binding affinity of
H2PO4− with a tert-butyl ammonium non-coordinating counter
cation were hindered by the salts lack of solubility in an
aqueous environment. One can however, assume with reasonable
accuracy that the binding of phosphate is principally achieved
through an oxygen–zinc interaction.6a,10 Therefore the binding
S
A
B
On this premise I now report the first example of positive ion
pair cooperativity exhibited by an artificial hetero-ditopic re-
ceptor in an aqueous environment. It is important to remember
the inherent difficulties associated with working in an aqueous
environment where hydrophillic receptors must overcome their
own solvation sphere and that of a polar substrate.
Fig. 1 gives the schematic structure of the ligand system.
It has been shown in previous work7 that the adoption of a
modular synthetic approach was preferable in terms of ligand
of the phosphate anion and the determination of DG◦ can
B
be obtained by using a model system which precludes cation
binding and its effects. Through the use of a zinc(II) cyclen
model we were able to measure the binding of the individual
phosphate anion. It can be seen from Table 1 that the three
different metal cations have negligible effects upon phosphate
anion binding to zinc(II) cyclen. Therefore, the model gives the
individual phosphate anion binding to the zinc metal centre. It
is important to realise that the findings are based on a model
† Electronic supplementary information (ESI) available: synthetic pro-
cedures, ITC data, NMR spectra. See http://dx.doi.org/10.1039/
b510262f
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 8 7 7 – 3 8 7 9
3 8 7 7
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Table 1 Thermodynamic data for the binding of ZnL and Zn cyclen in HEPES buffer at pH 7.4 at 25 ◦C
Ligand
Ligand : substrate binding ratioa
Substrate
K × 104
DG◦/kcal mol−1b
DH◦/kcal mol−1
−TDS◦/kcal mol−1
ZnL
ZnL
ZnL
Zn cyclen
Zn cyclen
Zn cyclen
1 : 1
1 : 1
2 : 1
1 : 1
1 : 1
1 : 1
Na[H2PO4]
K[H2PO4]
Li[H2PO4]
Na[H2PO4]
K[H2PO4]
Li[H2PO4]
3.90 0.81c
5.10 0.94c
3.13 0.21d
1.60 0.09c
1.52 0.19c
1.34 0.29c
−6.57
−6.42
−6.16
−5.74
−5.70
−5.13
0.39 0.02
0.29 0.02
1.71 0.01
−3.25 0.08
−3.89 0.24
−3.56 0.05
6.96
6.71
7.84
2.49
1.81
1.57
a Binding stoichiometry was also confirmed by 1H NMR titration experiments. b Calculated from DG◦ = DH◦ – TDS◦. c Units: mol−1 dm3. d Units:
mol−2 dm6
Scheme 1 Synthetic procedure for the synthesis of ZnL·(OTf)2.
system but offer an important insight into the mechanisms of
binding and the thermodynamic origins of cooperativity.
Having calculated both the individual and combined ther-
modynamic contributions, it was possible to determine the
ligand–ion pair interactions seems sufficient to facilitate positive
cooperativity. These experiments reiterate the findings of other
workers,8 that shows that in an aqueous environment it is often
the solvent effects which determine the nature of the binding.
The design of a receptor which complements the ion pair, is not
sufficient to guarantee high affinity between host and guests.
Exploitation of solvent spheres through their disruption holds
the key to highly efficient artificial hosts in water environments.12
three DG◦ values for the different metal phosphate salts. In
S
the case of Na[H2PO4], DG◦ was calculated to be +0.83 kcal
S
mol−1. For K[H2PO4] the DG◦ value was +0.72 kcal mol−1
S
and Li[H2PO4] was found to be +1.03 kcal mol−1. In the
three examples shown, cooperative binding of the ion pair
was found to be positive. This is the first example in which
a model system has exhibited ion pair positive cooperativity.
The nature of the binding and cooperativity is assigned almost
entirely to the entropy contribution. The favourable entropy is
associated with desolvation of both the phosphate guest and the
crown ether cavity. All three-phosphate species have significant
entropy contributions, the largest belonging to Li[H2PO4]. This
was attributed, principally with the desolvation of the crown
ether cavity and also the dispersion of the highly organized
water sphere around the Li+ metal.11 In terms of enthalpy,
the interactions are endothermic and slightly unfavourable.
Theoretically, the size of the crown ether favours the chelation
of potassium. The higher, more disfavoured, endothermic values
for the binding of the sodium and lithium phosphate were
associated with the poor complementarity of the cation with
the crown ether. The experimental results confirm that the
K[H2PO4] species is more tightly bound, exhibiting higher bind-
ing constants and the lowest unfavourable DH◦ of +0.29 kcal
mol−1.
Acknowledgements
I would like to thank my two supervisors Dr R. D. Peacock and
Dr A. C. Benniston for all their help. I would also like to thank
Professor A. Copper and Margaret Nutley for their assistance
and discussions regarding ITC.
Notes and references
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In conclusion it can be seen that positive cooperativity can
be achieved in model systems through a combination of good
complementarity between host–guest and efficient desolvation
effects. In the ZnL·(OTf)2 ligand system, positive cooperativity
was driven almost entirely by entropy. Despite the unfavourable
DH◦ contributions, highly efficient solvent expulsion due to the
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