Combinatorial approaches to synthetic receptors

Combinatorial approaches to synthetic receptors Natarajan Srinivasan and Jeremy D Kilburn
Combinatorial chemistry can be efficiently used for the synthesis and evaluation of binding properties of libraries of synthetic receptors. This approach has been applied particularly to ‘tweezer’ and other ‘multi-armed’ receptors, and has been used for the identification of receptors for peptides in aqueous media, and for the development of new sensors and sensor arrays. Addresses School of Chemistry, University of Southampton, Southampton, SO17 1BJ, UK
e-mail: [email protected]

Current Opinion in Chemical Biology 2004, 8:305–310 This review comes from a themed issue on Combinatorial chemistry Edited by A Ganesan and Anthony D Piscopio Available online 6th May 2004 1367-5931/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2004.04.014 Abbreviation MALDI matrix-assisted laser desorption ionization

Introduction The development of synthetic receptors continues apace. New molecular architectures, conferring selective binding properties, and able to bind ever larger guest molecules, are constantly being described, often providing fundamental insights into the nature of non-covalent interactions and molecular recognition [1]. And such receptor systems are being used for increasingly sophisticated purposes, often with real practical applications [2], particularly as specific sensors for single analytes and for sensor arrays for multi-analyte mixtures. However, as most practitioners in the field have found, the rational design and synthesis of novel receptors — and a stepwise, iterative approach to producing increasingly selective receptors — can be both time-consuming and frustrating. In the 1990s, an alternative to the iterative approach to the identification of selective peptide receptors was pioneered by Still [3]. Mirroring developments in medicinal chemistry, the combinatorial approach was used to prepare large libraries of possible receptor structures, which could then be screened to identify a receptor for a given substrate. In practice, Still used a steroidal core to attach two variable peptide strands to produce libraries, which were screened to identify receptors for enkephalin-like www.sciencedirect.com

peptides. Since the original publications by Still, several groups have pursued both this approach as well as the reverse process (i.e. screening a single receptor with a library of guests, allowing rapid evaluation of the binding properties of the receptor). Several reviews have described the earlier work from this area [2,4,5] and thus this short review focuses on more recent advances. The extremely powerful approach of using dynamic combinatorial libraries for receptor discovery is not discussed here, as several excellent, dedicated reviews have been published recently [6,7]. Similarly the application of combinatorial chemistry to molecularly imprinted polymers, which provides another approach to creating new supramolecular systems has recently been reviewed [8]. One should also note that the combinatorial chemistry approach is closely paralleled by more biological approaches to discovering new receptors, such as phage display and antibody technology.

Combinatorial receptor design The combinatorial approach to receptor chemistry has been most widely applied to the development of so-called ‘tweezer’ receptors or ‘two-armed’ receptors, and higher order multi-armed receptors. The structure of such receptors generally consists of a scaffold or ‘head group’ which is typically a conformationally restricted moiety that directs, or preorganises the functionalised substrate-binding ‘arms’. The combinatorial approach to these receptors proceeds by attachment of a suitably functionalized scaffold to a solid support, followed by library synthesis of the arms using a variety of monomer units and a split-and-mix approach (Figure 1). The arms may be synthesized simultaneously to give libraries of ‘symmetrical’ receptors in which the arms are therefore identical in any given receptor, or they may be synthesized sequentially to give structurally more diverse libraries of ‘unsymmetrical’ receptors. Still’s original steroid derivatives with two peptidic arms [3] are clearly a prototype for such receptors, although the original concept of tweezer receptors dates back to Whitlock [9]. The advantage of such structures is that they are relatively easy to synthesize (in comparison with more conformationally restricted macrocyclic structures) and, despite their conformational flexibility, are clearly capable of selective recognition, both in organic and aqueous solvents. Although the use of a variety of monomer units for the construction of the receptor arms have been investigated in the past, the use of amino acids (to give peptidic arms) remains the most popular approach, reflecting the easy availability of the monomer units and the tremendous reliability of amino acid coupling reactions on the solid-phase. Current Opinion in Chemical Biology 2004, 8:305–310

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Figure 1

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Combinatorial approach to tweezer receptors proceeds: attachment of a suitably functionalised scaffold to a solid support; library synthesis of the arms using a variety of monomer units and a split-and-mix approach. The arms may be synthesized simultaneously to give libraries of ‘symmetrical’ receptors, or they may be synthesised sequentially to give structurally more diverse libraries of ‘unsymmetrical’ receptors.

‘Tweezer’ receptors The scaffold or head-group to which the receptor arms are attached is an important component of the receptor structure and is normally chosen, or designed, to preorganise the receptor arms so that they align and create a binding cavity. The influence of subtle changes in the structure and conformation of the scaffold on the binding properties of derived receptors is nicely exemplified by recent results from the Wennemers group [10,11]. A diketopiperazine scaffold was used to create ‘two-armed’ receptors and their binding properties were assessed by screening with a library of peptide guests, using chloroform as solvent. The trans,trans diketopiperazine scaffold, with a rigid ‘U’ shaped conformation, and providing ideal separation between the two receptor arms, gave highly selective receptors. However, structures derived from the more linear cis,cis diketopiperazine were only moderately selective and structures derived from several other diamine scaffolds did not exhibit any binding at all (Figure 2). The receptor head-group can also be used to provide an additional binding site or ‘anchor point’ for the guest molecule. Using a diamidopyridine unit as the head group provides a binding site for carboxylic acid functionality and receptor libraries derived from this scaffold were synthesized attached to TentaGelTM beads. The libraries Figure 2

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Two-armed receptors based on (a) di(trans-4-aminoproline) diketopiperazine and (b) di(cis-4-aminoproline)diketopiperazine. Current Opinion in Chemical Biology 2004, 8:305–310

were incubated in organic solvents with dye-labeled peptide substrates with a free carboxylic acid terminus and ‘hit’ beads were identified by strong colouration from the dye-label [12,13]. Edman degradation of a linear peptide-coding strand on each bead led to the identification of receptors for the peptide substrates (Kass  104 M–1, CDCl3). As with the Wennemers’ system, small changes in the structure of the diamidopyridine scaffold led to significant differences in the observed binding selectivities with the peptidic substrates [13]. The use of dye-labeled guest molecules to visualize ‘hit’ beads is typical when screening receptor libraries of this type. Using the diamidopyridine libraries, the observed binding selectivities with a given peptide were very sensitive to the location and structure of the dye label [12]. Thus, although the dye on its own is not bound by any of the receptors, the expectation that the dye-label will be an innocent spectator in such screening experiments cannot be taken for granted. Receptor libraries using a guanidinium scaffold provide structures suitable for binding peptides with a carboxylate terminus [14]. The guanidinium scaffold was synthesized with orthogonal protecting groups for the guanidine and the two amine residues, in principle allowing the synthesis of libraries with the two arms randomized independently. A library was successfully screened, in aqueous solvent, to identify a selective receptor for dyelabeled Glu(OtBu)-Ser(OtBu)-Val (Figure 3). The guanidinium receptors are notable for having no real preorganisation conferred by the scaffold, but are still able to bind the peptidic substrate in very demanding aqueous solvent (Kass ¼ 8  104 M1), although receptors could not be found for less hydrophobic substrates such as the side chain deprotected analogue, dye-labeled Glu-Ser-Val. Schmuck and co-workers have also developed receptors for peptides with a carboxylate terminus and have prepared libraries of peptides terminating with a guanidinocarbonyl pyrrole moiety, which serves as a carboxylate www.sciencedirect.com

Combinatorial approaches to synthetic receptors Srinivasan and Kilburn 307

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O A tripeptide-based library of cationic guanidinocarbonyl pyrrole receptors was screened to identify receptors for the C-terminal sequence of amyloid b-peptide (L-Val-L-Val-L-Ile-L-Ala) in aqueous solvent.

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A receptor library derived from a guanidinium scaffold gives structures suitable for binding peptides with a carboxylate terminus. The library was successfully screened to identify a selective receptor for dye-labeled Glu(OtBu)-Ser(OtBu)-Val (Kass ¼ 8  104 M1 in H2O (pH 9)/15% DMSO).

binding site [15,16]. The libraries were prepared using standard Fmoc couplings and the split-and-mix approach in combination with IRORI-radio frequency tagging technology. These structurally simple ‘one-armed’ receptors were screened with dye-labelled L-Val-L-Val-L-Ile-LAla (the C-terminal sequence of amyloid b-peptide, which is implicated in Alzheimer’s disease) and receptors with good affinity for the amyloid b-peptide, even in neat water (Kass > 4000 M1), were obtained (Figure 4). The structures identified as strong receptors in organic solvents were quite different from those identified in water, and binding affinities varied significantly (by two orders of magnitude) with quite small changes to the receptor structure. It is interesting to note that www.sciencedirect.com

related structures, arrived at by a rational design approach, featuring a bicyclic guanidinium unit and a peptide arm have been found to be effective inhibitors of HIV protease dimerization, seemingly by binding simultaneously to the C- and N-terminal sequences of the HIV protease [17].

More complex, ‘three-armed’ structures can be accessed using tripodal scaffolds and several different scaffolds have been developed for this purpose [18–22]. Liskamp has used a triazacyclophane (with additional carboxylic acid functionality for attachment to a resin) to generate threearmed receptors. Libraries were successfully screened to identify vancomycin-like receptors for dye-labelled D-AlaD-Ala and D-Ala-D-Lac dipeptides, although binding was only observed in organic solvents [23]. The preparation of an orthogonally protected version of the triazacyclophane scaffold allowed the synthesis of libraries with three structurally distinct arms [24]. A 225-membered library derived from this scaffold [25] was screened with a range of substrates including Fe3þ. Incubation of the library with FeCl3 and staining with KSCN gave only three beads showing the red colour of the Fe(III) thiocyanate complex. Edman degradation allowed the receptor structure on each bead to be determined and the same structure was found for each of the three beads. Binding of Fe3þ by this receptor may well involve coordination of the Fe3þ by both carboxylates derived from the aspartic acid residues and the imidazole of the histidine, a situation found in several Fe3þ binding proteins (Figure 5). The library chemistry could be extended to give dipeptide arms and further Fe3þ binders could be identified, typically containing several aspartic and glutamic acid residues.

Sensors Substrates can be labeled with an appropriate dye molecule to visualize ‘hit’ beads when the substrate is screened Current Opinion in Chemical Biology 2004, 8:305–310

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Figure 5

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275-membered library (AAn ¼ amino acid) was screened to identify an Fe3þ binder, with a possible binding motif reminiscent of monooxygenase hydroxylase. Aloc, allyloxycarbonyl; Fmoc, fluorenyloxycarbonyl; o-NBS, ortho-nitrobenzenesulfonyl.

with a resin-bound receptor library. The approach can be problematic because binding to the dye label may interfere with selective recognition of the actual guest. An alternative approach is to attach a fluorescent reporter group to the tweezer structure, which detects the change of environment as a consequence of a binding event [26,27]. Using a conceptually similar approach, a peptide sequence has been optimized for strong binding of Tb3þ [28], hence generating new high-affinity lanthanide-binding tags with intense luminescence properties. Combinatorial libraries were designed, starting with a consensus peptide sequence derived from studies on calcium-binding motifs of EDF-hand proteins, replete with ligating aspartate, asparagines and glutamate residues, and incorporating tyrosine and tryptophan residues to sensitize Tb3þ luminescence. The peptide libraries were prepared on TentaGelTM macrobeads, using ‘split-and-mix’ synthesis and an 80:20 mixture of base labile and photochemically labile linkers. A laddering procedure using encoded peptide caps provided a coding strand for each bead that could be conveniently deconvoluted using matrix-assisted laser desorption ionization (MALDI) mass spectrometry analysis. Screening for lanthanide binding while the peptides are attached to the solid phase can lead to false-positive luminescent signals due to interference from the resin matrix, so the library beads were distributed in a buffered 2% agarose matrix that contained Tb3þ ions (50 mM). Photochemical cleavage released a portion of the peptide into the surrounding agarose and beads carrying a Tb3þ binding peptide had a luminescent halo on illumination with a short-wavelength transilluminator. Competing ligands such as citrate or phosphate moieties were added to the agarose to increase selective pressure in the later-generation libraries. ‘Hit’ beads from the screening experiments were treated with mild base to release remaining resin-bound peptide, which was then sequenced using MALDI MS. Following this approach, a novel peptide was identified with >100-fold increase in Tb3þ affinity (Kass ¼ 1:8  107 M1) compared with the starting peptide sequence (Kass ¼ 1:3  105 M1). IntroCurrent Opinion in Chemical Biology 2004, 8:305–310

duction of a disulfide bond to the optimal linear sequence gave a further increase in affinity (Kass ¼ 5  108 M1). An alternative approach to creating a highly selective individual receptor for each component of a complex mixture is to use a series of ‘differential’ receptors [2] so that any analyte in a complex mixture leads to a unique pattern of responses from the various sensors in an array. Analysis of complex mixtures is then possible by recording the response of the sensor array to the mixture and use of pattern recognition algorithms. Anslyn has used this approach to discriminate between the structurally similar compounds, ATP and GTP [29]. A two-armed receptor library was prepared on the solid-phase using an aryl scaffold that carries two guanidinium moieties. The guanidinium groups provide an affinity for triphosphates and the tripeptide arms provide differential binding selectivity. Thirty beads from a 4913-member library were randomly selected and placed on a chip-based array platform. Fluorescein was introduced to the array and the indicator was taken up by the cationic receptors to give orange stained beads. Exposure of the array to the triphosphates leads to binding of the analyte and displacement of the fluorescein (at different rates for each bead) and each bead loses colour. The red, green and blue transmitted light intensities from each bead were recorded via a charge-coupled device and analysed using principal component analysis. Each receptor bead responded differently to various nucleotide phosphate samples, allowing the samples to be clearly differentiated by the combined array (Figure 6).

Analytical methods Binding constants for individual receptors can be conveniently determined using NMR titration experiments, but require many data points (i.e. spectra) to be collected for each binding constant, and are not suitable for receptors that are NMR ‘silent’ (i.e. do not show a shift in the NMR signals on complexation). A calibrated competitive NMR method has been developed [30] that allows the binding constant of a substrate with any ‘unknown’ receptor, including those that are NMR ‘silent’, to be www.sciencedirect.com

Combinatorial approaches to synthetic receptors Srinivasan and Kilburn 309

Figure 6

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A sensing array created from a library of ‘differential’ receptors. (a) General structure of resin bound library of receptors and fluorescein. (b) Signal transduction scheme used to detect nucleotide phosphates within the resin-bound sensor. Initially, the receptors bind fluorescein and the beads are stained by the dye. On binding the analyte, the fluorescein is displaced and the bead loses the staining from the dye. AAn ¼ amino acid. Adapted with permission from [29]. Copyright 2003 American Chemical Society.

determined by recording a single 1 H NMR spectrum, and hence allows rapid determination of the binding constants for a library of receptors with a single guest substrate. The method requires initial calibration by characterizing the changes to the NMR spectrum of a single receptor (the calibrant) on addition of the substrate, and accurate determination of the binding constant for this complexation. Thereafter, a single 1 H NMR spectrum of a mixture of the ‘unknown’ receptor, the calibrant and the substrate is recorded and the observed chemical shifts of signals for the calibrant allow the direct determination of the binding constant for the ‘unknown’ receptor–substrate complexation. Direct determination of binding constants for resinbound receptors is readily achieved essentially by introducing the resin bound receptor to a solution of substrate and measuring the decrease in concentration of substrate in free solution (e.g. by UV/VIS) as it is absorbed onto the resin bead by the receptor. Wennemers has used dyelabeled diketopiperazine receptors, and a resin-bound peptide guest, to study the influence of the loading, and nature of the solid support, on the binding constants determined by this method [31]. Using solid supports with varying polarity and hydrophobicity did not appear to influence the binding constants, although the time www.sciencedirect.com

taken to reach binding equilibrium was quite variable (up to 48 h). Measured binding constants for a given receptor–substrate complex were lower with higher loadings of the peptide substrate, suggesting that lower loadings represent a more solution-like environment. However, relative binding constants were identical regardless of the loading or type of resin used, and although the method makes several assumptions, it provides a rapid method for assessing relative binding constants for a given receptor with a range of guests (or vice versa).

Conclusions The combinatorial approach to synthetic receptors has been applied to a range of new structures and the methodology allows rapid synthesis of libraries, with simple screening procedures. The use of amino acid building blocks for the library synthesis has led to an emphasis on using these receptors to bind peptide substrates. Clearly more diverse structures may be prepared when a larger range of building blocks and coupling reactions can be used in the library synthesis, but the amino acid building block approach has been used to create some sophisticated supramolecular systems as exemplified by Imperiali’s high-affinity peptide binder for Tb3þ, and Anslyn’s sensor array for nucleotide triphosphates. Application of these systems to the discovery of sensors and other, biological Current Opinion in Chemical Biology 2004, 8:305–310

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questions is likely to be a continuing theme as the combinatorial approach to receptors is developed further.

Structurally simple receptors consisting of a peptide sequence capped by a guanidinocarbonyl pyrrole were screened to identify receptors for the C-terminal sequence of amyloid b-peptide.

References and recommended reading

16. Schmuck C, Heil M: Using combinatorial methods to arrive at a quantitive structure-stability relationship for a new class of one-armed cationic peptide receptors targeting the C-terminus of the amyloid b-peptide. Org Biomol Chem 2003, 1:633-636.

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Hunter CA, Tomas S: Cooperativity, partially bound states, and  enthalpy-entropy compensation. Chem Biol 2003, 10:1023-1032. Hunter uses simple host–guest systems and a chemical double-mutant cycle to probe cooperative binding interactions between functional groups. The results suggest new rationales for cooperative phenomena, the enthalpic chelate effect and enthalpy–entropy compensation. 2. 

17. Breccia P, Boggetto N, Pe´ rez-Ferna´ ndez R, Van Gool M, Takahashi M, Rene´ L, Prados P, Badet B, Reboud-Ravaux M, de Mendoza J: Dimerisation inhibitors of HIV-1 protease based on a bicyclic guanidinium subunit. J Med Chem 2003, 46:5196-5207. 18. Choi HJ, Park YS, Yun SH, Kim HS, Cho CS, Ko K, Ahn KH: Novel C3V-symmetric tripodal scaffold, triethyl cis,cis,cis-2,5,8tribenzyltrindane-2,5,8-tricarboxylate, for the construction of artificial receptors. Org Lett 2002, 4:795-798.

Lavigne JJ, Anslyn EV: Sensing a paradigm shift in the field of molecular recognition: from selective to differential receptors. Angew Chem Int Ed Engl 2001, 40:3118-3130. An excellent review of recent advances in receptor development, both rational and combinatorial, and in which Anslyn introduces the concept of ‘differential’ receptors (i.e. receptors that each have different binding properties, but none of them is necessarily very specific or selective), and describes the application of such receptors to sensors.

19. Siracusa L, Hurley FM, Dresen S, Lawless LJ, Pe´ rez-Paya´ n MN, Davis AP: Steroidal ureas as enantioselective receptors for an N-acetyl a-amino carboxylate. Org Lett 2002, 4:4639-4642.

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21. Hennrich G, Lynch VM, Anslyn EV: Novel C3-symmetric molecular scaffolds with potential facial differentiation. Chemistry 2002, 8:2274-2278.

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22. Chamorro C, Liskamp RMJ: Approaches to the solid phase of a cyclotriveratrylene scaffold-based tripodal library as potential artificial receptors. J Comb Chem 2003, 5:794-801.

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Rowan SJ, Cantrill SJ, Cousins GRL, Sanders JKM, Stoddart JF: Dynamic covalent chemistry. Angew Chem Int Ed Engl 2002, 41:898-952.

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10. Wennemers H, Nold MC, Conza M, Kulicke KJ, Neuburger M:  Flexible but with a defined turn – influence of the template on the binding properties of two-armed receptors. Chemistry 2003, 9:442-448. Diamino diketopiperazines and several other diamines are evaluated as possible scaffolds for construction of tweezer receptors, highlighting the importance of using a suitable scaffold to preorganise the binding arms of the receptors. 11. Conza M, Wennemers H: Selective binding of two-armed diketo-piperazine receptors to side-chain-protected peptides. J Org Chem 2002, 67:2696-2698. 12. Braxmeier T, Demarcus M, Fessmann T, McAteer S, Kilburn JD:  Identification of sequence selective receptors for peptides with a carboxylic acid terminus. Chemistry 2001, 7:1889-1898. Screening experiments with diamidopyridine-derived receptor libraries, using dye-labeled substrates highlight how the dye label can influence the binding of the substrate. 13. Arienzo R, Kilburn JD: Combinatorial libraries of diamidopyridine-derived ‘tweezer’ receptors and sequence selective binding of peptides. Tetrahedron 2002, 58:711-719. 14. Jensen KB, Braxmeier TM, Demarcus M, Frey JG, Kilburn JD:  Synthesis of guanidinium-derived receptor libraries and screening for selective peptide receptors in water. Chemistry 2002, 8:1300-1309. Libraries of ‘tweezer’ receptors with a guanidinium head group and peptidic side arms lead to identification of receptors able to bind short peptidic substrates in aqueous media. 15. Schmuck C, Heil M: Peptide binding by one-armed receptors in  water: screening of a combinatorial library for the binding of Val-Val-Ile-Ala. ChemBioChem 2003, 4:1232-1238. Current Opinion in Chemical Biology 2004, 8:305–310

20. Ballester P, Capo´ M, Costa A, Deya` PM, Gomila R, Decken A, Deslongchamps G: Dual binding mode of methylmethanetriacetic acid to tripodal amidopyridine receptors. J Org Chem 2002, 67:8832-8841.

23. Monnee MCF, Brouwer AJ, Verbeek LM, Van Wageningen AMA, Liskamp RMJ: Biol-inspired synthetic receptor molecules towards mimicry of vancomycin. Bioorg Med Chem Lett 2001, 11:1521-1525. 24. Opatz T, Liskamp RMJ: A selectively deprotectable triazacyclophane scaffold for the construction of artificial receptors. Org Lett 2001, 3:3499-3502. 25. Opatz T, Liskamp RMJ: Synthesis and screening of libraries of  synthetic tripodal receptor molecules with three different amino acid or peptide arms: identification of iron binders. J Comb Chem 2002, 4:275-284. An orthogonally protected triazacyclophane scaffold was used to construct libraries of three-armed receptors that were successfully screened to identify new Fe3þ binders. 26. Iorio EJ, Shao Y, Chen CT, Wagner H, Still WC: Sequenceselective peptide detection by small synthetic chemosensors selected from an encoded combinatorial chemosensor library. Bioorg Med Chem Lett 2001, 11:1635-1638. 27. Hioki H, Ohnishi Y, Kubo M, Nashimoto E, Kinoshita Y, Samejima M, Kodama M: Synthesis of calyx[4]arene library substituted with peptides at the upper rim. Tetrahedron Lett 2004, 45:561-564. 28. Nitz M, Franz KJ, Maglathlin RL, Imperiali B: A powerful  combinatorial screen to identify high-affinity terbium(III)binding peptides. ChemBioChem 2003, 4:272-276. From a biologically inspired starting sequence, a peptide was optimised for high affinity for Tb3þ, using split-and-mix synthesis, orthogonal linkers with a two-step screening protocol, and a laddering procedure with encoded peptides caps to give a coding strand that was deconvoluted by MALDI MS. 29. McCleskey SC, Griffin MJ, Schneider SE, McDevitt JT, Anslyn EV:  Differential receptors create patterns diagnostic for ATP and GTP. J Am Chem Soc 2003, 125:1114-1115. An array of ‘differential’ receptors is created by randomly selecting beads from a split-and-mix library of tweezer receptors designed to bind nucleotide triphosphates. Binding of ATP or GTP can be distinguished by the pattern of responses from the different receptors in the array. 30. Heath RE, Dykes GM, Fish H, Smith DK: Rapid screening of binding constants by calibrated competitive 1 H NMR. Chemistry 2003, 9:850-855. 31. Conza M, Wennemers H: Determination of binding affinities on solid supports: influence of the loading and the nature of the solid support. Chem Commun 2003:866-867. www.sciencedirect.com