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The value of molecular libraries generated by combinatorialmethods is largely dependent on the ease and ability todeconvolute or decode the structure of compounds of interestafter screening the library. Following the introduction ofpromising concepts in the early 1990s, there has beenconsiderable progress in the development and refinement ofmethodologies to address this issue.

AddressesDepartment of Chemistry, University of Cambridge, Lensfield Road,Cambridge CB2 1EW, UK*e-mail: sb10031@cam.ac.uk

Current Opinion in Chemical Biology 2000, 4:346–350

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IntroductionCombinatorial approaches to molecular problem solvingare beginning to feature across a range of fields within themolecular sciences. Besides the major application of com-binatorial chemistry in drug discovery, areas such ascatalyst discovery and materials science [1,2] are now rou-tinely using combinatorial methodologies. This reviewdiscusses advances in encoding and deconvolution thathave appeared in the literature from June 1998 toNovember 1999. The split-and-mix or ‘one-bead-one-compound’ synthesis [3•], has continued to be a powerfulconcept within the field and the identification of activecompounds from such libraries is becoming increasinglystraightforward. Another powerful approach to diversitygeneration is the synthesis of mixed-compound libraries insolution [4], for which a number of improved deconvolu-tion procedures have emerged, permitting more facileidentification of hits. In addition, the continued improve-ment in analytical technology has provided further scopefor novel encoding methods.

Deconvolution of solution-phase mixturesThe screening and deconvolution of compound mixtureshas been primarily applied to libraries of oligomers, partic-ularly peptides, for which resynthesis andpositional-scanning approaches are well-known. Methodshave been demonstrated for iterative resynthesis and posi-tional scanning of small-molecule non-peptidic libraries,made both in solution and on solid phase, for the straight-forward identification of hits [5,6]. Particularly relevant tothis area is the HPLC deconvolution protocol used byGriffey et al. [7]. The method relies on fractionation of arelatively large quantity (20–100 mg) of a solution-phaselibrary of typically 25–1000 compounds by HPLC. Portionsof the library are collected and fractions showing interest-ing activity are further purified to give single compounds.Sufficient pure material can be isolated for multiple bio-

logical assays and mass spectrometry analysis for structuredetermination. The advantage over one-bead-one-com-pound libraries appears to be one of scale and the use ofsolution-phase synthetic chemistry.

In a subtle twist on positional scanning, Boger’s group [8••]have developed a deletion synthesis deconvolution proto-col for use in small-molecule libraries. To demonstrate thetechnique a simple 16 member 4 × 4 library was evaluated.The standard positional-scanning method requires eightlibraries of four compounds, which in ideal circumstancesgives only two mixtures containing hits, one for each activemonomer. For deconvolution by deletion, libraries are pre-pared by systematically omitting one of the monomers, inthis case to give 8 libraries of 12 compounds, of which onlytwo are inactive for the ideal case (Table 1). The examina-tion of absence of activity removes the chance of falsepositives, and is more suited to identifying the most potentlibrary member, rather than weak binders. The techniquewas used to identify cytotoxic agents from a self-dimerisedlibrary of more than 106 compounds by screening only 28libraries and has subsequently been used on a library of65,000 protein–protein interactive agents [9].

Analytical techniques that allow identification of a bindingligand without separation of a library mixture are the sub-ject of much research. With diffusion-encoded NMRspectroscopy (DECODES) it is possible to resolve mole-cules by the speed at which they diffuse and hence onecan pick up the signals from a small ligand bound to a tar-get and remove signals from the other small ligands, whichdiffuse more rapidly [10]. Recent studies have looked atthe binding of 10 similar tetrapeptides to the antibioticvancomycin to correctly identify the known bindingsequence and give an accurate evaluation of binding con-

Recent developments in the encoding and deconvolution ofcombinatorial librariesColin Barnes and Shankar Balasubramanian*

Table 1

Identification of hypothetical compounds A3–B2 from a 4 × 4solution-phase library by deletion synthesis deconvolution.

Library number* Monomers A Monomers B Screening resultpresent present

Del A1 2,3,4 1,2,3,4 ActiveDel A2 1,3,4 1,2,3,4 ActiveDel A3 1,2,4 1,2,3,4 InactiveDel A4 1,2,3 1,2,3,4 ActiveDel B1 1,2,3,4 2,3,4 ActiveDel B2 1,2,3,4 1,3,4 InactiveDel B3 1,2,3,4 1,2,4 ActiveDel B4 1,2,3,4 1,2,3 Active

*Each library contains 12 compounds and only the libraries that do notcontain monomers A3 or B2 will be inactive. If this technique is combinedwith standard positional scanning, large, complex libraries can be madeand deconvoluted without the need for resynthesis or rescreening.

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stants [11]. Further studies have selected small moleculesthat interact with DNA [12]. Techniques for studyingsmall-molecule–protein interactions from combinatorialmixtures are still the focus of significant research and havebeen reviewed by Shapiro and Wareing [13]. Mass spec-trometry techniques for studying the binding of librariesof small ligands to protein targets have also been subject toimprovements, and are detailed in a recent review [14].

Analysis of bead-based librariesDirect spectroscopic analysis of material bound to orcleaved from a single-bead is the simplest way to deconvo-lute a library and recent advances in single-bead analysishave made this a more realistic proposition. Such methodsavoid the need for tagging chemistry during synthesis andany associated chemical orthogonality issues. A potentialconstraint is that either the screening must be performedon-bead or the material must be isolated after screening forstructure determination.

Libraries of hexapeptides have been synthesised by Wells et al. [15] on relatively large (~250 µm), highlyloaded (2 mmol/g) beads. Sufficient material (~30 nmol)has been cleaved from a single bead to allow HPLC, elec-trospray mass spectrometry (ESMS) and NMR analysis forunambiguous characterisation. MS remains the mostwidely used technique for characterisation of the smallamount of material cleaved from a single bead (roughly1 µm 1 mmol/g bead). A problem with using MS to char-acterise a small-molecule library is that some of thecompounds can coincidentally have the same mass.Hughes [16] has addressed this problem using proceduresfor the selection of monomers to prepare libraries of sev-eral hundred compounds in which no two have the samemolecular mass. The use of isotopes further increases thesize of the sublibraries. The use of synthetically generat-ed ratios of stable isotopes such as 13C, 15N or deuteriumin the library can also be used to encode compounds forMS analysis [17].

A technique routinely employed for addressing the issue ofidentical masses in the synthesis of oligomers, particularlypeptides, is termed ladder sequencing. A small portion ofthe peptide is capped during each extension reaction togenerate a ladder of short peptide fragments that can beanalysed by MS to report the sequence. This techniquehas recently been extended to libraries of glycopeptides,using simple carboxylic acids of differing masses to codefor the glycan moiety [18•]. A library of over 300,000 com-pounds was prepared, screened on-bead anddeconvoluted. This technique should be applicable to anyoligomer library.

IR spectroscopy is routinely performed on single beads forreaction monitoring. Whilst direct IR measurement is lim-ited for unambiguous compound characterisation, a seriesof seven tags containing nitriles, phenols and alkynes(i.e. moieties with distinct IR fingerprints) have been

developed for use in an encoding scheme [19]. One dis-tinct advantage over other types of chemical encoding isthat the tags can be read on-bead, which simplifies andspeeds deconvolution. A similar idea for marking resinbeads involves the use of groups with distinct fluorineatoms, which can be read by single-bead fluorineNMR [20].

Molecular taggingTo fully encode a library, chemical tagging schemes rely onattachment of tags at some point during each split-and-mixcycle. A number of groups simplify the process using a par-tial encoding strategy in which the initial resin is markedin some way and the final reaction step is not pooled, andhence only the middle portions of the split-and-mix syn-thesis need be deconvoluted. For small libraries, this canoften be done by MS alone as the number of componentswith similar masses needing to be resolved is reduced. Thetechnique is, however, essentially limited to libraries withonly three diversity sites.

Several well-known methods for total chemical encodinghave been applied during the period under review. Theearly work using peptides and oligonucleotides as encod-ing moieties for small organic molecules has not beendeveloped further because of the limited chemical stabili-ty of these molecules. The combination of carbeneinsertion chemistry and cleavable tags that can be read byelectron capture gas chromatography (EC–GC) was firstdeveloped by Still and co-workers [21]. This approach hasbeen much used and some recent examples include encod-ing of catalyst libraries [22] and libraries of 20,000compounds screened as neurokinin antagonists [23]. Tanet al. [24••] have used the carbene-based tagging approachto generate a fully-encoded library of greater than 2 millionpolycyclic compounds. Such exemplifications have furtherconsolidated this approach as a robust and establishedencoding technology (Figure 1).

The other common chemical tagging method, developedby the Affymax group, uses orthogonal protecting groupsto enable coupling of secondary amines as tertiary amides[25]. Cleavage of the tags by acid hydrolysis after screen-ing liberates amines that, following a dansylation step, canbe decoded by HPLC. This technique has recently beenused to encode benzothiazepinone libraries, showing thegeneral orthogonality to a large variety of reagents [26•]. Arecent paper from the same group has shown significantoptimisation of the technique, studying all aspects fromtag attachment to the final HPLC methodology [27•]. It isnow possible to use the tags in a quantitative manner,rather than in binary fashion, which substantially increasesthe library size that can be tagged with a given number ofamines (only 15 amines are required to uniquely tag 105

compounds). The cleaved tags can now be detected byMS in both the fluorescent [28] and underivatised [29]forms, which has enhanced throughput to the point wheredeconvolution is no longer a rate-limiting procedure.

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Cleavage of the same secondary amine tags from a benzy-lamine rather than a tertiary amide linkage has also beenshown [30]. As yet, however, no protocol for the benzy-lamine formation during a split-and-mix synthesis hasbeen reported.

To avoid the problem of orthogonal attachment chemistry,Scott et al. [31•] have used a Friedel–Crafts alkylation todirect attachment of the tag to the resin backbone(Figure 2). Using this protocol, the resin is initially modi-fied with an electron-rich aromatic moiety that serves asthe attachment point for the tags throughout the synthesis.The library is synthesised in the normal way with littlerequirement for orthogonality (exemplified for a peptidelibrary on Wang resin). Alkylation of hydroxymethylpyrroles, mediated by a Lewis acid (Yb(OTf)3), allowsattachment of any choice of detectable tagging groups atevery stage of the split-and-mix cycle. The only limitationfound for this tagging chemistry was the inability to tagmethionine residues, probably because of S-alkylation bythe pyrroles.

‘Tea bag’-type technologyAny procedure for which a batch of resin can be isolated,marked and sorted has potential as an encoding approach.

The advantages of this type of library are that no post-screening identification is required as the identity of eachcompound is already known, and that the library can beprepared once on a large scale and the cleaved materialsstored as known single compounds. A widely-used repre-sentative of this type of tagging is the radiofrequencymicrocan approach. The technology for sorting resin batch-es each cycle requires a degree of automation and protocolsusing the overall process appear regularly in the literature[32]. A small-scale, simplified and somewhat more eco-nomical approach based on visual sorting of colouredreactors has also been described [33].

Other novel techniques with potential for encoding involvefluorescence bar-coding of individual beads. Properties oforganic fluorophores on resin have been studied by severalgroups who concluded that self-quenching of fluorophoreson resin can be problematic, particularly at higher levels[34,35]. Furthermore, the chemical instability of mostorganic fluorophores is a major limitation for tagging. Therecent development of inorganic quantum dots for markingindividual particles provides intriguing possibilities for afuture encoding strategy. To date, they have been shown tobe useful labels in biological systems and capable of under-going a level of synthetic derivatisation [36,37].

348 Combinatorial chemistry

Figure 1

Methodology used in the binary encoding of alibrary of over two million compounds. Thenumber of reagents chosen at each step was2n–1 to optimise use of each tag. The tagsare attached by a rhodium-catalysed carbeneinsertion into any electron-rich bond. Oxidativecleavage releases the hydroxyl group, which isthen silyated to facilitate gaschromatography/mass spectrometry (GCMS)analysis. Tag diversity arises from both thelength of the alkyl chain (p) and the numberand arrangement of halogens on the aromaticring (Xq). PEG, polyethylene glycol; RT, roomtemperature; THF, tetrahydrofuran;TMS, tetramethylsilane.

PEG

Library

OO

N2

O OXqp

PEG

Library

OO

O OXqp

Rh2(O2CCPh3)EtOAc, RT, 30 min

Ceric ammonium nitrate1:1 THF/H2O, RT, 10 min

HO OXqp

OTMS

NTMS

EC-GC analysis

+

Current Opinion in Chemical Biology

Figure 2

New method for the attachment of tags topolystyrene resins (shown as a grey sphere)via Friedel–Crafts chemistry. Using thismethod, the tags are directed into the resinbackbone with no requirements for orthogonalprotecting groups. The tag portion can be anymoiety that can be cleaved and detected(exemplified with secondary amines here).Yb(OTf)3, ytterbium triflate.

O O

NH

OHTAG

O

Library

O O

NH

TAGO

Library

Yb(OTf)3

Current Opinion in Chemical Biology

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MicroarraysThe microarray concept was pioneered by the Affymax groupfor the photolithographic generation of spatially addressedpeptides and oligonucleotides [38]. More recently, the con-cept of small organic molecules on a chip has been applied tothe screening of compound libraries [39•]. Components ofthe library are covalently attached to a glass slide in a spatial-ly encoded way simply by spotting each compound intoknown locations, allowing evaluation of interactions with flu-orescently labelled proteins. The potential advantage is thescale required for microarray formation, which can allow hun-dreds of assays from a single bead whilst leaving enoughmaterial for characterisation. A potential limitation is theeffect of the linker and surface on binding interactions withthe target, as is sometimes the case for on-bead screening.

ConclusionsThe research into encoding and deconvolution during thepast 12–18 months has consisted mainly of continuedimprovement and consolidation of existing concepts tothe point where several methods have become routine.The improvements in analytical instrumentation haveprovided some enhancement in throughput and librarysize. Whilst there remains every possibility of a funda-mental breakthrough technology in the future, it wouldappear that for now there are a number of robust encod-ing or deconvolution approaches to suit most applicationsof combinatorial libraries. No doubt, further refinementswill broaden the utility of encoded libraries in the mole-cular sciences.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Francis MB, Jamison TF, Jacobsen EN: Combinatorial libraries oftransition-metal complexes, catalysts and materials. Curr OpinChem Biol 1998, 2:422-428.

2. Hoveyda AH: Catalyst discovery through combinatorial chemistry.Chem Biol 1998, 5:R187-R191.

3. Furka A, Bennett WD: Combinatorial libraries by portioning and• mixing. Comb Chem High Throughput Screen 1999, 2:105-122.This general review covers the whole area of one-bead-one-compoundlibraries and is a useful introduction to the available technology and concepts.

4. Schriemer DC, Hindsgaul O: Deconvolution approaches inscreening compound mixtures. Comb Chem High ThroughputScreen 1998, 1:155-170.

5. An HY, Haly BD, Cook PD: Discovery of novel pyridinopolyamineswith potent antimicrobial activity: deconvolution of mixturessynthesized by solution-phase combinatorial chemistry. J MedChem 1998, 41:706-716.

6. Szardenings AK, Harris D, Lam S, Shi LH, Tien D, Wang YW, Patel DV,Navre M, Campbell DA: Rational design and combinatorial evaluationof enzyme inhibitor scaffolds: identification of novel inhibitors ofmatrix metalloproteinases. J Med Chem 1998, 41:2194-2200.

7. Griffey RH, An HY, Cummins LL, Gaus HJ, Haly B, Herrmann R,Cook PD: Rapid deconvolution of combinatorial libraries usingHPLC fractionation. Tetrahedron 1998, 54:4067-4076.

8. Boger DL, Chai WY, Jin Q: Multistep convergent solution-phase•• combinatorial synthesis and deletion synthesis deconvolution.

J Am Chem Soc 1998, 120:7220-7225.A fundamental paper on a novel deconvolution technique related to posi-tional scanning. The use of sub-libraries with systematically omitted

monomers allows deconvolution of mixtures without the need for resynthesisor re-screening. The methodology has the potential to rapidly decode anylibrary synthesised using pooled mixtures of reagents.

9. Boger DL, Jiang WQ, Goldberg J: Convergent solution-phasecombinatorial synthesis with multiplication of diversity throughrigid biaryl and diarylacetylene couplings. J Org Chem 1999,64:7094-7100.

10. Lin MF, Shapiro MJ, Wareing JR: Diffusion-edited NMR — affinityNMR for direct observation of molecular interactions. J Am ChemSoc 1997, 119:5429-5250.

11. Bleicher K, Lin MF, Shapiro MJ, Wareing JR: Diffusion edited NMR:screening compound mixtures by affinity NMR to detect bindingligands to vancomycin. J Org Chem 1998, 63:8486-8490.

12. Anderson RC, Lin MF, Shapiro MJ: Affinity NMR: decoding DNAbinding. J Comb Chem 1999, 1:69-72.

13. Shapiro MJ, Wareing JR: NMR methods in combinatorial chemistry.Curr Opin Chem Biol 1998, 2:372-375.

14. Swali V, Langley GJ, Bradley M: Mass spectrometric analysis incombinatorial chemistry. Curr Opin Chem Biol 1999, 3:337-341.

15. Wells NJ, Davies M, Bradley M: Cleavage and analysis of materialfrom single resin beads. J Org Chem 1998, 63:6430-6431.

16. Hughes I: Design of self-coded combinatorial libraries to facilitatedirect analysis of ligands by mass spectrometry. J Med Chem1998, 41:3804-3811.

17. Wagner DS, Markworth CJ, Wagner CD, Schoenen FJ, Rewerts CE,Kay BK, Geysen HM: Ratio encoding combinatorial libraries withstable isotopes and their utility in pharmaceutical research. CombChem High Throughput Screen 1998, 1:143-153.

18. St Hilaire PM, Lowary TL, Meldal M, Bock K: Oligosaccharide• mimetics obtained by novel, rapid screening of carboxylic acid

encoded glycopeptide libraries. J Am Chem Soc 1998,120:13312-13320.

Use of a ladder sequencing approach for encoding large glycopeptidelibraries that, in principle, could be applied to any oligomers. Use of cappingreagents with unique masses to code for the monomers in each pot of thesynthesis can block a small proportion of the strands in a way that allowscomplete mass-spectrometric sequence analysis of the cleaved material.

19. Rahman SS, Busby DJ, Lee DC: Infrared and Raman spectra of asingle resin bead for analysis of solid-phase reactions and use inencoding combinatorial libraries. J Org Chem 1998, 63:6196-6199.

20. Hochlowski JE, Whittern DN, Sowin TJ: Encoding of combinatorialchemistry libraries by fluorine-19 NMR. J Comb Chem 1999,1:291-293.

21. Nestler HP, Bartlett PA, Still WC: A general-method for moleculartagging of encoded combinatorial chemistry libraries. J Org Chem1994, 59:4723-4724.

22. Taylor SJ, Morken JP: Thermographic selection of effective catalystsfrom an encoded polymer-bound library. Science 1998, 280:267-270.

23. Appell KC, Chung TDY, Solly KJ, Chelsky D: Biologicalcharacterization of neurokinin antagonists discovered throughscreening of a combinatorial library. J Biomol Screen 1998, 3:19-27.

24. Tan DS, Foley MA, Stockwell BR, Shair MD, Schreiber SL: Synthesis•• and preliminary evaluation of a library of polycyclic small

molecules for use in chemical genetic assays. J Am Chem Soc1999, 121:9073-9087.

A library of over 2 million compounds was fully encoded in a binary mannerusing carbene attachment chemistry. The tags are randomly incorporatedinto the resin backbone and are oxidatively cleaved after a screening proto-col. Material photoreleased from single beads was active against a numberof different biological targets, the structure of compounds being fully identi-fiable by simple gas chromatography/mass spectrometry (GCMS) analysisof the cleaved tags.

25. Ni ZJ, Maclean D, Holmes CP, Murphy MM, Ruhland B, Jacobs JW,Gordon EM, Gallop MA: Versatile approach to encodingcombinatorial organic syntheses using chemically robustsecondary amine tags. J Med Chem 1996, 39:1601-1608.

26. Schwarz MK, Tumelty D, Gallop MA: Solid-phase synthesis of• 3,5-disubstituted 2,3-dihydro-1,5- benzothiazepin-4(5H)-ones.

J Org Chem 1999, 64:2219-2231.Use of secondary amine tags to encode a library of modified benzothiaza-penes. The ability to undertake complex multi-step synthesis whilst retainingorthogonality with the protecting group chemistry required for tag introduc-tion expands the potential of this tagging technique.

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27. Fitch WL, Baer TA, Chen WW, Holden F, Holmes CP, Maclean D,• Shah NH, Sullivan E, Tang M, Waybourn P et al.: Improved methods

for encoding and decoding dialkylamine-encoded combinatoriallibraries. J Comb Chem 1999, 1:188-194.

Extensive development of the secondary amine methodology. The ability toanalyse tags in a quantitative way gives the potential for an expansion of‘binary’ encoding, thereby simplifying analysis and reducing the number oftags required. Improvements in the cleavage and analysis protocols are alsodescribed.

28. Lane SJ, Pipe A: A single generic microbore liquidchromatography/time-of-flight mass spectrometry solution forthe simultaneous accurate mass determination of compounds onsingle beads, the decoding of dansylated orthogonal tagspertaining to compounds and accurate isotopic difference targetanalysis. Rapid Commun Mass Spectrom 1999, 13:798-814.

29. Spikmans V, Lane SJ, Tjaden UR, van der Greef J: Automated capillaryelectrochromatography tandem mass spectrometry using mixedmode reversed-phase ion-exchange chromatography columns.Rapid Commun Mass Spectrom 1999, 13:141-149.

30. Boussie TR, Murphy V, Hall KA, Coutard C, Dales C, Petro M,Carlson E, Turner HW, Powers TS: Parallel solid-phase synthesis,screening, and encoding strategies for olefin-polymerizationcatalysts. Tetrahedron 1999, 55:11699-11710.

31. Scott RH, Barnes C, Gerhard U, Balasubramanian S: Exploring a• chemical encoding strategy for combinatorial synthesis using

Friedel-Crafts alkylation. Chem Commun 1999:1331-1332.Novel Friedel–Crafts chemistry for tag attachment directly into the resinbackbone. The need for orthogonal protecting groups in tag attachment isremoved and resin directed attachment should give cleaner libraries and amore controllable level of tag release.

32. Andres CJ, Swann RT, GrantYoung K, Dandrea SV, Deshpande MS:A novel cleavage protocol for use with Rf-encoded split poolsynthesis technology: product cleavage and collection in standard96 well format. Comb Chem High Throughput Screen 1999,2:29-32.

33. Guiles JW, Lanter CL, Rivero RA: A visual tagging process for mixand sort combinatorial chemistry. Angew Chem Int Ed 1998,37:926-928.

34. Yan B, Martin PC, Lee J: Single-bead fluorescencemicrospectroscopy: detection of self-quenching in fluorescence-labeled resin beads. J Comb Chem 1999, 1:78-81.

35. Scott RH, Balasubramanian S: Properties of fluorophores on solidphase resins; implications for screening, encoding and reactionmonitoring. Bioorg Med Chem Lett 1997, 7:1567-1572.

36. Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP:Semiconductor nanocrystals as fluorescent biological labels.Science 1998, 281:2013-2016.

37. Mitchell GP, Mirkin CA, Letsinger RL: Programmed assembly ofDNA functionalised quantum dots. J Am Chem Soc 1999,121:8122-8123.

38. Fodor SPA, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D: Light-directed, spatially addressable parallel chemical synthesis.Science 1991, 251:767-773

39. Macbeath G, Koehler AN, Schreiber SL: Printing small molecules as• microarrays and detecting protein-ligand interactions en masse.

J Am Chem Soc 1999, 121:7967-7968.Shows the potential of microarray technology for screening large numbers ofcompounds simultaneously on glass surfaces.

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