generating and analyzing combinatorial chemistry libraries

109

Generating and analyzing combinatorial chemistry libraries Paul Wentworth Jr* and Kim D Jandat

The past year has seen developments in key areas of combinatorial chemistry, including new encoding and deconvolution strategies, techniques for analyzing library components and alternatives to the obligatory solid-supports utilized for library construction. Highlights include the utility of radiofrequency encoding strategies and the expansion of solution-phase combinatorial synthesis.

Addresses Department of Chemistry (BCC582), The Scripps Research Institute and Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jo[la, CA 92037, USA *e-mail: [email protected] re-mail: [email protected] Correspondence: Kim D Janda

Current Opinion in Biotechnology 1998, 9:109-115

htt p://biomednet.com/elecref/0958166900900109

© Current Biology Ltd ISSN 0958-1669

Abbreviations MS mass spectrometry REC radiofrequency encoded combinatorial

I n t r o d u c t i o n Originally developed as a method for producing huge arrays of peptides for epitope mapping, combinatorial chemistry has evolved into a key discipline for medicinal chemists involved in drug research in the search for both new lead compounds and for lead compound optimization.

The major focus of combinatorial chemistry at present is not just the development of new methods for generating molecular diversity, for example the portion-mix (split and pool) randomization method pioneered by Furka e ta / . [1], which allows the rapid generation of libraries of many millions of compounds on polymer support or in solution. New efforts are being directed towards library design and synthesis [2*,3,4], analysis of reaction progress and products on solid-phase supports, encoding and deconvolurion strategies and alternative strategies to solid-supports for library construction. In this review we will cover recent advances in a number of these areas.

L i b r a r y a n a l y s i s s t r a t e g i e s Arguably the most challenging facet of combinatorial chemistry is structural determination of a particular member, or members, within the library. This is of importance not only for determining the structure of active components of a completed library, but also to allow monitoring of the reaction progress especially where one is applying new synthetic methodology for library construction. Four main strategies are being pursued:

direct analysis; deconvolution; encoding; and spatially addressable synthesis.

Direct analysis Peptides and oligonucleotides can be cleaved from individual beads and, following work-up, analyzed by standard techniques, such as Edman degradation or Sanger's dideoxy sequencing, respectively [5,6]. A theo- retical approach for probing combinatorial library diversity dubbed 'massively parallel' mass spectrometry (MS) has been recently reported [7]. A number of methods are being developed for solution-based combinatorial libraries linking purification of constituent library members, via ligand binding, to analysis by MS. One of these, affinity capillary electrospray MS, allows simultaneous selection and identification of candidate ligands for a predefined receptor [8]. Using a method based on affinity selection of ligands for key receptors, Kaur eta/. [9] have developed a technique that rapidly highlights and analyzes drug lead compounds.

The most commonly used method for generating chemical diversity is still the 'split and pool' approach [1] whereby a library is generated on solid support beads which are subjected to a chemical reaction, then split and subjected to a different chemical reaction and then pooled again, this process can be reiterated as necessary. A major benefit of this approach is that each bead can contain multiple copies of the same molecule [10]. Analytical techniques that require cleavage of a library member from the bead risk the loss of key members. Also, during a synthetic procedure it is laborious, after each reaction, to perform a cleavage step before analysis of a reaction's progress can be performed: therefore, analytical on-bead analysis methods are being developed. Utilizing a photolabile linker, Fitzgerald eta/. [11] demonstrated the feasibility of photolytic cleavage with matrix-assisted laser desorption/ionization mass spectrometry for the direct analysis of molecules bound on solid support.

Fourier transform infrared spectroscopy is a standard technique for rapid determination of structural features of organic molecules. Yan et al. [12,13"] have modified this process so that rapid structural determination of resin-bound molecules can occur. In addition, a reaction course can be followed in real-time. It is a generally accepted philosophy that reaction kinetics on solid-phase supports are slower than their solution-based counterparts; however, this Fourier transform infrared method revealed that a simple heterogeneous esterification reaction was faster on Merrifield resin (chloromethylated polystyrene, 1% cross-linked) than in solution [14]. While the nature and scope of the chemistry tested clearly needs to be expanded, this is the first compelling kinetic evidence to

1 10 Analytical biotechnology

suggest that a re-evaluation of the major criticism of solid phase chemistry may be necessar3:

An extension of this IR approach has included the use of deuterium tagged compounds [15]. The benefit of which include the fact that the C-D stretching mode absorbances occur in the frequency region (2,300-2,200cm -1) which is clear of most other functional group absorption bands. To study the utility of this deuterium tagging approach dg-BOC e-protected lysine derivatives, were reacted iteratively by standard Fmoc peptide chemistry to produce a series of solid-phase materials 1-4 (Figure 1).

Standard NMR methods do not allow for detcrmination of molecules bound to resin beads. Due to the essential heterogeneity of the system, sevcrc line broadening occurs caused by the restricted molecular motion of the molecules bound to the polymer beads. Magic angle spinning NMR overcomes this by rotating the gel-phase sample at very high frequency in a special rotor. High-resolution magic angle spinning NMR is, therefore, emerging as a routine method for on-bead analysis of solid phasc organic synthesis products [16,17]. Using a conventional solid-state probe, solution quality 1H and 13C NMR data can be collected for intermediates throughout a synthetic scheme [18].

Analysis between the absorbance ratios of the C-D derivative to background styrene derivative peak for 1-4 showed that there is a linear correlation with increasing numbers of dg-BOC groups, highlighting this strategy as a potential tagging approach for combinatorial library construction with broad application to bond formation, bond cleavage or deprotection strategies.

Deconvolution Deconvolution is the process by which components within a combinatorial library are structurally determined. Two main strategies have evoh'ed: iterative deconvolution and positional scanning. Iterative deconvolution involves screening of compound pools, identification of the ac-

Figure 1

H N - - B o c - d 9

i i ~ NH2 ii ii

O NH 2

Polys tyrene , 2 % cross l inked 1 H N - - B o c - d 9 i

o%d

-o- O oc,

4 ~ ! ~ ~ N H F m o c

HN \ Boc-d 9

HN--Boc-d 9

HN \ Boc-d 9

Current Opinion in Biotechnology

Preparation of d9-BOC protected peptides 1-4 for deuterium tagged IR experiments. (i) Fmoc-Lys(BOC-d9)-OH, DIC, HOBt; (ii) 200/0 piperidine. BOC, t-butyloxycarbonyl; DIC, diisopropylcarbodiimide; Fmoc, fluorenylmethyloxycarbonyl; HOBt, hydroxybenzotriazole.

Generating and analyzing combinatorial chemistry libraries Wentworth and Janda 1 11

tire pool(s), resynthesis, and rescreening of sublibraries (smaller pools). Originally designed to resolve oligopeptide and oligonucleotide libraries, recent developments have focused on small-molecule libraries driven by the need for new molecules of pharmaceutical relevance [19]. The time consuming nature of having to resynthesize and assay sublibraries and the potential inconsistencies during resynthesis have lead to a search for alternative deconvolution approaches.

Positional scanning involves each of the sublibraries being synthesized with one position defined beforehand. This approach eliminates the need to resynthesize and assay sublibraries but does not benefit from activity enrichment during the procedure. Recent utility of this procedure include the generation of a chemically diverse tetrapeptide library modified by either alkylation or reduction of the amide bonds [20], and for epitope mapping of a hepatitis B virus binding monoclonal antibody with a hexapeptide library comprising of >3x 107 members [21].

Pirrung eta/. [22] have developed a methodology termed indexing, a hybrid of iterative deconvolution and positional scanning requiring no iterative resynthesis of sublibraries. Libraries of carbamates and tetrahydroacridines, potential inhibitors of acetylcholine esterase, have been produced bv this approach.

Encoding To overcome some of the problems associated with analytical techniques and deconvolution (see above), a range of encoding, also dubbed tagging, strategies have been applied during combinatorial synthesis. The underlying principle is the association of a unique descriptor, either chemical or non-chemical (Ta, Tb, Te), with each bead when each building block (A, B, C) is added (Figure 2). The structure of the library member is determined by analysis of this tag.

Chemical encoding Oligonucleotide [23], peptide [24] and molecular tags for 'binary encoding' [251 have all been used for combinatorial library encoding. While molecular tagging solves some of the stability issues associated with the oligonucleotide and peptide encoding strategies, there still exists the problem of increased synthetic steps required to incorporate and ultimately release the tag for decoding.

A recent strategy involves isotopically labeling resin beads during combinatorial library construction [26]. The tags are encoded as a controlled ratio of a number of stable isotopes and range from single to complex isotopic distribution. The isotopes are cleaved after assay and analyzed bv automated MS.

Radiofrequency encoding As discussed above, microanalysis, chemical encoding and deconvolution all contribute to the elucidation of

Figure 2

Split Tag a ~

Ta Ta Ta Tb Tb Tb Tc Tc Tc

Ta Ta Ta Tb Tb Tb Tc To Tc

A A A B B B C C C

Pool

Split

Tag a / ~ Y a g c

J Tagb l

TaTa TbTa TcTa TaTb TbTb TcTb TaTc TbTc TcTc

A B C A B C A B C

~ ~T~Ta< BA CA i~~ABTaTB TbTbTc:TbBB i~ CB AcTa~c Tb To Tc:~CiBB, CC

"Fag cleavage and analysis (deconvolution)

TaTa AA


3 x 3 tagged combinatorial library generated by the portion-mix strategy. Ta, Tb and Tc are chemically distinct tags and A, B and C are building blocks.

structural elements in a combinatorial library: however, they all possess problems. A strategy designed to overcome these limitations incorporates a non-chcmical, non-inva- sire approach and is dubbed radiofrequency encoded combinatorial (REC) chemistry [27]. Using small micro- electronic semiconductors (e.g. microchips), rather than

1 12 Analytical biotechnology

a chemical label, relevant information along a synthetic pathway is recorded through radiofrequency transmission. The stored information is then retrieved at any time by a radiofrequency detector. A recent report has utilized REC chemistry in an Ugi four component condensation reaction [28 °] (Figure 3).

Figure 3

A 64 member library was generated from a linear 3 step synthesis incorporating the Ugi four component condensation reaction with four inputs (1-4) per step (Figure 3a). Polypropylene mesh bags, containing Wang polystyrene resin and a single transponder per bag, were used in order to associate resin with unique IDs. The

(a)

(b)

~ OH i ii

Wang resin

I rf scan and sort

rf scan and sort

d rf scan and sort (deconvolution)

Spatially encoded library

0

O NH2 iv

R1

OH

v vi

OR 3


(a) Three step linear synthesis of a 64-compound REC library, rf, radiofrequency. (b) Synthetic route to the 64-membered library: (i) Fmoc-amino acid, DIC, HOBt, THF; (ii) piperidine; (iii) R2CHO, BnNC; (iv) p-hydroxyphenylacetic acid; (v) R3CO2H, DCC, pyridine; (vi) TFA/CH2CI 2. DIC, diisopropylcarbodiimide; Fmoc, fluorenylmethyloxycarbonyl; HOBt, hydroxybenzotriazole; TFA, trifluoroacetic acid.

Generating and analyzing combinatorial chemistry libraries Wentworth and Janda 113

final products of a microchip-encoded library are single compounds, per well, whose structure is easily decoded from the histogram of each transponder [29].

Parallel synthesis Parallel synthesis is a method by which library construction occurs in distinct spaces on an array, such that each member of a library is defined by its position on that array. An inherent property of this methodology is that the structure of a library member can be deduced from its position on the array. Because of this method's suitability for automation and ease of deconvolution it is increasingly becoming the basis of many research groups' approach to library construction. A recent report documents the breadth and applicability of the Parke-Davis Diversomer ® instrument, an automation machine for parallel library construction. Facile and high yielding construction of a hydantoin, benzodiazepine and benzisothiazolone libraries on solid-phase has been achieved [30].

A further method of this methodology was recently reported by Virgilio et al. [31] who synthesized and evaluated a parallel library of 1152 peptidomimetics based upon the 13-turn structural motif utilizing the Chiron Mimitope pin apparatus [32]. The flexibility and orientation of the sidechain functionality was altered by introduction of different backbone aminoalkylthiols, so as to provide 9- or 10-membered rings, or by preparing different combinations of the absolute configurations of each stereocenter introduced by either the i+l or i+2 sidechains (Figure 4).

Figure 4

(a) . 0 . (b) O R(i L , ~ R(I+I) R (i+2) ~\ R (i+1)

0=~ / H NH 0 / .-.,,od

H2N


Structural motif of a (a) ~-turn and (b) the I~-turn mimetic library generated by EIIman and co-workers [31].

A l t e r n a t i v e s to s o l i d - p h a s e suppor ts Liquid-phase combinatorial synthesis Tremendous efforts are sometimes required to transfer chemistry that originated in solution to solid-phase supports, and new technologies have to be developed to analyze on-bead compounds (see above); therefore alternative strategies for library construction based upon soluble polymer supports are being explored [33°,34]. A number of reports of soluble polymer supports for

chemical synthesis have appeared and have recently been reviewed [35]. Based on the preliminary work of Bayer and Mutter [36], the first liquid-phase synthesis of a combinatorial library was a PEG-supported pentapeptide library [37"]. The library was screened for binding an anti [3-endorphin monoclonal antibody and library construction was routinely followed by 1H and 13C NMR. A small-molecule library of aryl sutfonamides, a well characterized pharmacophore, was also generated on liquid-phase. In the search for new biomimetic oligomers, a novel peptidomimetic, based on an azatide backbone synthesized on a liquid-phase support has recently been reported [38] (Figure 5).

Figure 5

M e O ~ F - O H

PEG5~0

R O R 3

H II I H H I O R2 O R4

Azatide scaffold

H O 2 C - ~ o B u t

MeO ~ O O - ~ ~ O H

O H t -Bo

iii ~-/ .~ O R,-n

F5

M e O ~ O \

O ~ O - ~ O R,-n NH--N

o~-°Su ,


Liquid-phase syntheis of polyazatides. (i) Dicyclohexylcarbodiimide (DCC), dimethylaminopyridine (DMAP); (ii) trifluoroacetic acid (TFA); (iii) dimethylaminopyridine (DMAP).

Azatides should be resistant to protease degradation as they possess the stereochemical orientation of the R-group at the oc-nitrogen atom intermediate between the L- and D-enantiomers of naturally occurring amino acids.

It should be stressed that the generation of molecular diversity within soluble polymer-supported libraries is sub- ject to the same principles as its solid-phase counterpart. That is, library generation can be performed via the portion-mix [1] strategy, as was demonstrated with the pentapeptide and acylsulfonamide libraries [37"]. There is no direct analogy, however, with the one bead one compound [10] strategy and, therefore, one is always screening library components as a mixture, with the associated problems of finding a 'needle in a haystack'. With the philosophy of library construction moving rather to a parallel spatial array format, however, soluble polymer

114 Analytical biotechnology

supports do not suffer from any significant disadvantages over their solid-phase cousins.

Solution-based combinatorial libraries Tile majority of small-molecule libraries generated to date have followed a solid-phase format for ease of isolation and purification of products, coupled with the ability to drive a reaction to completion by use of excess of reagents. l 'he solution phase, however, does have advantages: few reactions have bcen adapted to solid-phase, and some reactions are incompatible with the heterogeneous nature of insoluble polymer supports. With the driving demand for ever increasing molecular diversitx; chemists are modifying strategies to incorporate solution-based chemical synthesis of libraries.

A numbcr of approaches have been developed to address the problem of removing unwanted side-products and unrcacted starting materials from the mixture containing thc library required. Resin capture allows for library synthesis in solution with a subscquent transferral of the required products to a solid support for further transformation if necessary [39]. By this strateg'~; difficult to monitor or low yielding reactions can be performed in solution, and the subsequent reactions can be performed on solid support. This approach has been used fox the purification of a cych~hexanamide [40"] and a vinyl boronate library [411. A soluble-polymer supported reagent approach has been developed by Wentworth et a t [42]. A PEG-supported triphenyl phosphine reagent produced facile and efficient homogeneot, s Staudinger reduction of azidcs and Mitsunobu ether formation. The products were isolated by a simple process of precipitation of the spent rcagent by ether and filtration.

A recent strategy has targeted tile removal of excess rcagcnts by solid-phase quenching agents [43]. Resin-supported metlLvlisocyanate and tris(2-methylaminoethyl)an~ine have been used to neutralize excess solution-phase isocwmate and amine reagents respectively in an approach dubbed polymer-suppurted quench.

In an analagous approach Flvnn etaL [44"1 have developed a purification of solution-based libraries by principles of molecular reactivity and recognition. Specificall> parallel solution-phase reactions are purified by resins containing molecular recognition or molecular reactivity function- alities complementary to those of the solution-phase rcactants. In this way, solution-phase synthesis of amides, carbamatcs and sulfonamides lead to purities of >94 % aftcx simple filtration of the resin-reagents.

Conclusions The past year has seen significant strides in tile development of technologies for both the analysis of combinatorial library components and deconvoluting combinatorial libraries on solid-supports and strategies for solution or liquid-phase combinatorial synthesis. The ability to

routinely follow reaction progress on-bead should reduce the time taken to complete a given reaction scheme and minimize potential side-products formed due to extended reaction times.

New encoding strategies, such as isotope tagging and radio-frequency encoding seem to offer considerable advantages over the normally invasive and time consuming chemical tagging techniques. What still remains to be seen is whether the REC strategy can be successfully applied to a large library in a one-bead one compound format. The cost of the individual radiofrequency transponder may make this methodology prohibitive from an economics basis.

As more limitations arm found with solid-phase chemistry; and as library construction moves inextricably to a spatially addressable format, perhaps there will be a re-evaluation of the monopoly of solid-supports in combinatorial synthesis.

Acknowledgements Our work in this area has been supported by thc Ska tes Institute fur (~hcmical Bioh~gy.

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

• of special interest • • of outstanding interest

1. Furka ,g,, Sevestyen F, Asgedom M, Dibo G: General method for rapid synthesis of multicomponent peptide mixtures. Int J Protein Peptide Res 1991, 37:487-493.

2. Thompson LA, EIIman JA: Synthesis and applications of small molecule libraries. Chem Rev 1996, 96:555-600.

his is an excellent review which chronicles the recent expansion in small- molecule library development and solid-phase chemistry.

3. Fruchtel JS, Jung G: Organic chemistry on solid supports. Angew Chem Int Ed 1 996, 35:17-42.

4. Krchn&k V, Lebl M: Synthetic library techniques-subjective (biased and generic) thoughts and views. Mol Divers t 996, 1:193-216.

5. Lebl M, P&tek M, Kocis P, Krchn&k V, Hruby VJ, Salmon SE, Lam KS: Multiple release of equimolar amounts of peptides from a polymeric carrier using orthogonal linkage-cleavage chemistry. Int J Peptide Protein Res 1993, 41:201-211.

6. Weinberger H, Lichte E, Griesinger C, Kutscher B: Small peptide libraries-combinatorial split-mix synthesis followed by combinatorial amino acid analysis of selected variants. Archly der Pharmazie 1997, 330:109-111.

7 Demirev PA, Zubarev RA: Probing combinatorial library diversity by mass spectrometry. Anal Chem 1997, 69:2893-2900.

8. Chu YH, Dunayeskiy YM, Kirby DP, Vouros P, Karger BL: Affinity capillary electrophoresis-mass spectrometry for screening combinatorial libraries. J Am Chem Soc 1996, 118:7827-7835.

9. Kaur S, Meguire L, Tang DZ, Dollinger G, Huebner V: Affinity selection and mass spectrometry-based strategies to identify lead compounds in combinatorial libraries. J Prot Chem 1997, 16:505-511.

10. Lain K, Lebl M, Krchn&k V: The 'one-bead-one-compound' combinatorial library method. Chem Rev 1997, 97:411-448.

11. Fitzgerald MC, Harris K, Shevlin CG, Siuzdak G: Direct characterization of solid-phase resin-bound molecules by mass spectrometry. Bioorg Med Chem Lett 1996, 6:979-982.

12. Yah B, Kumaravel G, Anjaria H, Wu A, Petter R, Jewell CF Jr, Wareing JR: Infrared spectrum of a single resin bead for real-

Generating and analyzing combinatorial chemistry libraries Wentworth and Janda 115

time monitoring of solid-phase reactions. J Org Chem 1995, 60:5736-5738.

13. Yan B, Kumaravel G: Probing solid-phase reactions by = monitoring the IR bands of compounds on a single 'flattened'

resin bead. Tetrahedron 1996, 52:843-848. This article describes a routine and general method for analyzing reaction progress, in real-time, on solid-phase.

14. Yan B, Fell JB, Kumaravel G: Progression of organic reactions on resin supports monitored by single bead FTIR microspectroscopy. J Org Chern 1996, 61:7467-7472. •

15. Russell K, Cole DC, McLaren FM, Pivonka DE: Tools for combinatorial chemistry. In Molecular Diversity and Combinatorial Chemistry Libraries and Drug Discovery. Edited by Chaiken IM, Janda KD. Washington DC: ACS Conference Proceeding Series; 1996:246-254.

16. Fitch WL, Detre G, Holmes CP, Shooreley JN, Keifer PA: High- resolution 1H NMR in solid-phase organic synthesis. J Org Chem 1994, 59:7955-7956.

17. Anderson RC, Jarema MA, Shapiro M J, Stokes JP, Ziliox M: Analytical techniques in combinatorial chemistry: MAS CH correlation in solvent-swollen resin. J Org Chem 1995, 60:2650-2651.

18. Dhalluin C, Pop I, D6prez B, Melnyk P, Tartar A, Uppins G: Magic- angle spinning NMR spectroscopy of polystyrene-bound organic molecules. In Molecular Diversity and Combinatorial Chemistry Libraries and Drug Discovery• Edited by Chaiken IM, Janda KD. Washington DO: ACS Conference Proceeding Series; 1996:256-272.

19. Gordeev MF, Patel DV, Gordon EM: Approaches to combinatorial synthesis of heterocycles: a solid-phase synthesis of 1,4- dihydro-pyridines. J Org Chem 1996, 61:924-928.

20. Houghten RA, Blondelee SE, Dooley CT, Dorner B, Eichler J, Ostresh JM: Libraries from libraries-generation and comparison of screening profiles. Mo/Divers 1996, 2:41-45.

21. Appel JR, Muller S, Benkirane N, Houghten RA, Pinilla C: Highly specific cross-reactive sequences recognized by an anti- hbsag antibody identified from a positional scanning synthetic combinatorial library. Peptide Res 1996, 9:174-182.

22. Pirrung MC, Chau JH-L, Chen J: Indexed combinatorial libraries: nonoligomeric chemical diversity for the discovery of novel enzyme inhibitors. In Combinatorial Chemistry: Synthesis and Appfication. Edited by Czarnik AW, Wilson SR. New York: John Wiley & Sons; 1997:191-206.

23. Nielsen J, Brenner S, Janda KD: Synthetic methods for the implementation of encoded combinatorial libraries. J Am Chem Soc 1993, 115:9812-9813.

24. Youngquist RS, Fuentes GR, Lacey MP, Keough T: Generation and screening of combinatorial peptide libraries designed for rapid sequencing by mass spectroscopy. J Am Chem Soc 1995, 117:3900-3906.

25. Ohlmeyer MHJ, Swanson RN, Dillard LW, Reader JC, Asouline G, Kobayashi R, Wigler M, Still WC: Complex synthetic chemical libraries indexed with molecular tags. Proc Nail Acad Sci USA 1993, 90:10922-10926.

26. Geysen HM, Wagner CD, Bodnar WM, Markworth C J, Parke G J, Schoenen FJ, Wagner DS, Kinder DS: Isotope or mass encoding of combinatorial libraries. Chem Biol 1996, 3:679-688.

27. Nicolaou KC, Xiao X-Y, Parandoosh Z, Senyei A, Nova MP: Radiofrequency encoded combinatorial chemistry. Angew Chem Int Ed Engl 1995, 34:2289-2291.

28. Armstrong RW, Tempest PA, Cargill JF: Microchip encoded • combinatorial libraries-generation of a spatially encoded library

from a pool synthesis. Chimia 1996, 50:258-260. Radiofrequency encoding of beads may potentially solve most of the problems of library deconvolution. This article describes its application in the construction and analysis of a 64 membered library

29. Armstrong RW, Brown DS, Keating TA, Tempest PA: Combinatorial synthesis exploiting mutiple-component

condensations, microchip encoding, and resin capture. In Combinatorial Chemistry: Synthesis and Application. Edited by Czarnik AW, Wilson SR. New York: John Wiley & Sons; 1997:1 ?7- 180.

30. Hobbs Dewitt S, Czarnik AW: Parallel organic synthesis using Parke-Davis Diversomer technology. In Combinatorial Chemistry: Synthesis and Appfication. Edited by Czarnik AW, Wilson SR. New York: John Wiley & Sons; 1997:25-38•

31• Virgilio AA, Bray AA, Zhang W, Trinh L, Snyder M, Morrissey MM, Ellman JA: Synthesis and evaluation of a library of peptidomimetics based upon the ~-turn. Tetrahedron 1997, 53:6635-6644.

32. Maeji N J, Valerio RM, Bray AM, Campbell RA, Geysen HM: Grafted supports used with the multipin method of peptide- synthesis. Reactive Polymers 1994, 22:203-212.

33. Gravert D J, Janda KD: Organic synthesis on soluble polymer • supports: liquid-phase methodology. Chem Rev 1997, 97:489-

509. This review describes the evolution of soluble-polymer supports in organic synthesis and combinatorial chemistry. As more limitations become appa- rant with solid-phase polymer supports, liquid-phase alternatives may offer a number of potential advantages.

34. Vandersteen A, Han H, Janda KD: Liquid-phase combinatorial synthesis: in search of small-molecule enzyme mimics. Mol Divers 1996, 2:89-96.

35. Geckeler KE: Soluble polymer supports for liquid phase synthesis. Biopolymers 1995, 35:32-79.

36. Bayer E, Mutter M: Liquid-phase synthesis of peptides. Nature 1972, 237:512-513.

3?. Han H, Wolfe MM, Brenner S, Janda KD: Liquid-phase • combinatorial synthesis. Proc Nat/Acad Sci USA 1995,

92:6419-6423. This article is the first to report a soluble-polymer supported combinatorial library and opens up a whole new area of combinatorial chemistry research.

38. Han H, Janda KD: Solution and liquid-phase synthesis of a new peptidomimetic. J Am Chem Soc 1996, 118:2539-2544.

39. Armstrong RW, Brown DS, Keating TA, Tempest PA: Combinatorial synthesis exploiting mutiple-component condensations, microchip encoding, and resin capture. In Combinatorial Chemistry: Synthesis and Application. Edited by Czarnik AW, Wilson SR. New York: John Wiley & Sons; 1997:180- 186.

40. Keating TA, Armstrong RW: Postcondensation modifications of • the Ugi four-component condensation products: 1-isocyano

-cyclohexene as a convertible isocyanide. Mechanism of conversion, synthesis of diverse structures, and demonstration of resin capture. J Am Chem Soc 1996, 118:2574-2583.

This article is the seminal report outlining solution-based synthesis followed by solid-phase purification- resin-capture.

41. Brown SD, Armstrong RW: Synthesis of tetrasubstituted ethylenes on solid support via resin capture. J Am Chem Soc 1996, 118:6331-6332.

42. Wentworth P Jr, Vandersteen AM, Janda KD: Poly(ethylene glycol) (PEG) as a reagent support: the preparation and utility of a PEG-triarylphosphine conjugate in liquid-phase organic synthesis (LPOS). Chem Commun 1997:759-760.

43. Booth R J, Hodges JC: Polymer-supported quenching reagents for parallel purification. J Am Chem Soc 1997, 119:4862-4886.

44. Flynn DL, Crich JZ, Devraj RV, Hockerman SL, Parlow J J, South • MS, Woodward S: Chemical library purification strategies

based on principles of complementary molecular reactivity and molecular recognition. J Am Chem Soc 1997, 119:4874-4881.

With the expansion in solution-based combinatorial synthesis new methods are required for purification of unwanted components of mixtures• This article describes an elegant approach utilizing resin-supported reagents to trap these impurities via a range of methodologies.

generating and analyzing combinatorial chemistry libraries

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