Proc. Nad. Acad. Sci. USAVol. 91, pp. 10779-10785, November 1994

Review

Tagged versus untagged libraries: Methods for the generation andscreening of combinatorial chemical librariesKim D. JandaDepartments of Molecular Biology and Chemistry, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037

ABSTRACT Over the past two dec-ades the pharmaceutical industry has beendriven by the biological sciences. The dis-covery and description of the biologicalmechanisms that underlie disease statesaccompanied by an unraveling of thesemechanisms has provided drug, and morerecently biotechnological, companies witha barrage of new therapeutic targets. Par-adoxically, as a result of such biologicaland biochemical advances, new sources ofdrug leads are in short supply. Consider-able effort in trying to create potentialdrug candidates has led to the parturitionof combinatorial chemical libraries. In thisreview I will examine some of the maintechnologies for generating and deducingactive components from combinatorial li-braries that have been segregated into twoschools of thought: (i) the creation anddecoding of combinatorial libraries by so-called tagged methodologies, and (it) theproduction and deconvolution of chemicallibraries by untagged protocols.

The random screening of natural prod-ucts from microbial fermentations, plantextracts, and marine sources for possibleactivity as therapeutic agents has been arich source of new drug discoveries foryears in the pharmaceutical sciences (1).However, with the advent of molecularbiology and progress in crystallographyand computational chemistry, "rationaldrug design" has found many advocates.The knowledge of the three-dimensionalstructure ofreceptors or enzymes and themanipulation of this information has leadto the development of a number of drugcandidates including angiotensin con-verting enzyme (2, 3) and renin inhibitors(4-6). But even such "rational" successstories, initiated from first principles andbased solely on structural informationabout the macromolecule alone, have notproved as fast or reliable as once claimed.In recent years there has been a renais-sance in drug screening with an ingress ofnew technologies based on combinatorialchemical libraries (for review, see ref. 7).These methods expose many compoundsto a target and allow the compounds thatbind the target with the highest affinity tobe filtered out from a pool of statisticalsequences. In its purest form, a combi-natorial chemical library can be defined

as any ensemble of molecules, whereasthe production and ultimately the meth-ods of screening these combinatorialchemical libraries determine the "hitrate"-i.e., the success or failure ofthese collections of molecules (8).

Untagged Approaches to GeneratingCombinatorial Chemical Libraries

The two general procedural methodolo-gies in formulating untagged combinato-rial libraries are "mixture synthesis" and"portioning-mixing." Mixture synthesisis exactly what it sounds like, in thatmixtures of chemical units are coupled toan activated support to produce chemicaldiversity (9-12). The advantage of suchan approach is that combinatorial librar-ies of vast complexity, in theory, shouldbe accessible. The problem of achievingsuch diversity is that product dispersal isstrongly influenced by the relative kineticrates of each competing chemical unitbeing coupled. Deviating from reactionswhere the kinetic constants for the addi-tion of each individual component areunknown could be disastrous to the prod-uct distribution. Portioning-mixing (13-15), or what has come to be known ascombinatorial library split synthesis, is atwo-step operation based on a divide-couple and recombine procedure. Theessentials of this strategy are that a poly-meric support is divided into equal por-tions for coupling to modular units [suchunits have typically been amino acids;however, in principle, any chemical moi-ety (say X) could be appended to thesupport]. The matrices are combined in asingle vessel for washing and/or depro-tection and then divided again for thenext coupling. Repeating this protocolfor a total of n cycles can produce astochastic collection of up to Xn differentmolecules. More important, this strategyallows for an equal distribution of thecoupled chemical units and for uniformcouplings to occur.

Various approaches for generating andscreening untagged combinatorial chem-ical libraries have been developed. Eachhas advantages and disadvantages in itsefforts to create chemical diversity. Sev-eral of the most prominent will be con-sidered below.

Mimotope Strategy

Geysen and coworkers (11, 16, 17) havepresented an amide linkage strategywherein peptides can be synthesized in areusable format. The peptides are syn-thesized on polymer matrices, "pins,"positioned within a microtiter 96-wellplate. In this approach, natural L-aminoacids, as well as unnatural D-amino acids(we will term either enantiomer X), canbe incorporated into the library; thus, XApeptides are conceivable. However, tomanage such large numbers with this pinformat dipeptide units are initially heldconstant, whereas the rest of the se-quence is formed from randomly incor-porated amino acids. To synthesize ahexapeptide library in this regime re-quires NNX[3]X[4]NN, where now X[31and X[4]* are defined amino acids at po-sitions 3 and 4, respectively, and N rep-resents positions where residues are ran-domly incorporated by using reactionmixtures containing natural or unnaturalamino acids. If a primary set is madeusing an alphabet of 20 amino acids, thenthe size of this set will be 400 (20 x 20preparations), each of which is now amixture of peptides theoretically consist-ing of 160,000 different peptides. Thederivatization level will be at =z100 nmolper pin or an average of -4 x 1011 copiesof each of the individual peptides.Examining this primary set of 400 pep-

tide mixtures for binding with an antibodyor receptor allows identification of theoptimum dipeptide sequence for X[3JX[4J.A second screen (40 peptide mixtures)NNX[3JX[41D[5JN and ND[2JX[3JX[4JNN,wherein the amino acids X[3JXM4J are fixedand D equates to positions where singleamino acids are to be incorporated, allowsresolution or absolute identification ofad-ditional residues. This cycle of synthesisand screening is reiterated until the entirehexapeptide sequence is optimized forbinding to the target of interest.

Originally this mimotope strategy wasused to determine discontinuous epitopeswith a protein antigen (12). Recently, itsscope has been expanded to include thescreening of other relevant receptors

*Subscripts in brackets throughout text rep-resent residue positions rather than multi-ples.

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Lithogrphic *Mas I IL ' I

t tNVOC NVOC NVOCI I I

H-N H-N H-N

0 H

HOAXI11-N-NVOCCoupling

l ho

a ,I ,

MASK t f L

NVOC

H-N

--I

NVOC NVOCI I

H-N H-NI I

NH NH

0 H

HOAX121-N-NVOCCoupling

Deprotechou

NVOC

H-N NH2 NH2

NVOC NVOCI

H-N H-N

NVOC 0 x11, o X111H-N NH NH

I I IX[n] represents an amino acid whileNVOC represents the 3,4-Dimethoxy-6-nitrobenzyloxycarbonyl appendage

DeprotectionW.

ho

NVOCH-N

O I

NH

NVOCH-N

HNH

I

NVOCH-N

lO=<XIN

NH2 NH

NH2HI

NH

NVOCH-N

0HN

NH

FIG. 1. Scheme of how peptide combinatorial libraries can be constructed using thelight-directed, spatially addressable parallel chemical synthetic approach developed by Foderet al. (19).

(18). The positive aspects of the mimo-tope scheme are that modular syntheticstrategies are ultimately used in second-ary, tertiary, etc. . . . screens. The li-brary diversity can also be increased byapplying a-disubstituted and P-amino ac-ids to the process.

Light-Directed, Spatially AddressableParallel Synthesis of CombinatorialLibraries

next NVOC-protected amino acid or oli-gonucleotide, wherein coupling of eitherunit occurs only in the regions exposed tolight. The procedure is repeated until allthe building blocks are coupled to thesupport. The pattern of masks and thesequence ofreactants define the productsand their locations. In other words, theidentity of the sequence and its locationare known.

Screening for activity within these syn-thesized libraries is done via a fluores-cent reporter-group assay, wherein a flu-orescently tagged receptor or enzyme isallowed to interact with the library. Thefluorescent intensity at each site will de-pend on the affinity ofthe receptor for thecompound, the concentration of the re-ceptor, and the number and density ofthesites resulting in the binding event.The strengths of this approach are that

high-density arrays of chemical com-pounds can be created in a very smallarea. A 10-step binary synthesis withamino acids results in the formation of1024 peptides in 1.6 cm2. Furthermore,the chemistry is not limited to just pep-tide and oligonucleotide synthetic strat-egies. As evidence, the Schultz group(20) has extended the Fodor technologyto an oligocarbamate library (Fig. 2).

One-Bead, One-Peptide Solid-SupportTechnology

A one-bead, one-peptide combinatoriallibrary using split synthesis was pio-neered in a collaborative effort by work-ers at the University of Arizona and theSelectide Corporation (21). Their ap-proach is defined by a noncleavablelinker moiety that attaches the peptideunit to the bead. Library screening isaccomplished by using receptors conju-gated to fluorescein or an enzyme; eitherentity distinguishes an association eventbetween relevant library members and anacceptor molecule from the rest of thepopulation. Individual library memberbeads that are stained by this process areremoved and analyzed by Edman micro-sequencing to deduce the sequence ofthecorresponding peptide ligand.Using this methodology, the Tucson

group has synthesized a 19-amino acid(less cysteine) pentapeptide library(2,476,099 members). The library wasscreened against a monoclonal antibodyknown to bind the f3-endorphin epitope(YGGFL) with a Kd of 17.5 nM. The

Fodor and his colleagues at Affymax (19)have demonstrated a technology that in-tertwines solid-phase synthesis with pho-tolithography in the, preparation of pep-tide and oligonucleotide combinatoriallibraries (Fig. 1). Their general procedurewas to derivatize an aliphatic amino-terminated matrix with the light-sensitiveamino-protecting group 3,4-dimethoxy-6-nitrobenzyloxycarbonyl (NVOC). Pho-tolysis of the surface removes the pro-tecting group and thereby activates thearea for further synthesis. The syntheticscaling process that ensues depends onthe photolithographic masking patternused. Thus, after photolysis (deprotec-tion) the entire surface is exposed to the

Y0 R2 H 0 R4

Rr R3

Peynideb

FIG. 2. A peptide and the corresponding oligocarbamate. R represents any standard aminoacid side chain.

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antibody 3E7 conjugated to alkalinephosphatase retrieved six new pentapep-tide-binding sequences with Kd valuesfrom 15 to 8780 nM.The power of this one-bead, one-

peptide split synthesis concept lies in thefact that any solid-phase chemistry cantheoretically be applied and investigatedin a receptor-based and/or enzyme as-say. However, this advantage also leadsto drawbacks, one ofwhich is that librar-ies must be screened attached to thesupport. Subsequent work has been di-rected at using multicleavable linker ap-pendages to allow for screening of solu-ble peptide mixtures (22).

Dual-Defined, Positional Snning, andRobotis Library Technokogy

Houghten et al (23-25), using what theytermed a "dual-defined iterative" meth-odology, have assembled soluble combi-natorial peptide libraries via split synthe-sis. In the seminal paper (23) a dual-defined hexapeptide library containing 18amino acids was constructed as follows:Four cycles using solid-phase peptide syn-thesis and "portionig-mixing" provided104,976 protected tetrapeptide resin se-quences (184). This partial library ofNNNN-resins (where N is a randomizedamino acid position) was divided into 324aliquots, so that the synthesis of the nexttwo positions X[lXrt2J NNNN-resin couldbe defined (X is an amino acid positionthat is defined; the bracketed subscriptindicates residue position); upon depro-tection and cleavage from these respec-tive resins, a now complete solublehexapeptide library of >34 million mem-bers was obtained. These 324 pools areassayed, and positive results for the firsttwo residues (say A[l]B[21) were noted.Next, 18 new libraries were synthesizedwith the formula AtI]B[2jX133NNN, one foreach amino acid at position 3, and testedto define X[3]. The process is repeateduntil all positions are defined. Essentially,this methodology is an iterated searchprocess that consists ofmaking the libraryin a number of segregated pools, findingthe active pool that defines the entity forthe position on the molecule, and thenrepeating the process until the active com-ponent has been identified. A similar iter-ative process called SURF (synthetic un-randomization of randomized fragments)was used by ISIS Pharmaceuticals (Carls-bad, CA) for an oligonucleotide library(26).A virtue of this dual-defined iterative

technology is that the multiplicity ofcom-ponents decreases with each step, so thatan enrichment process occurs, and be-cause molecules can be assayed in solu-tion, it permits functional, as well asbinding, assays. An application of thisiterative strategy includes the discoveryof antimicrobial peptides with activities

against Gram-negative and Gram-posi-tive bacteria (23, 24).The positional scanning format is again

based on soluble combinatorial libraries;however, its proof of concept has beenshown to be viable using mixture synthe-sis (27, 28); now for the same hexapeptidelibrary of 18 amino acids, six differentlibraries (i.e., 108 positional scanningsublibraries) of the general formulaX[lIN[2]N[3]N[4JN[5]N[61, N[l]X[2]N[31-NM4JN 5JN[6J, N[lJN[2]XX[3N[4JN[s N[6J,N[,lN[2]N[3]X[4]N[51N[6], N[1]N[2]N[31-N[41X[,N[61, and N[l]N(2]N[3JN(4]N[5JX[6]must be synthesized and assayed. Thistechnology defines the preferred residueat each position of the sequence. Thetechnology also alleviates the unwieldyiterative synthesis and selection stepsrequired in the dual-defined methodol-ogy. However, this strategy, unlike thedual-defined methodology, is not en-dowed with an enrichment process or aprogressive improvement of the signal-to-noise ratio.The Chiron group has developed a fully

automated peptide synthesizer that al-lows combinatorial peptide libraries to becreated in the split-synthesis format (29,30). The instrument consists of an arrayof reaction vessels, solenoid valves, anda Zymark robot that is computer con-trolled. The use of this instrument wasshown by synthesizing a 361-memberdecapeptide library. Through competi-tive ELISA and an affinity-selectionmethodology decapeptide library mem-

p(1)A .a Siave EA

2/3

Couple with: A

p(2)A Save I N[11A[2j I

1/2

Couple with: A

L N 11N[2A[31 |

bers were identified that bound an anti-gpl20 monoclonal antibody.

A Recursive Deconvolution Strategy

The final "nontagged" combinatorial li-brary methodology to be described is thatof my group (31). The essence of ourmethod is to build and hold a set of par-tially synthesized combinatorial libraries.A formal example ofour method is shownin Fig. 3 for a library of degree 3, madefrom an alphabet ofthree components, A,B, and C. In our process we define threechannels of synthesis, and each involvesonly the addition of a single component.Initiating the process requires the makingof three pools, in which A, B, and C areadded to a matrix adapted with alinkerforcoupling of the components. A portion ofthis library is set aside and labeled aspartial library p(l). This portion or frac-tion to be saved and catalogued is theinverse of the degree of the library in thefirst step. Hence, in the example pre-sented, a degree of three, one-third ofeach pool is saved and labeled; for sub-sequent steps the amount saved and cat-alogued is the inverse of degree minusone. The remaining material is combinedand separated into three portions, eachchannel is loaded, and A, B, and C areattached as before. Again, an aliquot ofthis library is set aside as partial libraryp(2), which now is three pools made up ofN[tlA(2], N[11B[2j, and N[lJC[2]. The re-mainder is again pooled and split, and the

B - p(l)B

2/3

mbine, Randomize, and Split

B

- 2

| N~1B[2] S_

11/2

[i _p(1)C

2/3

p(2)B |_N1JC[21 |Sa p(2)C

1/2

Dmbine, Randomize, and Split

Nt1tN[2]B[3 1 I NI1JN[21C[3J |

FIG. 3. General scheme of how a chemical combinatorial library using the concept ofrecursive deconvolution is synthesized and cataloged. Each final pool contains 9 molecules;there is a total of 27 unique molecules in this example. Subscripts denote residue position.

Co

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third step of addition is carried out to givethe final library N[11N[21A[31, N[11N[2]B[31,and N[11N[2]C[31-

Screening against a receptor, ligand, oreven an enzyme and then determining themost active library member(s) is donesimply by examining the final three poolsfirst and proceeding backward to the par-tial libraries saved. Thus, for the examplepresented in Fig. 3, we have three pools,N[11N[2]X[31 (X is A, B, or C), nine com-pounds in each pool (i.e., a total of 27different compounds), which are tested byan appropriate assay, and the active poolis determined. Suppose N[l]N[2]B[3] fromthe final library shown in Fig. 3 is positive.We then go back to library p(2) and add Bto an aliquot of each of the three pools,P(2)A, P(2)B, P(2)C, to give three newlibraries of the general formula N[11X[21-B[31. These three new libraries now con-tain only nine components, so a 3-foldenrichment has been achieved. Again, af-ter testing, suppose pool N[11A[21B[31 isactive. We proceed to partial library P(1)and add A to each followed by B to givethree new pools with the structureX[11A[2,B[31, which can be tested to findX[11. Again, a 3-fold enrichment has beenachieved; the structure is synthesized,and the sequence is deduced.We have examined this recursive de-

convolution in a peptide combinatoriallibrary that was tailored to contain penta-peptide sequences that display binding tothe commercially available anti-f3-endor-phin monoclonal antibody 3E7. In thefinal analysis, the native epitope YGGFLwas found to be the most extensivebinder; however, other weaker binderswere also deduced through this strategy(31).There are a number of advantages in

using this recursive deconvolution strat-egy. (i) Split synthesis, a rather cumber-some process, need only be done once foreach combinatorial library made. In starkcontrast is the dual-defined method,which requires numerous split syntheticoperations. (ii) The deconvolution pro-cess recursively defines the synthesis ofthe active component, so that in the lastcycle, the active compound is synthe-sized. In addition, this methodology al-lows the deduction of alternative activemembers, as each deconvolution pathwaycan be followed either in parallel or suc-cessively. (iii) Any chemistry is applicablewith this technology, which, as we willsee, can be problematic in generation ofencoded combinatorial libraries.

Tagged Methodologies to GeneratingCombinatorial Chemical Libraries

Evident from the discussion presented isthat there are three critical aspects in anycombinatorial library: (i) The chemicalunits that go into the library, (ii) the tech-nique for generating the library, and (iii)

identification of library members that in-teract with the biological target ofinterest.Although these three points have beenaddressed in several of the "untagged"combinatorial library protocols de-scribed, limitations do exist. In this nextsection alternative strategies will be pro-vided, which are termed "tagged" com-binatorial libraries.

Phage Technology

Arguably, one of the most powerful tagtechnologies is that of strictly biologicalorigin. The general concept is one inwhich a library of peptides is presentedon the surface of a bacteriophage suchthat each phage displays a unique peptideand contains within its genome the cor-responding DNA sequence (32-34). Indetail, foreign DNA can be inserted intothe minor coat protein locus (gene III) offilamentous phage to create fusion phagethat express these corresponding pep-tides at the N terminus of the absorptionprotein (pIll), which is displayed on thephage surface (Fig. 4). The diversity ofdisplayed peptides is generated by clon-ing randomly synthesized oligonucleo-tides that are inserted into a specificregion of gene III. These phage librariesencoding peptide units can possess asmany as 109 unique sequences, which canbe screened for binding to any type ofimmobilized receptor in a selection pro-cess known as "panning." This methoduses an affinity capture technique to se-lect peptide display phage that bind to thereceptor of interest. Selected phages areamplified by infecting E. coli, whereaseach cycle of panning and amplification

Helper Phage

enriches certain peptide display phagesequences that bind most tightly to thereceptor molecule. After the panningprocess, DNA from the isolated phage issequenced, and the peptide responsiblefor binding is elucidated.The application of phage technology to

the binding of receptors (antibodies) hasbeen demonstrated by several groups(35, 36), including Smith's group (34),who have shown that an epitopehexapeptide library (-107 members) dis-played within the pIII protein could pro-vide individual members that bound totwo different antibodies that were specif-ically made to the DFEKI peptide unitfound on the surface of myohemerythrin.In a library designed by Devlin et al. (37)phage display epitope libraries with 15,rather than 6, amino acids were made.This approach not only increases theeffective size of the library, it also allowsthe possibility of the display of discon-tinuous epitopes.The strengths of phage technology are

that large combinatorial libraries of pep-tide fragments can be generated veryquickly and efficiently. Of greater impor-tance is that these peptide libraries arephysically linked to their own encodingtag (DNA), which allows for user-friendly amplification, enrichment, anddecoding of pertinent binding sequences.At first glance, this methodology seemsvery appealing; however, a glaring weak-ness inherent in this technology and other"genetic" library methodologies is thatthe overall diversity of the chemical unitsthat can be applied within these systemsis finite. Exploitation of these librariesfor drug discovery may thus be limited.

mExctrusion

Periplasmic pillSpace-

LibraryPhage DNA

Phagemid

FIG. 4. Scheme illustrating the proposed pathway for peptide display on filamentousbacteriophage. Helper phage infects Escherichia coli cells harboring phagemid DNA thatcontains the genes for the peptide libraries. Helper phage DNA is used to express native pIll,whereas phagemid DNA is used to express peptide-pIll. Single-stranded (ss) phagemid DNAis packaged into phage particles through the aid of helper phage-encoded proteins.

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Peptides-on-Plasmids

An alternative to phage technology, whichalso relies on a biological tack is a geneticcombinatorial library approach termed"peptides-on-plasmids." What Schatzand his coworkers at Affymax (38) havedone is establish another efficient meth-odology for a physical connection be-tween a peptide and nucleic acid thatencodes for it. In their procedure, a li-brary ofpeptides is constructed so that thegenetic material encoding them is linkedthrough the DNA-binding activity of thelac repressor protein. The random pep-tides are fused to the C terminus of therepressor by cloning degenerate oligonu-cleotides at the 3' of the repressor gene(lac I) present on a plasmid. This plasmidalso has lac repressor-binding sites, so thefusions bind the same plasmid that en-codes them. Assay for peptide-receptorbinding in the Schatz approach is quitesimilar to the panning process in phagemethodology; however, now cell lysis isused to liberate the peptide-lac-plasmidcomplexes that are screened repeatedlyfor affinity enrichment.A proof of principle of this work came

from the construction of a random dodec-amer library. This library was used toprobe for potential peptide members thatcould bind to IgG D32.39, a specific hy-bridoma to the dynorphin B epitope. Asexpected, a consensus sequence was dis-covered that corresponded to similarbinding studies of phage libraries (35).More recently, this peptides-on-plasmidapproach has been extended to a morechallenging system-the discovery ofnewsubstrates for E. coli biotin haloenzymesynthetase. The results were that smallerpeptide units (13-residue sequences)could be used as substrates (39).Two features distinguish the peptides-

on-plasmid approach from phage tech-nology. (i) Random peptides are dis-played with a free C terminus. (ii) Therepressor fusions are cytoplasmic,whereas phage fusions are periplasmic.However, as with phage technology thismethodology is restricted in its repertoireof chemical building blocks to oligopep-tide units.

Peptide Coded Libraries

In an attempt to address the diversityproblem inherent in genetic libraries whilestill retaining the advantages of a taggingunit, the Chiron Corporation group de-vised a chemical approach with peptidesas the encoding unit (40). The chemicalcombinatorial strategy uses resin-splittingpeptide synthesis to alternately synthe-size a "binding" strand and a "coding"strand. An orthogonal protecting-groupscheme is also used to allow for the par-allel synthesis of both chemical units onthe resin shown in Fig. 5.

The isolation of receptor-binding li-gands from a tagged library of this typecan be done by affinity-selection or bead-staining techniques. The identification ofthe binding sequence can be determinedby Edman sequencing. However, whenusing such sequencing technology, thebinding strand, if ofpeptide composition,must be made nonsequenceable. In ananalogous fashion, the Selectide Corpo-ration group (41) has also described apeptidic coding approach. In their report,protecting-group schemes are used thatallow an assay for library receptor bind-ing, either on the matrix or in a solubleformat.The most desirable feature of this type

of peptide encoding strategy is that itgrants a potential for alternative chemicalunits other than the natural amino acids(or nucleotides) to be incorporated intothe binding strand. However, this meth-odology does not allow for enrichment byserial selection, the cornerstone uponwhich genetic library selection methodsare founded.

Electrophoric Polyhalobenzene CodedLibraries

Another solution to the chemical codingof combinatorial libraries has been dis-closed recently by Still and coworkers(42). In their approach, a combinatorialpeptide library attached to beads was as-sembled using the split synthesis method,while simultaneously being indexed bywhat they refer to as electrophoric tag-ging. In their scheme, a series of aromaticelectrophores varying in hydrocarbonchain length (Fig. 6) are used as taggingunits. Tagging is done by an alternatingsynthetic process that coincides with theaddition ofeach library binding unit; how-ever, the tagging process here does notrequire sequential connection. To furthersimplify the entire scheme, the tags areused in a binary code to record the addi-tion of each building block and, thus, thereaction history of each bead.

Screening of the library is done by areporter-group assay (vide infra), and

identified beads with affinity to the re-ceptor are individually picked out by amicropipetter. Use of the o-nitrobenzyl-carbonate moiety on the linker portion ofeach coding unit allows release of tagalcohols through photolysis, which canbe analyzed by electron capture gas chro-matography and identified by the binarysynthesis code. Borchardt and Still (43)have advanced this technology to thesynthesis of a combinatorial N-acylatedtripeptide library using both D- andL-amino acids. This library was used toprobe the binding requirements of a syn-thetic receptor. The outcome of thisstudy was that it allowed new types ofhost-guest interactions to be observedthat might not have been discovered byconventional studies.A clear advantage of the Still coding

technology is that numerous types ofchemical processes are amenable to it.Thus, the potential to create highly di-versified chemical libraries is well withinits purview. The tagging methodology iselegant, as no cosynthesis is required.Again, however, this methodology, likethe peptide-tagging technology, does notallow for amplification or enrichment tooperate.

Encoded Combinatorial Libraries

If the breadth and versatility of chemicalsynthesis could be linked to the power ofgenetics, a potentially more diverse ap-proach to tagged combinatorial librariescould be achieved. A conceptual methodto bridge the gap between these two dif-ferent realms and apply it to combinatoriallibraries was advanced by Brenner andLerner (44) and termed encoded combi-natorial chemistry. In their theory, tocarry out encoded combinatorial chemis-try (Fig. 7) two alternating parallel com-binatorial syntheses must be done so thata genetic tag is chemically linked to thechemical structure being synthesized.Thus, addition of a chemical unit to amatrix is followed by addition of an oli-gonucleotide sequence that codes thechemical unit appended. As with all chem-

HI

FMOC N 1%

FIG. 5. Resin and bifunctional linker unit containing orthogonal protecting groups to allowfor alternating synthesis of the binding and coding strands in peptide coded libraries. FMOC,9-fluorenylmethyloxycarbonyl functionality; MOZ, 4-methoxybenzyloxycarbonyl.

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HOOG // O~lf/O\V(~%

NO,

Linker

cl\

Ar==l

cI CI

Electrophoric Tag

Cl H

CI H

Cl H

I H F

FIG. 6. Tagging units used to create binary synthesis code for electrophoric polyhaloben-zene coded libraries.

ical tagging technologies, split synthesis isapplied in making the library. However,where this encoded approach differs fromother coded synthetic methodologies is inthe selection process between librarymembers and receptors. Now active li-brary molecules are affinity-selected to areceptor, and amplified copies of theirretrogenetic tags are obtained via PCR.DNA strands that are amplified can beused to enrich fora subset ofthe library byhybridization with the matching tags, andthis type of panning process can be re-peated with this subset. Thus, serial en-richment is achieved by a process of pu-rification. Ultimately, the DNA ofbindingmembers can be decoded to provide thechemical history and, hence, the structureof the binding unit.

The general principles of this technol-ogy are straightforward. The chemicalmanipulations to create such libraries canbe complex. If one just considers synthe-sizing a chemical library of peptides, newlinker technologies, protecting-groupschemes, and, moreover, synthetic pro-tocols must be contemplated and devised.At the time when this encoded combina-torial chemical library approach was dis-closed, the alternating parallel synthesisof peptides and oligonucleotides had yetto be described. My group recently dem-onstrated the synthetic methods neededfor implementation of this type of mixedchemical synthesis (45, 46). The gist ofourtack was to make solid-phase peptide andoligonucleotide synthesis compatible. Weaccomplished this through the use of9-flu-

orenylmethyloxycarbonyl (Fmoc) chem-istry for the peptide portion of the mole-cule, whereas a methyl phosphate schemewas engaged for the oligonucleotide-encoding unit. Following our lead, similarsynthetic methodologies applied to beadshave also been published by the group atAffymax (47). In their program, an oligo-nucleotide-encoded D, L peptide librarywas made on a 10-lm support. Screeningthe library for binding to an epitope ondynorphin B provided a number of pep-tide members after amplification and de-coding.The fervor that encoded combinatorial

libraries brings to the chemical libraryfield is evident, as it exploits the best ofboth worlds-chemical diversity and thepower of genetics. The main constraintsin using this technology come from itsstrength-namely, the tagging unit. Oli-gonucleotides can be chemically labileand incompatible with certain syntheticprocedures. However, even with theselimitations, the elegance of this approachis hard to surpass.

Concluding Remarks and FutureDirections

Within a very short time span, a largebody ofwork has accumulated within thefield of combinatorial chemical libraries.As with any new scientific endeavor,different paths of research are exploredin trying to advance the subject. I havepresented what has come to be a philo-sophical divide within the scientific com-munity on combinatorial libraries: the

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FIG. 7. Scheme that describes the general format used in obtaining encoded combinatorial chemical libraries. CPG, controlled pore glass.Subscripts denote residue position. The figure is reproduced with permission from ref. 45 [copyright (1993) American Chemical Society].

CPG BeadA Linker UnitPCR PrimerAmino Acid,Amino Acid2

PA3 Amino Acid3Encoding Nucleotidesfor Amino Acid,Encoding Nucleotidesfor Amino Acid2Encoding Nucleotidesfor Amino Acid3

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Proc. Nati. Acad. Sci. USA 91 (1994) 10785

notion of tagging the combinatorial enti-ties or leaving them as untagged compo-nents. Clearly, strong arguments can bemade in defense of either technology, butultimately the deciding factor as to whatmethodology will be engaged depends onuser need. Addressable libraries or onesprocured by split synthesis for an itera-tive or recursive deconvolution approachhave the potential to create the mostdiverse combinatorial libraries. In con-trast, coded libraries, specifically thoseof genetic origin, allow for the screeningof receptors that are rare and/or of lim-ited concentration.

Currently, most available combinato-rial libraries (tagged or untagged) are ofeither peptide or nucleotide origin. Itappears that the next wave of combina-torial research will be directed at thedesign of libraries that are devoid of therepetitive backbone linkage found withinpeptides or nucleotides. It is here thatstructural/stereoelectronic variation andunconstrained motifs will be allowed toexpand to unparalleled combinatorialchemical diversity. Already importantadvances in this area by Bunin and Ell-man (48) and Hobbs Dewitt et al. (49)have provided us with a glimpse of pos-sible developments.

Finally, it should be noted that combi-natorial chemical libraries can provide uswith another paradigm for drug discoveryand development. Although the field isstill young, the methods/technologies forgenerating and screening these librariesis already quite diverse and is becomingincreasingly more sophisticated. Thequest for the future is to find whethercombinatorial chemical libraries can pro-vide us with more than just "lead"'sources for drug discovery. As long asthe essence of drug discovery is to findquick and cost-effective new drug candi-dates, combinatorial chemical librariesprovide a unique and ever changingsource of chemical diversity.

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