drug discovery by dynamic combinatorial libraries

© 2001 Macmillan Magazines Ltd26 | JANUARY 2002 | VOLUME 1 www.nature.com/reviews/drugdisc

R E V I E W S

Since its formulation in the early 1980s1, COMBINATORIAL

CHEMISTRY has matured to become a key tool in manyaspects of chemistry and biology in general, and in drugdiscovery processes in particular2,3. Not only are combi-natorial arrays of compounds generated as potentialsources of drug leads as well as for lead refinement, butcombinatorial approaches are also gaining importancein the development of synthetic processes in catalystdiscovery and in materials science.

From the beginning, chemical combinatoriallibraries have been developed primarily using PARALLEL

SYNTHESES and specific techniques. Although manyprotocols have implemented RESIN-based chemistryand compartmentation of separate syntheses, the useof pools of discrete soluble substances in the samecompartment has not progressed as fast. In thesecases, the compounds are usually prepared as discrete,reasonably stable entities, which are tested individuallyor in small sets for their effects. Although combinato-rial techniques have allowed the synthesis of verylarge arrays of compounds in a short time, each indi-vidual compound needs to be prepared, often overseveral synthetic steps, and then sufficiently charac-terized. Automation techniques in combination withsolid-phase synthesis and high-throughput analytical

methods have enabled the development of suchprocesses.

If, however, the target substance itself could be usedto select an active ligand/inhibitor directly from alibrary pool, then the screening process would be moreefficient and greatly simplified. In addition, if the librarypool itself were able to undergo changes in compositionduring this process, so as to adapt to the target con-straints, then the screening signal would be amplified,facilitating detection and characterization. Further-more, if the active species could be analysed directlywhile bound to the receptor site, several synthetic stepscould be avoided.

Dynamic combinatorial chemistry (DCC) is a newparadigm in drug discovery that aims to produce suchflexible, adaptive libraries4–10. Such an approach is of asupramolecular nature, being driven by the interactionsof the library constituents with the target site, and relieson reversible reactions or interactions between sets ofbasic components to generate continually interchangingadducts. This gives access to virtual combinatoriallibraries with potentially accessible constituents that areall possible, latent combinations of the componentsavailable. The basic tenets and prospects of thisapproach have been outlined4–6,8–10 in relation to the

DRUG DISCOVERY BY DYNAMICCOMBINATORIAL LIBRARIESOlof Ramström and Jean-Marie Lehn

Dynamic combinatorial chemistry is a recently introduced supramolecular approach that usesself-assembly processes to generate libraries of chemical compounds. In contrast to thestepwise methodology of classical combinatorial techniques, dynamic combinatorial chemistryallows for the generation of libraries based on the continuous interconversion between the libraryconstituents. Spontaneous assembly of the building blocks through reversible chemical reactionsvirtually encompasses all possible combinations, and allows the establishment of adaptiveprocesses owing to the dynamic interchange of the library constituents. Addition of the targetligand or receptor creates a driving force that favours the formation of the best-bindingconstituent — a self-screening process that is capable, in principle, of accelerating theidentification of lead compounds for drug discovery.

Laboratoire de ChimieSupramoléculaire, ISIS —Université Louis Pasteur,4 rue Blaise Pascal, F-67000,Strasbourg, France.Correspondence to J.-M.L. e-mail:[email protected]: 10.1038/nrd704

COMBINATORIAL CHEMISTRY

The generation of largecollections, or ‘libraries’, ofcompounds by synthesizing allpossible combinations of a set ofsmaller chemical structures, or‘building blocks’.

PARALLEL SYNTHESIS

Strategy by which sets of discretecompounds are preparedsimultaneously in arrays ofphysically separate reactionvessels or microcompartmentswithout interchange ofintermediates during theassembly process.

© 2001 Macmillan Magazines LtdNATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JANUARY 2002 | 27

R E V I E W S

building blocks that are capable of interacting reversiblywith one another; second, establishment of library gen-eration conditions, in which the building blocks areallowed to form interchanging, individual molecular‘keys’ (for example, ligands); and third, subjection of thelibrary to selection pressure, which results from bindingstrength to a molecular ‘lock’ (for example, a receptor).So, if the receptor itself can act as a trap for a given ligand,the ensemble of candidates will be forced to rearrange inorder to produce this species. Obviously, the conversesituation, in which a specific synthetic receptor is selectedfrom a pool of interconverting receptors by the additionof a certain ligand, can also be envisaged. These two caseshave been termed ‘substrate casting’ and ‘receptormoulding’, respectively12.

All building blocks contain functional groups thatcan form reversible bonds to one or more componentsin the combinatorial ensemble, allowing the formationof more or less complex molecular entities. Essentially,this gives rise to true dynamic combinatorial libraries, aseach library constituent is a combination of buildingcomponents, and each member of the library intercon-verts with every other possible counterpart over time.

Selection of building blocksFor a dynamic combinatorial library (DCL) to be efficiently produced, the building blocks need to fulfilseveral important characteristics. First, they must havefunctional groups that can undergo reversibleexchange. Second, they must cover as completely aspossible the geometrical and functional space ofpotential target sites, in particular by means of recog-nition groups that are potentially able to interact withthe molecular features of the binding site (FIG. 2). Similarto static combinatorial library design, the choice ofinteracting groups can rely on experience, or on carefulstudy of the (crystal) structure of the target species.Third, these recognition groups need to be organizedgeometrically for optimal binding to occur, and for thisreason organizational units (FIG. 2) must be devised.These structural elements can either be part of therecognition groups, or preferably represent separatecomponents that are based on various types of molecularSCAFFOLD, which undergo dynamic decoration byreversible reaction with the recognition components.The organizational components may be able to establishboth the core geometry and the topicity (the numberof reversible connections) of the DCL constituents.The use of separate components for organization andinteraction allows the same set of recognition elementsto be used for various targets. Furthermore, the geometryand topicity can easily be varied, and more pro-nounced amplification effects may be produced. Theeasier external control of the library is also an advantage,as tags/reporter groups or ‘handles’ can be attached tothe scaffold.

Generation of dynamic diversityMolecular/supramolecular DCLs. The generation ofdynamic libraries can, in essence, be accomplished usingany type of reversible physical or chemical mechanism, as

work of our group5,10. It allows for the target-drivengeneration or amplification of the active constituent(s)of the libraries, thus performing a self-screening processby which the active species is (are) preferentiallyexpressed and retrieved from the library.

The DCC principleA highly schematic presentation of the DCC concept,inspired by Emil Fischer’s lock-and-key metaphor11, isindicated in the top part of FIG. 1. Here, the process hasbeen divided into three steps: first, selection of initial

Receptor

Receptor selection

Librarygeneration

Selection of best binder

Initial building blocks

Receptor

Library of interchanging species

Figure 1 | Schematic representation of the concepts behind dynamic combinatorialchemistry and virtual combinatorial libraries. Top: a true dynamic library of interchanging‘keys’ (for example, ligands), which are formed through reversible exchange of a limited numberof initial key building blocks. On addition of a molecular ‘lock’ (for example, a receptor), the bestbinder is selected, forcing the library to rearrange so as to produce more of this member. Bottom:generation of a virtual combinatorial library, the constituents of which become detectable only inthe presence of the selector.

X Y Z

Y

X

Z

Linear scaffold Globular scaffold

Recognition componentsConnectors Organizational units

Figure 2 | Essential elements of a dynamic combinatorial library. All building blocks musthave functional groups that can form reversible linkages. These are represented as tubularconnectors: X, Y and Z. These linkages can be covalent/molecular or non-covalent/supramolecular. The building blocks must cover as completely as possible thegeometrical and functional space of potential target sites, in particular by means of recognitiongroups that are potentially able to interact with the molecular features of the target site. Theserecognition groups need to be organized geometrically for optimal binding to occur, and for thisreason, organizational units must be devised. These structural elements can either be part of therecognition groups, or represent separate components that are based on various types ofmolecular scaffold (for example, linear or globular) that are undergoing dynamic decoration byreversible reactions with the recognition components.

RESIN

Synthesis of compounds on thesolid surface of an insolubleresin support allows them to bereadily separated (by filtration orcentrifugation) from excessreagents, soluble reaction by-products, or solvents.

SCAFFOLD

Core portion of a molecule thatis common to all members of acombinatorial library.


R E V I E W S

either covalent or non-covalent (including metalcoordination) character.

Transition-metal coordination represents a versatile,and often easily controlled, class of reversible connec-tions that can be successfully used in DCC13–22. Thestability and geometry of the complexes can be mod-ulated by choice of metal ion and ligand type. The com-plexes can be rapidly disassembled by adding efficientcompeting binding agents, and could, in principle, beswitched by oxidation/reduction processes. The maindrawback might lie in the size of the coordinationcentres, which require a binding site that is able toaccommodate the library constituents. In addition, assome biological target species are sensitive to coordinatingligands, which can extract essential metal-ion cofactors,special caution in the choice of template ligands has tobe observed. Other non-covalent interactions couldalso be used23–26, although they are more difficult tomaster in aqueous media.

Functional groups that stabilize reversible covalentconnections are crucial for the generation of dynamiclibraries, and several are available (TABLE 1), each of whichhas particular characteristics, advantages and drawbacks.Addition–elimination reactions at carbonyl groups (orderived groups) are by far the most important class ofreaction, especially imine exchange12,27–37, and to a lesserextent (hemi)acetal/aminal38, transacylation39–44 and aldolreactions. Fine-tuning of the formation and exchangekinetics can be achieved by changing the electronicproperties of the carbonyl compound and the nucle-ophile. For example, primary amines undergo rapidimine formation and exchange with common aldehydes,but the equilibrium in this case is towards the startingmaterials in aqueous medium.With hydroxylamines andacyl hydrazides, the situation is the opposite: the stabilityof the imines is high, whereas the kinetics of the reac-tion are slower30,33. Conjugate addition to carbonylcompounds could also be considered, although this par-ticular type of reaction has not yet been exemplified. Inaddition to these reaction types, pericyclic reactionsrepresent another class of reversible processes that couldbe used to generate dynamic libraries. However, thereactivities of the starting materials vary substantiallywith the substitution pattern, inhibiting to somedegree the generation of near iso-energetic systems. Inthe case of alkene metathesis45–48, recent advances incatalyst development have enabled the use of this reac-tion in dynamic systems, even in aqueous solutions49.Exchange reactions at non-carbon centres could alsobe used, such as alcohol–bor(on)ate50 and, to someextent, alcohol–vanadate exchange, and especiallythiol–disulphide interconversion51–53.

A highly desirable feature of molecular/supramolecu-lar systems is that they should allow the potential fixationof the DCLs — that is, the freezing of the exchangeprocess — either by changing the surrounding conditions(for example, pH, temperature, solvent composition), orby adding quenching reagents (for example,oxidation/reduction reagents). In this way, such librariescan be more easily subjected to various analytical schemes,and the best binders more rapidly identified.

long as the respective interconverting states can beproperly controlled and the final products identified.The most important processes involve molecular/supramolecular interchanges, where chemical bondsare continuously formed and broken. They can makeuse of several reversible connections (TABLE 1), of

Table 1 | Dynamic processes for potential use in DCC systems

Reversible covalent bond formation

Carbonyl reactions

Imine formation

Hemiketal formation

Transacylation

Aldol formation

Michael reaction

Disulphide formation

Diels–Alder reaction

Metathesis reaction

Boronic ester formation

Reversible interactions

Metal coordination

Electrostatic interaction

Hydrogen bonding

Donor–acceptor interaction

Reversible intramolecular processes

Configurational

Cis–trans isomerization

Conformational

Internal rotation

Ring inversion

Structural

Tautomerism

Fluxionality

DCC, dynamic combinatorial chemistry

O H2N R NR

HO ROH

O R

XR1

OY R2

YR2

OX R1

S SSHHS

R1 R2R1 R2

R B(OH)2

HO

HO

R1

R2

O

O

R1

R2

BR

O OH

H

OH X

OX

R COO–H3N+ R'

ON

OH O N

OH

R COO– H3N+ R'

X Y YX

NO

B

AN

O

A

B

A

B

A

B

NH

OH

R2R1

R2R1

Mm+ nL [MLn]m+

O

D A [D,A]

O

XX

N

O O

A

B

A B

A

BM

A

M

B


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FLUXIONAL MOLECULES

Molecules that show rapidintramolecular rearrangementsamong their component atoms.At equilibrium, fluxionalmolecules can manifest manydifferent isomers and fluctuaterapidly among them (forexample, bullvalene).

TAUTOMER

One of two or more structuralisomers that exist in equilibriumand are readily converted fromone isomeric form to another.

Fluxional DCLs. Structural changes also occur in FLUXIONAL

systems, such as TAUTOMERISM and intramolecular-rearrangement reactions (for example, the continuousCope rearrangement in bullvalene). Although these sys-tems have an intrinsic dynamic character that is poten-tially useful for the generation of DCLs, the limitedvirtual library space that they offer, as well as their con-trol and possible fixation, have so far inhibited theirexploration.

Types of DCC systemSo far, three approaches to DCL generation and screeninghave been developed, which have a common firstreversible generation step, but differ in the screening/selection phase.

Adaptive DCLs. The first and foremost among theseapproaches are the true adaptive DCLs, in which gener-ation of the constituents is carried out in the presence ofthe target, resulting in amplification of the best-boundspecies so that screening takes place simultaneously inthe same compartment. All dynamic characteristics ofthe system can be used here, and adaptation and ampli-fication can be obtained. Given the reversible, dynamiccharacteristics of a DCC system, it can respond to dis-turbances and adapt to internal changes or to externaltriggers. These changes could be either physical orchemical in nature, such as addition or removal of othercomponents, and changes in pH, temperature or electricpotential, all potentially forcing the system to adjust tothe new prerequisites. This adaptability, which isenabled by the reversibility of the processes, gives thesystem the potential to be amplified. For example, if oneconstituent in the DCL interacts better than the otherswith a certain target species, then this constituent will bewithdrawn from the equilibrating pool, and all of thecomponents that make up this constituent will also bemasked by the binding. Because of the equilibrium situ-ation, the system has to rearrange so as to produce moreof this constituent at the expense of the other species inthe library. On re-equilibration, the most active con-stituent (the best binder) will therefore experience acertain degree of amplification, in comparison with thesituation in which no target molecule was added.

The degree of amplification depends on several para-meters, notably binding strength and the design of thelibrary. Although an analysis of an oligomeric library,composed of linear combinations of monomers, indi-cates that this amplification effect might be limited to afactor of about two56 (that is, twice the amount pro-duced than in the corresponding situation in theabsence of any target species), much larger amplifica-tions can be, and have been, reached in actualsituations12,13,35. For instance, if one of the organizationalcomponents (a scaffold component) is added in a lim-ited amount (less than or stoichiometric to the targetspecies) compared with all of the interactional com-ponents (near stoichiometric amount each), a truepotential library will be produced. Initially, all con-stituents will be present according to stability, but mostof the interactional components will be free in solution,

The generation of DCLs can also involve morethan one type of connection chemistry; two or morereactions or interactions can be used to vastly extendthe diversity of the library29,34. Dynamics can thenoperate in several dimensions — one for everyreversible reaction type used — giving rise to highlycomplex multidimensional DCLs. The pharmaco-chemical space will obviously be covered much moreefficiently in this case, given a similar number of origi-nal building blocks. The chemistries that are usedshould preferably be chosen in an orthogonal manner,so that they can be controlled separately at will, as, forexample, in combinations of metal coordination andimine exchange. Also, the rapid increase in diversitywill allow such libraries to be made from fewer build-ing blocks, resulting in yet more compact, easily con-trolled sets of compounds.

Configurational/conformational DCLs. All of the con-figurational and conformational changes in a moleculeare inherently dynamic, and, in principle, these can beused to produce DCLs15,28,54,55. This is notably the casewhen each configuration or conformer is sufficientlystable to be studied, either isolated or in complex with aligand/receptor. Examples of such processes are cis–transisomerization of double bonds, ring inversions andinternal bond rotations. Double-bond isomerization inparticular lends itself to producing stable DCLs, inwhich one isomer can be transformed into the other byphotochemical and/or thermal means. More rapidlychanging species (rotamers, and so on) that are difficultto isolate can sometimes be studied in situ by solution-phase methods.

++ nn + + n-mer

Virtual dynamic library

Cl– SO42–Fe2+

N N N N

N N

Figure 3 | Dynamic combinatorial library of circular helicates. Addition of iron (II) chloride to asolution of a tris-bipyridyl ligand (which interacts with the Fe2+ ions through the nitrogens on thearomatic rings, shaded in blue) could, in principle, allow the assembly of any member of acollection of circular helicates, from which the pentagonal species was produced in quantitativeyield, with the chloride acting as a template13. When the chloride was replaced by sulphate, ahexagonal species was the main species formed. This is an example of a virtual dynamic library,as none of the circular helicate constituents exists before the actual experiment.


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however, such deconvolutions can be significantlysimplified as further mentioned below. The pre-equi-libration procedure must also be used when the con-necting reaction requires equilibration conditionsthat are incompatible with the biological target. Inboth cases, the dynamic nature of the process makes itpossible to rapidly generate the library constituents.

Iterative DCLs. The third approach is to use an iterativepre-equilibration protocol. In this case, the DCL is gener-ated in one compartment under appropriate conditions,then, in a subsequent step, constituents of the DCL areallowed to interact with the target species either in thesame reaction chamber or separately. It is necessary tobe able to separate bound species from unbound onesby using immobilized or entrapped target entities. Theunbound species are then re-transferred to the reactionchamber, re-scrambled and again allowed to interactwith the binding site. After several rounds of such a‘dynamic panning protocol’, the accumulated activespecies can be analysed54.

Features of DCC processesControl of the DCC process. The dynamic nature of theprocess is a key feature of the DCC approach. In theideal case, all reversible interconnections should besufficiently fast for an equilibrium state to be attained ina short time,and the library should be put under thermo-dynamic control, to minimize the risk of stalling atmetastable local minima. Equally desirable, the con-nective groups should show equivalent or similar reac-tivities in order to produce iso-energetic libraries. Allconstituents can then potentially be formed in compara-ble amounts, avoiding an unfavourable discriminationbetween various members. Also, when applying DCC tosensitive biological macromolecules, a rapid process isimportant to avoid degradation effects of the targetspecies. As several reversible reactions are catalysed byacid or base, being slowest at neutral pH, it is preferableto use reversible connections that equilibrate undermildly acidic or basic conditions when working withbiological macromolecules in aqueous media. However,external control over the kinetics of the library becomespossible if the conditions can be adjusted so that arapidly equilibrating system slows down, or even com-pletely stops, after a change in conditions. This is the caseif a freezing step is applied, in which a fast (relative to theexchange) fixation of the library is required. Controlover the rates of the reversible reactions is preferablymade by chemical or physical means, such as changes inpH, redox state, temperature or light irradiation.Alternatively, a quenching reagent can be used to stopthe reaction, in this case giving an extra control tool, as inthe reduction of imines to amines. In this case, thereaction that is used should not disturb the equilibriumstate that is reached.

Virtuality. The dynamic nature of the DCC processgives access to all possible combinations of the compo-nents, which form the full set of constituents of thelibrary that could be generated; whether or not they are

and all scaffolds will be part of library constituents. Onaddition of a thermodynamic sink, the scaffold com-ponent (and associated interactional components) willbe removed from solution, rearrangement will occur toproduce more of the best binder,and a large amplificationeffect might result.

Pre-equilibrated DCLs. The second approach,denoted pre-equilibrated DCLs, involves generatingthe dynamic libraries under reversible conditions, andperforming the identification/screening under staticconditions. No amplification can take place in thiscase, but this type of protocol is, nevertheless, very useful when working with sensitive and delicate bio-logical target species that are unavailable in largeamounts. In this case, the DCL is produced underreversible conditions that are equivalent to the adaptivesituation, but before biological testing is carried out,conditions are changed so as to quench the reversiblereactions, rendering the library static. Screening canthen be accomplished according to classical combina-torial methods, and identification of active componentscan be achieved using various DECONVOLUTION proto-cols. Because of the dynamic nature of the library,

DECONVOLUTION

The process of optimizing anactivity of interest byfractionating a pool with somelevel of the desired activity togive a set of smaller pools. Thisstrategy can be appliediteratively to identify singlemembers with (ideally) a highlevel of activity.

G G FY L

GY

G G LF L

G G LF L

F Y

F YF Y

G GL

G GL

GL

GL

GG

F

Y

L

GG

F

Y

L

G GY

GY

Selection of pentapeptide(YGGFL) by antibody

Library generation through protease-mediated amide hydrolysis and synthesis

Thermolysin Endorphin- selective antibody

Semipermeable membrane

F L

G GY F L+

Figure 4 | Generation and screening of peptides generated by reversible proteolysis.Incubation of the peptides Tyr-Gly-Gly (YGG) and Phe-Leu (FL) with the protease thermolysin(yellow) produces a library of peptides. The library was generated in the presence of an antibody(blue; protected from proteolysis by a semipermeable membrane) that specifically binds theamino terminus of β-endorphin, which has the sequence YGGFL. Peptides with this sequencewere selected, and amplification was observed relative to the quantities in a correspondingantibody-free experiment.


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DCL in the ideal case. So, with a ‘kth-order’ dynamiclibrary, in which each reversible reaction results inm × np species, a final library size of (m × np)k will bethe result. In these examples, symmetry effects (forexample, if A

1–B–A

2equals A

2–B–A

1) have not been

accounted for; when such effects occur, the numberswill be reduced to some extent.

Development of the DCC conceptIn essence, dynamic combinatorial libraries existed inthe chemist’s flasks long before the concept was actuallyenvisaged; especially in reversible supramolecular recog-nition systems. In supramolecular chemistry, molecularassemblies are created that are held together by ‘weaker’reversible interactions, and often non-covalent andmetal-coordination interactions between the differentunits are used. Many of these systems consist of mixturesof various interactional partners in solution, and thecontrol and understanding of the outcome of suchinteractions have been, and still are, fundamental issuesin the field. In many cases, such supramolecular mix-tures are reasonably well defined, and all discreteassemblies can be fully identified. For many years, suchsystems were regarded as more or less impure mixtures,and protocols were normally optimized with respect toacquiring as high a yield as possible of the desiredspecies. However, as a combinatorial library can beregarded as a controlled mixture, in which each inde-pendent constituent is known and can be addressedindividually, many such supramolecular mixtures mightactually be considered to be precursors of dynamiclibraries . Predecessors of this kind include, for example,metal-ion-assisted imine macrocycle synthesis57,58, andthe reversible assembly of short nucleotide stretches byusing a complementary oligonucleotide strand as atemplate59. Self-selection in the reversible formation ofhelical metal-coordination complexes provided an earlycase that involved the generation of a specific set ofconstituents from instructed mixtures in dynamic equi-librium under ‘internal’ selection60. When it becameapparent that such instructed mixtures could actually beunder the control of target species — that is, be subjectto ‘external’ selection — the forest was seen where therehad previously been just a collection of trees, and theconcept could be formulated. So, mixing a tris-bipyri-dine ligand with an octahedrally coordinating metal ion,such as Fe2+, led to reversibly interchanging libraries ofcircular helicates, which, in principle, could have anysize, ranging from squares (four metal ions), to hexagons(six metal ions) and larger constructs13,61 (FIG. 3).Whereas in the presence of chloride ions, the pentagonalhelicate was expressed in quantitative yield, the hexago-nal species was obtained with other anions. In this case,the chloride ion was acting as a template, controllingthe assembly of the library components, and shifting thesystem to the expression of the pentagonal species,which acts as a receptor that binds very strongly to thechloride substrate. The process was perceived as givingaccess to a dynamically accessible virtual library of circularhelicates13. This represents a case of a true virtual dynamiclibrary, as none of the circular helicate constituents exist

actually formed depends on the system and the condi-tions used. These dynamic libraries are thereforevirtual5,13, the optimal situation being that in whichnone of the potential constituents is formed in theabsence of the target, and only addition of the targetleads to formation; that is, expression of the best-boundspecies among the latent ones (FIG. 1). Amplification of areal (that is, already present) constituent of a library cantherefore be formally distinguished from expression of alatent one. It is worth noting that, in view of the strongpH dependence of the imine formation equilibrium inaqueous solution, the corresponding libraries mightspan these two types of behaviour as a function of theacidity of the medium.

Combinatorics. Until now, DCLs have usually beenmade on a small scale, with libraries ranging from afew species up to a few thousand compounds.However, the sizes of dynamic libraries can easily bemade very large, and the library sizes that can beaccomplished with dynamic library systems are by nomeans unimpressive. In a simple example, startingfrom n components of type A and m components oftype B, a ditopic library of n × m constituents A–B caneasily be formed. With 10 components each(n = m = 10), a library made up of 100 constituents isproduced. Larger and more diverse libraries can easilybe created using linkers or scaffolds. So, n componentsof type A, interacting with m type B linkers/scaffoldsof topicity p, result in a library of m × np constituents.Using the same formula as above, with 10 compoundseach of A and B, if B has a topicity of 2, a library of1,000 compounds can be made, and when the topicityincreases to 3, 10,000 compounds can be accessed, andso on. For multidynamic libraries, the diversity growseven faster, leading to the product of each individual

OS

OOH

O

O

O

HOO

OS NH2

O

O

HN

O

NH2

NH2

O

NH2

R

O

H+ R' NH2

R

NR'

HNR'

NaBH3CN

Aldehydes Amines

NH2

HN

NH2

O

OH

H

H

Carbonic anhydrase

NH

S

NH2

OO

a

b c

R

OHO

Figure 5 | Dynamic combinatorial library of imines interacting with carbonic anhydrase.a | Imines were reversibly formed from a mixture of aldehydes and amines (structures shown inpanel b) in the presence of carbonic anhydrase12; the equilibrium was then halted by addition ofsodium cyanoborohydride (NaBH3CN) to reduce the imines to amines. One of theamine–aldehyde combinations was selected and amplified; it could easily be analysed onreduction of the imines to the corresponding amines (structure shown in c).


R E V I E W S

peptides could be selected and amplification could bedetected. In this case, the target antibody was protectedfrom digestion by the protease using a semipermeablemembrane. So, if the scrambling conditions might bedisadvantageous for the target species, it can be protectedby compartmentalization.

ExamplesSince the formulation of the concept, numerousreports describing the DCC technique have appearedin the literature. Many of these have dealt primarily withthe generation chemistry, in an effort to find suitableconditions for initiation and control of the reversiblereactions, and the resulting libraries have not been sub-jected to any target-directed screening23,25,30-32,38–44,46,53,64.For the most part, synthetic model systems have beenstudied, often in non-aqueous media, in order toestablish basic system design and analysis, and thesesystems will not be discussed further in the presentcontext. However, several studies have addressed boththe generation and the screening phases in the pres-ence of a target, and various protocols have beendeveloped12,13,15,16,24,27,29,49,51,52,54,61–63,65–69.

In particular, several applications that target biologicalmacromolecules or biogenic ligands have beenreported. Biological molecules are both the most inter-esting and the most challenging target molecules. Theyare often available only in scarce amounts, they areunstable to harsh treatment for long periods of time,and all studies have to be made in well-defined buffersystems. So, several reversible chemistries that are veryefficient in the organic phase are catalysed by acids orbases, and cannot be used. Instead, reversible reactionsoccurring under mild conditions that do not interferewith the sensitive target molecules have to be used. Sofar, mainly reversible covalent connections, in particularimines, but also disulphides, and to some extentmetathesis and metal-coordination interactions, havebeen successfully used in these systems.

Enzymes as targets. As mentioned above, imine formationand exchange (transimination) are especially attractivereactions that are fairly compatible with water, and arecharacterized by rapid formation equilibria and fastexchange rates. So, as a first example, an imine systembased on three different aldehydes and four different pri-mary amines was used with the enzyme carbonicanhydrase, resulting in a library of twelve different con-stituents12 (FIG. 5). The building blocks were chosen toresemble known inhibitors of this enzyme,and the librarywas composed of sulphonamide-groups, potent in inter-acting with the Zn2+-binding site, as well as lipophilicmoieties for potential interactions with the neighbouringhydrophobic site.An excess of amine was applied to over-come the effect from potential amino groups at thesurface of the enzyme,and, in addition,cyanoborohydridewas added to freeze out the formed imines by reduction tothe corresponding secondary amines. One of the iminecombinations was found to bind preferentially to theenzyme, and its formation was markedly amplified withrespect to the concentration in the absence of any enzyme.

before the actual experiment, and the process is adaptive,as any member of the library can be generated at anytime from any other component, as demanded by thesubstrate anion.

Another early example of such receptor assemblyemanated from work on macrolactonization fromhydroxy-acid precursors40. In this case, a mixture oflinear and cyclic esters could be dynamically formed, in acontrolled manner, using base-catalysed transesterifica-tion. Although no selection pressure was applied in theinitial system, it was in a subsequent modified version62.

Biological dynamic libraries have also been reported.For example, in a study in which trypsin was targeted forinhibition by tripeptides that carried a terminal boronategroup, crystallization of the enzyme in the presence ofthe inhibitor candidate, with either methanol, ethanol orisopropanol, resulted in selective ester formation50.Another case involved the generation of ligands for anantibody by reversible proteolytic digestion of peptides63

(FIG. 4). Thermolysin, a soluble protease that has broadspecificity,was used to generate libraries of short peptides.When this process was carried out in the presence of atarget receptor — a monoclonal antibody that was specificfor the amino-terminus of β-endorphin — binding

N

HNNH2

O

HNNH2

O

HNNH2

O

N

H3C

CH3

HNNH2

O

N

H3C

CH3

CH3

CHO

NH3C

CH3CH3

OH

OH

CHO

N

CHO

CHO

OH

CHO

CHO

CHO

CHO

N

CHO

CHO CHO

CHO

CHO

CHO

N

NHN

N

NHN

O

O

Enz

yme

activ

ity

a

b c

Buffer All 1 2 3 4 A B C D E F G H I

Hydrazides

Aldehydes

1 2 3 4

A B C D

E F G H I

4-I-4

Figure 6 | Dynamic deconvolution of acyl hydrazone libraries of potential inhibitors ofacetylcholinesterase. a | Sequential removal of each building block (structures shown in b) results in identification of individual activities. c | The combination of molecules 4 and I yieldedthe best inhibitor33.


R E V I E W S

VANCOMYCIN

Vancomycin is an antibiotic thatacts by binding to cell-wallprecursors that terminate in thesequence D-Ala-D-Ala, therebyinhibiting cell-wall synthesis.

redox properties of the system, without significantlydisturbing other components in the solution. This wasshown in the case of a small disulphide-based carbo-hydrate library that was tested against the lectinConcanavalin A52 (Con A; FIG. 7). Generation of thelibrary was performed at neutral to slightly basic pH, atwhich level the redox exchange is rapid, and by loweringthe pH, the exchange could efficiently be stopped.Starting from 6 different thiol-derivatized carbohydratehead groups, ditopic libraries of up to 21 differentdisulphide species were generated. A bis-mannosideunit could be selected from binding to the immobilizedCon A, in accordance with known ligands for thislectin. Obviously, this bond type is especially advanta-geous for proteins that are devoid of any disulphidelinkages, such as Con A, and internal disulphidebridges in the proteins studied could present a problem.However, in an example in which such a protein wasused (acetylcholinesterase), no anomalies could beseen. (T. Bunyapaiboonsri and J.-M.L., unpublishedobservations). Most probably, internal disulphidebridges are protected in the interior of the protein,and are not easily accessible to thiols at low concentrationin the surrounding pool of solvent.

Nucleotides as targets. Metal-coordination interactionscover a wide range of stabilities, and several of themare sufficiently prone to scrambling in aqueous media.In one such example, Zn2+ was used in conjunctionwith a library of salicylaldimines and probed againstbinding to immobilized duplex DNA: oligo(dA)bound to solid-phase poly(dT)27 (FIG. 8). Thirty-sixdifferent combinations of Zn2+ complexes were generatedfrom six starting elements in buffered aqueous solution.Two of the salicylaldimines were active, one of whichshowed a higher binding to the double-strandednucleotide than all of the other library constituents. Abinding constant in the lower micromolar range couldalso be recorded.

Ligands for biological receptors. Not only biologicalmacromolecules, but also cell-surface ligands or othersmall biogenic species could be targeted with DCLs. In arecent example, the bacterial cell wall building-block pep-tide D-Ala-D-Ala was probed with a library of VANCOMYCIN-derived elements49 (FIG. 9). As the vancomycin dimer isknown to bind to its ligand much more efficiently thanthe monomer, DCLs were made by linking two peptide-binding units by a linker chain. Disulphide interchangeor alkene metathesis were used in the linker to introducereversibility into the system, and in this way, libraries ofup to 36 members could be constructed. The finallibrary components were tested for antibacterial activityagainst a series of vancomycin-resistant bacterial strains,and several components were found to be active.

Analysis and screening of DCLsSimilar to pure solution-phase combinatorial libraries,the analysis of DCLs is not a trivial challenge. As the sizeof the library increases, the identification of discrete con-stituents becomes more demanding. For smaller libraries,

However, because of the imine instability, other typesof C=N bond have been studied that can be isolatedwithout reduction. Among these, acyl hydrazones seemparticularly attractive; they are reversible by mild acidcatalysis, and stable enough at higher pH. This chemistrywas used in another example, this time of a pre-equili-brated DCL33 (FIG. 6). A DCL composed of interconvert-ing acyl hydrazones was generated and screened for inhi-bition of acetylcholinesterase from the electric rayTorpedo marmorata. This enzyme has two binding sitesin close proximity to each other: one active site at thebottom of a deep gorge, and a so-called ‘peripheral’ sitenear the rim of this gorge. Both sites are selective forpositively charged functionalities, such as quaternaryammonium groups, and by bridging the two sites, veryefficient inhibitors can be found. So, starting from asmall set of 13 initial hydrazide and aldehyde buildingblocks, some of which contained quaternary ammo-nium groups, a library of 66 different species could beobtained in a single operation. Of all possible acylhydrazones formed,active compounds that contained twoterminal cationic recognition groups separated by aspacer of appropriate length could be rapidly identifiedusing a dynamic deconvolution procedure that wasbased on the sequential removal of starting buildingblocks. A very potent bis-pyridinium inhibitor wasselected from the process (K

i= 1.09 nM, αK

i= 2.80 nM),

and the contribution of various structural features couldbe evaluated.

Receptor proteins. Receptor-type proteins, such aslectins, can also be targeted with DCLs. In one example,a prototype library of four different interchangingstereoisomers could be generated from the Fe2+-assistedassembly of a carbohydrate-decorated bipyridine unit(N-acetylgalactosamine-bipyridine, GalNAc-bpy)15. Oninteraction with a range of GalNAc-selective lectins, thedistribution of these isomers was adjusted dependingon lectin.

As mentioned previously, another type of aqueous-phase-compatible reaction is thiol–disulphide inter-change. Disulphides can be scrambled by means of the

OHO

HOOH

O

OHOHO

HO

OH

O

OH

SS

SS

+ SS

SS

SS

+

Figure 7 | Dynamic library of disulphide-containing carbohydrate structures. Starting from6 different thiol-derivatized carbohydrate head groups, libraries of up to 21 different disulphide-linked carbohydrate-dimer species were generated by thiol–disulphide exchange; an exampleexchange reaction is shown. A bis-mannoside was selected in the presence of the Jack bean(Canavalia ensiformis) lectin, Concanavalin A (yellow)52.


R E V I E W S

Challenges and future prospectsDynamic combinatorial chemistry has, in just a fewyears, become increasingly established as an efficientmeans of simultaneously producing molecular diversity,directly addressing the target, and self-screening thelibrary. However, some challenges still persist, especiallyfor applications to biological targets and so for drugdiscovery. Although such DCC protocols have beenestablished on several occasions, as exemplified above,new and controllable reversible chemistries that areentirely compatible with ‘biological conditions’ — thatis, aqueous buffers, a specific range of temperature andpH, and so on — are nevertheless greatly needed.Reversible reactions that show no interference withproteins and other essential components in the systemwould add to the DCC toolbox and greatly enhance thechances of success. In addition,powerful analytical proto-cols are required for the evaluation of larger libraries.

So far, the constituents of most DCLs that have beendeveloped are characterized by having rather extendedstructures, being composed of reversible connectors ofsome size; for this reason, more compact libraries arealso of interest. Many biological receptor sites are lim-ited in size, capable of accommodating only rather smallligands. On the other hand, extended binding sites arehighly appropriate targets for DCLs, as larger ligands canbe used. One such example of high interest in biologicalcommunication and regulation mechanisms is pro-tein–protein interactions.

In conclusion, DCC is a new tool that can be usedto generate adaptive libraries that can respond to agiven selection pressure by virtue of their ability toexpress all latent, virtual constituents of the library.Not only can such libraries be produced in syntheticorganic systems, in themselves highly interesting, butthey can also be implemented with biological entities,such as enzymes and receptors. The concept hasopened new perspectives for drug discovery processes,offering a versatile and rapid targeting methodendowed with a self-screening capability. It emphasizesthe generation of informed diversity, pointing towardsthe development of instructed, ‘smart’ combinatoriallibraries for which the desired target species drives theassembly process.

The DCC/virtual combinatorial library approach is,however, by no means limited to drug discovery. Rather,

various separation methods, such as high-performanceliquid chromatography (HPLC), high-performancecapillary electrophoresis (HPCE), and gas chromatog-raphy (GC), can be used relatively easily to isolate andquantitate single library constituents. Similarly, massspectrometry (MS) techniques allow the identificationand, to some extent, quantitation of (smaller) librariescontaining constituents of discrete mass units.However, for larger libraries, combinations of separationtechniques coupled to modern MS equipment would,in principle, allow sufficient identification of the mainconstituents, the separation step greatly facilitating theidentification step. In addition, if amplification can begenerated in the library, resulting in the increased con-centration of one or a few species, analysis would behighly simplified. Ideally, the best solution would be toanalyse directly what is selected by the target species, andprogress in this direction is continuously being made.For example, some MS techniques, such as quadrupoletime-of-flight electrospray mass spectrometry (Q-TOF-ESMS), are sufficiently mild to measure complexesbetween proteins and ligands70, and differential nuclearmagnetic resonance (NMR)-techniques (for example,transfer nuclear Overhauser effect (NOE) protocols)have also been established for the same purpose71.

As mentioned, screening can also be effectivelyperformed with pre-equilibrated libraries. Particularlypowerful is the dynamic deconvolution procedure, inwhich removal of a given component will lead to theredistribution of the generated constituents. By preparingseveral sub-libraries, in which one (or several) buildingblocks have been removed from the library soup, andtesting these in comparison with the full library, largelibraries can be screened in a short time. All species thatcontain this (these) unit(s) will be deleted from thelibrary, and a decrease in inhibitory effect will indicatethat the removed component is an important elementin the generation of an active compound in thedynamic mixture.

NR1

O

N

R1

OZn2+

NR1

OH

Zn2+

NRi

O

NRj

O

Zn2+

NR1

O

N

R2

OZn2+

NR6

O

N

R6

OZn2+

NR2

OH

NR6

OH

DNA

Figure 8 | Library of Zn2+ complexes interacting with duplex DNA. Thirty-six differentcombinations of Zn2+ complexes were generated from six starting elements in buffered aqueoussolution. Selection by incubation with a dA oligonucleotide bound to solid-phase poly(dT) allowedthe identification of a complex with an affinity of ~1 µM (REF. 27).

L-Lys-D-Ala-D-Ala

Vancomycin buildingblocks carrying linkersof varying length

Selection of bis-vancomycin with optimal linker length

Figure 9 | Receptor library screened against thebacterial cell wall building block D-Ala-D-Ala.Vancomycin units were connected by spacers of varyinglength and their dimer formation and interaction with thetarget peptide were probed49.


R E V I E W S

polymeric structures72. It therefore has an importantrole in the emergence of adaptive chemistry73. Finally, ina completely different vein, combinatorial procedureswith dynamic features have also been used outside theimmediate scope of natural sciences, as illustrated; forinstance, in fine arts, literature and music74–77.

rapid and vigorous exploration of the potential ofdynamic combinatorial systems and of virtual diversitypresentation might be expected in various directions. Ofspecial significance is that DCC can be extended to thevast field of materials science, for example, resulting inthe property-driven generation of new, adjustable

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