CA2160457A1 - Random chemistry for the generation of new compounds - Google Patents

Abstract

Methods for the generation of new compounds are disclosed. The present invention eliminates the need to know in advance the structure or chemical compounds of a compounds having a desired property. The disclosure of the present invention provides that diversity of unknown compounds may be produced by "random" chemistry, and such a diversity of unknown compounds may be screened for one or more desired properties to detect the presence of suitable compounds. In one aspect, a starting group of organic compounds is caused to undergo a series of chemical reactions to create a diversity of new organic compounds that are screened for the presence of organic compounds having the desired property. In another aspect of the present invention, a diversity of compounds is generated from a group of substrates which are subjected to a group of enzymes representing a diversity of catalytic activities.

Description

2 ~ ~ O ~ ~ 7 PCTIUS94/04314 RANDOM CHEMISTRY FOR
THE GENERATION OF NEW COMPOUNt)S

Cross-Re~erence to Relate A lication The present application is a continuation-in-part of U.S.
patent application 08/049,268, filed April 19, 1993, the entire disclosure of which is hereby incorporated by reference.

Technical Field The ~resent invention relates generally to the generation of new compounds without predeLe",)ining a desired structure or composition, and the screening of such compounds for one or more desired properties. This invention is more particularly related to the use of a random chemistry, with or without enzymes, to ge"erate a variety of new compounds from which those with a desired property may be cl,aracteri~ed or identified, e.g., for subsequent production in batch quantities by conventional methodologies or otherwise.

Background of the Invention Humankind's attempt to acquire new and useful compounds has been one of the more interesLin~ but problematic endeavors, especially with respect to medically useful compounds. In general, the traditional approaches to the acquisition of new compounds have been either isolation of natural products (i.e., isolation of compounds found in nature) or synthetic preparation. Discovery of new WO 94/24314 216 0 ~ 5 7 PCT/US94/04314 and useful compounds via naturai products is hampered by a variety of problems including the availability of source materials from which to isolate compounds. Further the variety of compounds via natural products is not unlimited as plants and other living organisn,s do not make every compound theoretically possible.
All~r"~ ely new compounds have been prepared synthetically i.e. by the creation of compounds in the laboratory rather than through the isolation of naturally occurring compounds. The synthetic generation of compounds utilizes the principles and methodologies of organic cl,e,nisl,y especially reaction mechanisms.
Compounds are clealed by deliberate "rational" approaches in which the structure of a desi,ed compound is first determined or conceived and a synthesis strategy is then developed. This approach appears to be reaching its zenith in the field of drug design where computer-~ssisted.
structure-activity stu~lies are performed to generate rational drug design.
However in general rational drug design has not achieved the successes initially envisioned.
Once a desired compound structure is identified a strategy for synthetic preparation is developed. Traditional st,alegies for organic sy,lti,esis are serial sy"ll,esis or assembly of subunits in parallel, or a combination II,ereof. Serial synthesis involves the modification of one compound to form another compound which in turn is chemically I,a"s~or",ed (and so on) until the desired compound is synti,esi~ed.
Assembly of subunits in parallel involves the sy"l~,esis of "portions" of a desired compound with production of the desired compound resulting from the joining of the individual portions.
More specifically current techniques to sy"ll ,esi~e desired compounds through a sequence of catalyzed reactions are based on the control of each reaction step in a sequential synthesis to optimize the yield of each intermediate compound used along the pathway of synthesis of the s~ecific dedr~d termi~ ~roduct compound. The logic ~ WO 94124314 21~ 0 ~ ~ 7 PCT/US94/04314 of this established procedure rests on the fact that the structure of the desired le""inal product molecule is known berorel)and, and that a thermodynamically efficient reaction pathway leading from substrates to the desired product exists. As noted above, two major ge,)eral strategies to synthesize a desired target compound are common in the art. In the first, the terminal target is built up sequentially by successive modification of a starting subsl,ale, acted upon in conjunction with other possible suL,sl-ales, either by enzymes or careful choice of reaction conditions. A simple example is the sequential chemical synthesis of a desired peptide by cycles of protection and deprotection of the growing peptide chain as a succession of activated amino acids is added one by one. A second major allernali~e strategy in the art is the synthesis of a desired cl,emical compound by the s~ccessive sy"ll,esis of increasingly complex sets of building blocks which are finally joined to make the desired target. A simple example is the synthesis of a specific hPY~rertide (ABCDEF) from the amino acid mGnG"~ers A, B, C, D, E, F, by the sy,lti,esis of the dipeptides AB, CD, EF, then the joining of the dipeptides to form the hexapeptide. The same two general strategies are utilized in many areas of synthetic chemistry with a variety of di~dre,ll organic compounds. Both slrateyies are hindered by a variety of problems, including the necessity for knowledge about, and use of, prespecified reaction pathways.
In summary, current approaches to the ~c~ sition of new and useful compounds are subject to a variety of limitations. Thus, there is a need in the art for a method for ge neralil~g new compounds without the necessily for predeler"~ining chemical structures, compositions, or synthesis pathways. The present invention fulfills these needs and further provides other related advantages.

WO 94/24314 21~ ~ 4 5 7 PCT/US94/04314 ~

Summar,v of the Invention In contrast to the current approaches to the ~ctluisition of new and useful compounds, the present invention el;",inales the need to know the structure or chemical composition of the desired compound prior to its synthesis. The disclos~re of the present invention provides that a diversity of unknown compounds may be produced by "random"
chemistry, and such a diversit,v may be screened for one or more desired properties to detect the presence of suitable compounds. It is central to the subject methods that one does not need to know in advance the structure or composition of the useful compound sought.
Briefly stated, the present invention provides methods for the production of an organic molecule having a desired property, or for the generation and chara~Aeri~alion of an organic molecule having a desired property.
In accolda"ce with a first aspect of the present invention, the method comprises first providing a starting group of different organic moleclJles. At least one chemical reaction is cAused to take place with at least some of the clirrere"l organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecu'es dir~-e~l from the organic molecules in the starting group.
This step of causing at least one cl ,e"~ical reaction to take place is repeated at least once. Each repetition uses the reaction mixture of the previous step, and in the end prod~ces a final reaction mixture as a result of the last repetition. The final reaction mixture is screened for the I.resence of the organic molecule having the desired property.
In accordance with an embodiment of the first aspect, the method for the production of an organic molecule having a desired property as described above is performed. If the screening step of this aspect is successr.ll in detecting the organic molecule having the desired propert,v in the final reaction mixture, then the following A~J~tiol ,al s~ ue pe~med. n~ starting group of different organic WO 94/24314 2 ~ 6 0 4 ~ 7 PCT/US94/04314 molecules is divided into at least two subgroups, each containing less than all of the different organic molecules in the starting group. The chemical reactions are performed on each of the subgroups in the same way as with the starting group to produce a final reaction submixture cor.es,~)~nding to each of the subgroups~ Each of the final reaction submixtures resulting from this step is screened for the presence of the organic molecule having the desired property. These additional steps are repeated at least once for each of the sl~ccess~ul subgroups from which the organic molecule having the desired property is pro~uce~ by substit~lting the successtul subgroup as the subgroup in the first ad~itional step to thereby identify a narrowed group of dir~ere"t organic moleu ~les from which the compound having the desired property can be prod~ ~ce~
In one embodiment, the method comprises the steps of:
(a) reacting a group of dirrerent sul~slrales, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehydldtin5~ agent to yield a first reaction mixture;(b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture;
(c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture;
(d) ,uel~o~ ing a concle,lsalion reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture;
(e) exposing the fourth reaction mixture to light within a wavelength of about 220 nanometers to 600 nanometers, thereby producing one or more organic molecules different from the substrates and agents;
(f) screening the exposed fourth reaction mixture for the presei-ce ot an organ c mdec~ he desired property; and WO 94/24314 21~ ~ 4 ~ 7 PCT/US94/04314 ~

(g) isolating from the exposed fourth reaction mixture the organic molecule having the desired propertv.
In an alternative embodiment, any subset of steps a-e above may be performed in any order prior to steps f and g. Further, steps a-e or any subset of these may be repeated in any order prior to steps f and g. Similarly, exposure to other reagents, singly, sequentially, or simultaneously, may be substituted for steps a-e, prior to steps e and f.
In another embodiment of the first aspect, the method comprises the steps of:
(a) reacting a group of dir~e"~ subsl-a~as, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable colldiliGns with a deh~ aliny agent to yield a first reactio mixture;
(b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture;
(c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture;
(d) performing acondensaLion reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture;
(e) exposing the fourth reaction mixture to light within a wavelength of about 220 nanometers to 600 nanGI"elers, thereby 2~ producing one or more organic molecu~es di~re"l from the SUb~lrdl~S
and agents;
(f) screening the exposed fourth reaction mixture for the presence of an organic molecule having the desired property; and (g) determining the structure or functional properties characte,i~i"y the organic molecule having the desired property.

~ WO 94/24314 ~16 0 4 5 7 PCT/U594/04314 Any subset of steps a-e above may be performed in any order prior to steps f and 9. Further, steps a-e or any subset of these may be repeated in any order prior to steps f and 9. Similarly, exposure to other reagents, singly, sequentially, or simultaneously, may be substituted for steps a-e, prior to steps e and f.
In accordance with a second aspect of the present invention, the method comprises the steps of:
(a) reacting a group of dirrele"l enzymes representing a diversity of catalytic activities under suitable conditions with a group of clifrere,lt substrates, thereby producing one or more organic molecl~les .li~relenl from the enzymes and subsl.ales in the reaction mixture;
(b) screening the reaction mixture for the presence of an organic molec~b having a desired property; and (c) isolating from the reaction mixture the organic molecule having the des;led property.
In one embodiment of the second ~spect the method comprises the steps of:
(a) reacting a group of dirrere,1l enzymes replese~ y a diversity of catalytic activities under suitable conditions with a group of dirrere"l sul)~lrdles, thereby producing one or more organic molecllles dirre,e"l from the enzymes and subslrales in the reaction mixture;
(b) screening the reaction mixture for the presence of an organic moleulle having the desired property; and (c) dele" "ining the structure or functional properties characterizing the organic molecule having the desired property.
Other aspects of the invention will become evident upon rererel,ce to the following detailed description.

WO 94/24314 :~16 0 ~ 7 PCT/U594/04314 Detailed DescriPtion of the Invention In the first aspect of the present invention, the method comprises first providing a starting group of cJifferel,l organic molecules.
At least one chemical reaction is c~lsed to take place with at least some of the different organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecules difrere"l from the organic molecules in the sla,li~,g group. This step of causing at least one chemical reaction to take place is repeated at least once. Each repetition uses the reaction mixture of the previous step, and in the end produces a final reaction mixture as a result of the last repetition. The final reaction mixture is screened for the presence of the organic molecule having the desired property.
As noted above, in another aspect, a diversity of compounds is generated from a group of substrates which are subjected to a group of enzymes represeulirlg a diversity of catalytic activities. In still ~"~tl,er aspect of the ,uresenl invention, a diversity of compounds is ge"erdled from a group of subslrales which are subjected to a variety of conditions, in the absence of enzymes. An embodiment of either aspect utilizes a group of substrates with dirrere"l core structures. Another embodiment of either aspect utilizes a group of sut,sLra~es with similar or identical core structures, but a variety of differellt functional groups as substituents. The latter embodiment permits the creation of a diversity of compounds centered around a particular compound or a particular class of compounds.
The methods of the present invention are employed to generale new compounds having a desired property. Examples of ,I~refer,ed desired properties include the ability to function as drugs, vaccines, liganding agents, catalysts, catal,vtic cofactors, structures of use, ~letector molecllles, and building blocks for other compounds. A
liganding agent may bind, for example, to protein, DNA, RNA, carbohydrate, enzyme, receptor, or membrane. Liganding agents ~ WO 94/24314 216 0 4 5 ~ PCT/US94/04314 include agonists and antagonists, such as competitive inhibitors of enzymes or hormones. Structures of use include low energy structures (e.g., structures capable of self assembly) and material structures, like silk. Detector molecules include compounds having optical reporter properties of inleresl. A new compound may mimic, mod~ tei enhance, antagonize, modify, or simulate a substance. Specific molecules of i"lerest include molecules: (1) able to bind to a helper T cell receptor of specific clones of helper T cells (e.g., such binding leads to amplification or leletion of specific helper T cell clones); (2) able to be incorporated into DNA or RNA in place of normal nucleotides (e.g., such incorporation alters biological activity); and (3) able to act as a sub:,L,ale for an enzyme or modify the activity of an enzyme (e.g., may modify the binding activity of a biological molecule). Such molecl~les are useful for a variety of diagnostic and therapeutic purposes. Other specific molecules of in~eres~ include oral contraceptives and molecules with improved properties over analgesics like naproxen, or protease inhibitors like captopril, antitumor agents like mitomycin, antibiotics like vancomycin, and antifungals like amphotericin.
Sul.~lrales for the processes described herein include all organic compounds. A prer~r,ed group of suL,slra~es includes alkanes, alkenes, alkynes, arenes, alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds, organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleotides. A more preferred group of subslra~es incl~ es 2~ acids, amines, alcohols, amino acids, nucleotides, and unsaturated compounds, such as alkenes and alkynes. The most prerer.ed group of subsL,ales is amino acid-based compounds (e.g., amino acids, peptides and polypeptides), nucleotide-based compounds (e.g., nucleotides and nllcleosides), and combinations thereof. These substrates may include additional functional groups as substituents and may be acyclic, cyclic, and heterocyclic in nature. The acids, amines and alcohols can be primary, secondary, carboxylic, phosphoric, sulfonic, aromatic, heterocyclic, aliphatic, etc. For increased reactivity, primary amines and alcohols are prerer,ed.
An aller"dli~/e to the selection of clir~erent substrates with a wide variation in their overall structures is to choose subsLrates that include compounds which are clir~erenl but share one or more common structural features with a molecl~e of i.,le,est or a class of molecules of inleresl. Thus, the diversity of compounds to be ge"erated would be created around a molecule of interest or a class of molecules of inle~esl.
For example, a ringed compound, such as a steroid, may be selected and then a variety of dir~ere, ll derivatives obtained. Derivatives include the addition and/or deletion of functional groups, and acyclic compounds with ringed s~lbstituents similar to a portion of the original cyclic compound. Such derivatives are subjected to the la~ldoll, cl,e,l,islly processes described herein to generaLe a greater diversity, from which a compound having a desired property may be ~letected for further chara~,1t;ri~dlion, with or without issl~tion. For example, a group of subsllates COI)Si~lS of related compounds, which are then subjected to the methods without enzymes as described herein. Alltr"dli~ely, a group of sul~slrales co~,sisls of related compounds plus reagents, which are then subjected to the methods with enzymes as described herein. A
variation upon these embodiments of the present invention is to yel~erale derivatives using the random chemistry processes described herein, and then subject such derivatives to these processes to generate a g,ealer diversity, from which a compound having a desired property may be ~etected for further characleri~alion, with or without isolation.
Classes of molecl~les, which are pre~ened focal points from which to obtain derivatives to serve as substrates, include heterocycles, steroids, alkaloids, and peptides/mimetics (including cGnslrained molecu'es, e.g., constrained by S-S disulfide bonds). Examples of heterocycles include purines, pyrimidines, benzodiazepins, beta-lactams, ~ WO 94/24314 21~ 0 ~ S 7 PCT/US94/04314 tetracyclines, cephalosporins, and carbohydrates. Examples of steroids include estrogens, androgens, co,liso,)e, and ecdysone. Examples of alkaloids include ergots, vinca, curare, pyrollizidine, and mitomycines.
Examples of peptides/mimetics include insulin, oxytocin, bradykinin,captopril, enalapril, and neurotoxins (e.g., from snails, snakes, etc.).
In one aspect, the present invention provides methods for gene~alion of new compounds wherein a group of subslrales are acted upon by a group of "enzymes," such that a diversity of product molecules are formed. As used herein, the term "enzyme" incl~ ~des enzymes (e.g., naturally or non-naturally occurring or produced), catalysts (e.g., catalytic surfaces), candidate catalysts and candidate enzymes (e.g., antibodies, RNA, DNA or random peptides/polypeptides).
In one embodiment, the method comprises the steps of: (a) reacting a group of dif~e,e,)l enzymes representil)g a diversity of catalytic activities under suitable conditions with a group of different subslrales, thereby producing one or more organic molecules dirre~e"l from the enzymes and subslrales in the reaction mixture; (b) screening the reaction mixture for the ,~.rese"ce of an organic molecule having a desired property; and (c) isolating from the reaction mixture the organic molec~ ~le having the desired property.
In a"oll)er embodiment, the method comprises the steps of: (a) reacting a group of cJirrerel,l enzymes representing a diversity of catalytic activities under suitable conditions with a group of dirrere, suL~Irales, thereby producing one or more organic molecules dirrere"l from enzymes and subslrates in the reaction mixture; (b) screening the reaction mixture for the presence of an organic molecule having the desired property; and (c) determining the structure or functionalproperties characte,i~i,)g the organic molecule have the desired property.
From a library of product moleuJles produced by the methods provided herein, those of practical interest are characterized.

WO 94/24314 21~ 0 ~ ~ 7 PCT/US94/04314 ~

As noted above, it is central that, in the present procedures, one does not need to have prior knowledge of the structure or composition of the useful molecule sought. This aspect of the present invention rests on catalysis of, or otherwise causing, a sufficient diversity of reactions among a group of initial substrates, such that a diversity of further products are formed. In order to more fully appreciate the diversities of products which may be generaled by the methods of the present invention, it may be helpful to consider a st~tistic~l analysis of ~he average properties of reaction graphs among a set of moleu~les, as well as the average properties of the catalyzed reaction subgraph among these molecules which is formed when the molecules are incub~ted in the presence of candidate enzymes or catalysts which may catalyze one or more of the reactions.
A reaction graph is the proper mathematical description of a set of organic molecules and all the reactions that those molecules can undergo. Organic reactions can be categorized into cl~sses by the number of sub~t.ale and number of product molecule species. A first class l,a"~ror",s a single substrate into a single product. An isomerization reaction, catalyzed by an isomerase, is an example. A
second class joins two su~slrales to form one product. A dehydration reaction joining two nucleotides by an ester bond, is an example. Such reactions are commonly catalyzed by ligases. A third broad class cleaves one subsl,ale into two products. Cleavage of a polynucleotide by a p hos~l ,odiesterase is a familiar example, as are many steps in inler",e~3iate metabolism. Finally, a fourth class t,a,-sror",s two subsl-dLes into two products. Often this occurs by t.a":,rer of a reactive group from one of the two initial subsl.ates to the second subsl.ate.
A convenient replesenlalion of a reaction graph denotes each organic molecule species as a point in three dimensional space.
One or two lines lead from the one or two substrate molecules derived from the reaction of the substrates. Arrows on the lines leaving the ~ WO 94/24314 216 0 4 5 7 PCTIUS94/04314 substrates point into a box denoting the reaction. Arrows leaving the reaction box for the products point toward the products. Since reactions are reversible, the arrows merely indicate one possible direction of the reaction. The set of all such arrows and boxes, representing all the reactions among all the organic molecules in the system, comprises the reaction graph.
An important feature of reaction graphs is that, for almost any initial set of organic molecl ~les, the reaction graph in which that set is considered as substrales will also require addition of new organic mo'ecules (i.e., molecules not in the initial set of subslrates) where those new organic molecules are the products of one or more of the possible reactions among the initial set of subslrales. In a mathematical process, called the "growth of the reaction graph", the reaction graph "grows" by iterations. At the first step, a set of initial sul,slrale molecl l'es is listed.
At the next step, the reaction graph among those s~,bslrates is formed ",a~her"alically, and a seco"J iterate of the reaction graph is formed by listing both the initial substrates plus any new organic molecule products of the possible reactions. At the third step, all the possible organic reaclio,)s a",o"g this now enlarged set of organic molecllles is written down. This new reaction graph may indicate that still further novel organic molec~lles are products of the reactions now possible. Over a succession of iterations of this mathematical graph growth process, the set of organic molecules incl~ er~ in the graph may increase enormously compared to the initial set of subslrates. This sllccessive increase is called "supracritical behavior." Another possible mathematical behavior of the reaction graph growth processes is that a few new products may be formed on the first graph growth cycle, and sllccessively fewer on the s~ccessive graph growth cycles, until no further new product molecules are generated. Behaviors in which graph growth is limited are termed "subcritical.~

2~4~ ~

lf a set of organic molecl ~les and a set of "enzymes," as defined herein, are present in reaction conditions allowing the enzymes to act on the organic molecules, then the natural mathematical leprese"talion of the total system is the reaction graph, as defined above, plus an accounting of which enzymes catalyze which reactions.
The latter accounting of enzymes and the reactions catalyzed comprises the catalyzed reaction subgraph of the reaction graph. This mathematic re~.rese"lalion is formed by noting, for each candidate enzyme, which reactions if any it catalyzes. An arrow may then be drawn from that enzyme to the reaction box representing the reaction catalyzed, and the arrows into and out of the box representing transformations of subsL,ate(s) into product(s) can be noted in a convenient way, e.g., by coloring those arrows "red." The set of all red arrows represents the reactions which are catalyzed by one or more of the candidate enzymes present in the system.
Just as the reaction graph itself may be subcritical or supracritical in its behavior, so too may the catalyzed reaction subgraph among the organic molecllles. In this case, one considers only the catalyzed reactions among the initial "founder" sul,sl,ates. The catalyzed reactions lead to products not in the founder set of substrates. These new products are available, together with the initial founder set of substrates, to allow further reactions, some of which might be catalyzed by the set of candidate enzymes present in the system. Over a successicn of iterations, this process of catalyzed reaction growth may increase vastly in diversity, in a supracritical mode. Alternatively, the set of novel molecules formed via catalyzed reactions may dwindle over successive iterations of the growth of the catalyzed reaction graph. This is a form of subcritical behavior.
The total behavior of the system is represenled by the behavior of the reaction graph plus the catalyzed reaction subgraph, over iterations. The uncatalyzed reactions represent reactions that occur spontaneously. Whether a reaction graph behaves subcritically or supracritically depends upon the diversity of founder substrates and the diversity of candidate enzymes present in the system. In addition, the behavior is dependent upon factors including conce"L,dlions of all reagents, solubility of the organic molecules, and directions of deviation from equilibrium across each reaction.
In general, a phase transition from subcritical to supracritical behavior of a reaction system is governed by the diversity of organic molecule!s and the diversity of enzymes in the system. Systems with low diversity of both organic molecllles and enzymes are typically subcritical. Systems with high diversities of either organic molecules alone, low diversities of organic compounds together with high divel~ilies of enzymes, or high diversities of both are typically su~,ra.;-itical. Systems of organic molecl~les alone without addition of exogenous enzymes can be supracritical, because the spontaneous reaction graph is supracritical, or bec~use some of the sul sl,ales or products are enzymes themselves in the sense defined above. In one of its forms, the present invention takes advantage of the mall ,emalical phase transition between subcritical and supracritical behavior to choose reaction conditions which yield high diversity libraries of organic molecllles from a founder set of organic molec~es~
The general character of the phase transition from subcritical to supracritical behavior can be illustrated, by way of a non-lillliling example, based on the prefer.ed use of a cloned library of antibody molecu'es as the candidate set of enzymes, and a set of suLsl,ales which, without loss of generality, can be taken to be peptides containing mixtures of D and L amino acids and nonnatural amino acids, or can be taken to be small polynucleotides, or a wide variety of other organic molec~ ~ies. To illustrate the general character of the phase transition it is useful to estimate the number of reactions in a reaction graph with a given number of small organic molecules. In general, the WO 94/24314 216 0 ~ ~ 7 PCT/US94/04314 number of reactions is not known. However, minimum realistic estimates are obtainable. For example, a founder set of peptides made of D and L
amino acids and non-natural amino acids, each with 10 amino acids, may be used as substrates. The number of possible subsLrates is very large, and given by the number of kinds of amino acids raised to the tenth power. Any two peptides length ten can undergo transpeptidation reactions cleaving and excl,a"~ing the terminal amino acid(s) subsequences at any of the internal peptide bonds of each of the dec~reptides. Since there are 9 internal bonds in each, any pair of lec~reptides can undergo 81 such transpeptidation reactions. In each case, two substrates yield two products. Since any pair of decapeptides can undergo 81 transpeptidation reactions, it is clearly an uncJereslir"ate to suppose that the two peptides can undergo only 1 transpeptidation reaction. But even with this clear undere~li",ale, the number of reactions in a system with a diversity of N types of peptides is equal to the number of possible pairs of peptides, hence equal to N squared.
The same general features occur with many classes of organic mobcl~'Qs undergoing reactions with two sut slrdLes and two products.
For most pairs of organic molecu~es, it is conservative to estimate that the two can undergo at least one reaction to form two products. Thus, in general, N squared is a conservative e~limale of the diversity of reactions in a reaction graph with N kinds of organic molecu!es Again, as a non-limiting example to illustrate the general character of the phase transition in catalyzed reaction graphs, a set of 100,000,000 cloned human antibody molecules is used as the set of candidate enzymes. Based on the statistics of generating catalytic antibodies (as described below), the probability that a randomly chosen antibody molecule is able to catalyze a randomly chosen reaction is between 10-5 and 104 (Pollack et al., Science 234:1570, 1986;
Tramontano et al., Science 234:1566, 1986; Tramontano et al., Proc.
Natl. Acad. Sci. USA 83:6736, 1986; Jacobs et al., J. Am. Chem. Soc.

~ wo 94/243l4 21 6 0 ~ ~ 7 PCT/US94/04314 109:2174, 1987; Pollack and Schulk, Cold Spring Harbor Symposium on Qu&n(its~li,/e Biology Vol. 52, 1987; Tramontano et al., Cold Spring Harbor Symposium on Quantitative Biology Vol. 52, 1987). The more - c~"se,~/ative estimate, 10-8, may be used for illustration. Reaction systems in which the diversity of subsl,ale molecl~les is varied are considered, and this diversity noted on the Y axis of the Cartesian coordi"dle system. Simultaneously, the diversity of the candidate set of enzymes is varied and noted on the X axis. Low diversity of substrates and candidate enzymes almost certainly yields subcritical reaction systems. To be concrete, a system with two subslrales, hence one reaction, and a single randomly chosen antibody molecule is considered. The clla,lce that this antibody molecule acts as a catalyst for any of the four single reactions afforded by the sul)slr-dles is 10-8. Thus, almost certainly, no catalyst for the reaction is present in the system, and the formation of no novel product is catalyzed. The system is subcritical. A high diversity of subsl,ates and candidate enzymes will be supracritical with high probability. A diversity of 1,000 organic mol~cu'es is incl~b~ted with a diversity of 1,000,000 antibody molecules in an appropriate reaction vessel. The number of reactions among the 1,000 organic molecl~les is at least, by the conservative estimate, 1,000,000. Each reaction might be catalyzed by any one of the 1,000,000 candidate antibody enzymes, and each antibody has a chance of one in a hundred million of being able to act as a catalyst for each reaction. Thus, the exrecte~ number of reactions for which antibody catalysts are present in the system is 106 X 106/108 = 104. Thus, 1û,000 reactions among the million possible should be catalyzed by one or more of the antibodies present. Thererore, as these catalyzed reactions occur, the products of the 10,000 reactions will be formed. Most of these will differ from the 1,000 substrate molecllles initially present.
Thus, the diversity of the set of organic molecules has increased. After sufficient time has elapsed for the cGnce~ ,I,dlions of these novel WO 94124314 21 i~ 0 ~ 7 PCTIUS94/04314 ~

molecl ~les to increase sufficiently, the new system has a diversity of substrates on the order of 10,000 rather than 1,000, hence, now a diversity of 10,000 squared reactions are possible among the enlarged set of subslr~les. The expected number of reactions which now find catalysts among the antibody molecl ~les is thus 1o8 x 106/1o8 or 1,000,000. Thus, within two reaction steps of the founder set of 1,000 organic molecules, the diversity of organic molecl l'es has increased to about 1,000,000. Over s~ccessive reaction cycles, diversity will increase.
This is supracritical behavior.
In general, in the X-Y plane, a roughly hyperbolic curve separates a subcritical regime near the origin, represe, Itil ,9 low diversities of founder substrates and enzymes, and a supracritical regime with high diversity of initial subslrales, enzymes or both. With a fixed low diversity of substldles, the system can cross into the supracritical regime if a high enough diversity of enzymes is present. Conversely, if the enzyme diversity is fixed rather low, the system can cross into the supracritical regime if a high enough diversity of subsl,ates is present.
The actual shape of this roughly hyperbolic curve depends upon the specific way the number of reactions increases as subsLIate diversity increases, which in turn depends upon the particular set of organic mcleclJIQs used as founder subsl-ales. The curve also depends upon ~he distribution of probabilities that antibody molecules cataîyze the dirr~ren~ reactions arrorded by the founder subsLra~es and their prodllcts.
However, for all these cases, a sufficient diversity of both subslrales and enzymes leads to supracritical behavior. The diversity of organic molecl ~les in the system will increase dramatically via the catalysis of connected webs of reactions leading from the founder set of organic moleu~les to an increasing diversity of their pro~lucts It is important to em,chasi~e that a system of substrates alone can explode into a diversity of products, even in the absence of exogenously supplied enzymes, if the substrates are present in sufficient ~ WO 94124314 216 0 4 S 7 PCTIUS94/04314 diversity and high enough concentrations to interact on a reasonable time scale. For example, a large diversity will be generaled if the spGnlaneous reaction graph is supracritical. However, systems with exogenously added enzymes are preferred.
The human antibody repertoire is used herein as a non-ilin~ example of a set of candidate enzyme moleclJ~es. As is known in the art, the combinatorial diversity of human antibody molecu'es due to genomic lear,d"yement is on the order of 100,000,000 (prior to the onset of somatic mutation during maturation of the immune response which further increases potential diversity). As is also known in the art, antibody molecules can function to catalyze a wide variety of reactions with a rate (\/max) ~cceleration of three to eight orders of magnitude compared to the spontal,eous reaction. Such catalytic antibodies are commonly gel,eraled by immunization of an immune competent animal with a molecule that is a stable analogue of the transition state of the desired reaction. MonoclGI)al antibodies are generated from this immunization, and each is tested for its capacity to catalyze the desired reaction. Rec~use the stable analogue is similar chemically to the transition state of the reaction, typically on the order of 5% to 10% of the ",onocloilal antibodies tried are able to catalyze the desired reaction.
Presumably the catalysis rerle~;ts high affinity for the transition state and lower affinity for sub:,lrales and products.
It is possible to estimate the probability that a randomly chosel) antibody molecule will be able to catalyze a given, randomly chosen reaction. The fraction of B cells which respond to immunization with an arbitrary epitope bearing antigen is on the order of one in a hundred thousand. B cells which respond to an antigen typically have modestly high affinities for the antigen to be triggered to divide. Thus, the probability that a randomly chosen antibody molecule can bind with modest affinity, 104 M-', to an arbitrary antigen is about one in a hundred thousand. The monoclonal antibodies used to create catalytic WO 94/24314 216 0 ~ ~ 7 PCT/US94/04314 ~

antibodies may have undergone further somatic mutation that increased amnity for the antigen. It is reasonable to estimate the probability that a Idlldolllly c:l,osen antibody has high affinit,v for an arbitrary antigen is about 10~ to 10~. -It is further well known in the art that a cloned high diversity library (10~ or more) of antibody molecules can be and has been created in a variety of ways. Thus, such antibody libraries are a non-li,~ iny example of a high diversity set of candidate enzymes.
The use of a repertoire of human antibodies as a set of candidate enzymes is a ~.ret~r,ed, but non-limiting example of the sets of molecules which can advantageously be used as sets of enzymes.
Additional candidate sets include the following:
(1) Libraries of fully ,anclo"l or partially slocha~lic polynucleotide sequences, DNA or RNA, which, upon translation yield libraries of fully or partially stochastic pe~liJes, polypeptides or protei.ls.
These libraries can be cloned in prokaryotic or eukaryotic hosts to amplify the polynucleotide sequences and obtain ~.roleil, products which col,~lil.lte the candidate enzyme library. Allerllali./ely, the polynucleotide sequences can be amplified in vitro and translated in vitro to obtain the candidate enzyme library. If needed, the candidate protein library can be isolated from other molecular components by means known in the art. For example, an advantageous means to do so uses libraries of fusion prolei,ls with stochastic peptides, polypeptides, or proteins fused Adj~cent to, for example, ubiquitin. Antibodies to ubiquitin allow affinity purification of the librar,v of fusion protei,ls which then serves as the set of candidate enzymes.
(2) A library of antibody molecules can be derivatized by cloning partially stochastic DNA sequences into the hypervariable region of the antibody molecules. A refinement of this involves cloning such stochastic sequences into one or more of the complement delel"lining regions (CDRs), of the antibody molecule. Each CDR has on the order ~ WO 94/24314 21~ 0 ~ 5 7 PCTJUS94/04314 of 5 to 10 amino acids. This modified library is a set of candidate enzymes.

(3) Partially stochastic DNA sequences or RNA
- sequences can be cloned into a gene encoding any protein, e.g., h;;,lo"e 1 or any other protein, to create a fusion protein with the novel DNA or RNA at one end, or in the middle of the host protein sequence.
The well folded host protein serves as a framework to aid folding and stability of the cloned sequences. The set of such proiei. Is is a library of candidate enzymes.

(4) Libraries of DNA sequences in themselves, or RNA
sequences in themselves, co,lslilute libraries of candidate enzymes. The e~isLence of ribozymes and of DNA sequences able to bind arbitrary ligands, such as thrombin, show that both kinds of polymers are strong can~ tes to bind transilioll states and catalyze reactions.

(5) Other libraries of combinatorial molecular diversity, linear sequences or otherwise, as known in the art, may be used as candidate catalysts. Sets of known enzymes alone, or together with a small or large variety of mutant variants of those enzymes, can serve advantageously as the ca"didale set of enzymes. More specifically, and as a non-li.)~iling example, the sut~sLrales of i"leresl in generaling a library of molecules may be D and L amino acids, including "onnal.lral amino acids, and some small dipeptides, tripeptides, and tetrapeptides formed of these building blocks. It is known in the art that larger peptides can be synthesized from amino acids and small peptides using prote~ses, peptidases, lipases, hydrolases, and eslerases (Schellenberger and Jakubke, Chem. Int Ed. Engl. 30:1437, 1991).
Thus, such a set of enzymes can be used jointly in a common milieu, or sequentially, acting on a set of substrates. Further, it is known in the art that it is possible to select mutant variants of proteases which are able to alter substrate specificity, or alter catalytic activity in unusual solvents, such as low water dimethylformamide solvents (Arnold, Proc. Natl. Acad.

WO 94124314 216 0 ~ ~ 7 PCT/US94/04314 ~

Sci USA, in press 1993). Thus, in a preferred embodiment of the present invention, libraries of mutants of each of a set of enzymes of i"Leresl are used. For each enzyme, length N, there are 19N one mutuant variants, and on the order of that number squared of two mutant variants. Hence, a library of severai million mutant proteins of a given enzyme, obtained by means known in the art, can be readily prepared. For a diversity of ten dir~t rent initial enzymes, lip~ses, hydrolases, eslerases, and proteases, the resulting library of candidate enzymes has on the order of 100,000,000 cJirrere"l protein species, each a candidate enzyme. These are then incllb~ted with the founder sul.~l,a~e library of inlerest.
Increase of the diversity of candidate proteins from 1,000,000 (described below in a non-limiting example based on antibody molecl~les) to 100,000,000, tog~her with a maximum 10mg/ml solubility of these ploteins, implies that product molecu!es will form more slowly. Hence in a 1,000 microliter volume, it would require about 1 second to geneiale a 1 nano",olar conce"l-alion of a product molecule from saturated enzymes using a diversity of 1,000,000 candidate enzymes, and about 100-fold longer using a library of 100,000,000 candidate enzymes. The example of small D and L peptides is non-limiting. Other core building blocks, carbohydrates, heterocyclic compounds, a variety of ~d~ts, and otherwise, can be used as the starting library of substrates in all the methods of the invention.
Where traditional protein-based enzymes are used to effect a diversity of catalytic activities, such enzymes include oxidore~ ct~ses;
l,~nsrerases; hydrolases; Iyases; isomerases; and ligases.
Oxidoreduct~ses catalyze oxidation and reduction reactions. Examples of oxidore~uct~qses include dehydrogenases; redlJct~ses; oxidases (monooxygenases and dioxygenases); and peroxidases. Trar~s~erases catalyze the transfer of functional groups. Examples of transrerases include aminotral)srerases (transaminases); phosphotransferases;
pyrophosphokinases; and nucleotidyltransferases (RNA and DNA

polymerases). Hydrolases catalyze the hydrolytic cleavage of bonds, such as ester, glycosyl, and peptide bonds. Examples of hydrolases include phosphodieslerases; amylases; proteases (peptidases, ~roLei.,ases); nucle~ses (exo- and endo-; ribo- and deoxyribonucle~ses);
and phosph~l~ses Lyases catalyze double bond formation by non-hydrolytic removal of groups from subsLrales. Examples of Iyases include decarboxylases; anhydrases; and sy"Ll,ases. Isomerases catalyze geometric or structural changes within one molecule. Examples of isomerases include racemases; epimerases; tautomerases; and m~ ~t~ses. Ligases catalyze the joining together of two molecules coupled with the hydrolysis of pyrophosphate bond. Examples of lig~ses include synthet~ses Generation of useful high diversity libraries requires that the suL,slrales be soluble in the solvent, that the candidate enzymes be soluble in the solvent, that the volume be sufficiently small and c~"ce"trdLions sufficiently high that subslrdtes and enzymes encounter one arlulller rapidly, and at high enough co"cenL,dLions to occupy a sufficient fraction of enzymatic sites to enhance reaction velocities, and that the high diversity product library be present in high enough cGIlcel llrdLions that useful molecu~es can be rletected. All these requirements have been considered for the present invention. For example, enzymes typically can tolerate some percenLage of organic solvents such as ethanol, methanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or combinations thereof, in aqueous (water based) solutions (Gupia, Eur. J. Biochem. 205:25, 1991). Thus, where not all the substrates are water soluble, it is desirable to include water-miscible organic solvents.
SubsLr-dLes of the types indic~ted vary in solubility. In general, it is reasonable to obtain millimolar conce"L,dLions of on the order of 1,000 substrate species in small reaction volumes, on the order of 1 to 100 microliters. Under reaction conditions such that the diversity WO 94/24314 21 ~ 7 PCT/US94/04314 of these 1,000 sub~L-ates increases by a factor of 1,000,000, yielding 1,000,000,000, or a library of small molecules with a diversity of 10~, the average co"ce"~ralion will have fallen by a factor of 10~, hence have fallen from millimolar to nanomolar, 10~ M. The detection methodologies discl ~ssed below to identify a molecule of interest are able to detect readily in the ,a"Gi"olar range, and typically are able to detect in the picomolar, 10-l2 M, range. Thus, even with a 1,000-fold decrease in co"ce, It.alions of some products below the mean when diversity is one billion, the detection means can detect molecllles of i"~elesl. Other detection procedures allow detection at 10 t5 to 10 20 molar.
The diversity of candidate enzymes in a reaction mixture is limited by the solubility of the enzymes. For example, for proteins in ~lueolJs media, a 10mg/ml col)cenl,alion is typically attainable. For candidate enzymes with 200 amino acids, on the order of 10X protein molecules can be in solution in 1,000 microliters. Thus, if a diversity of 106 candidate enzymes is used, each will be present in 1014 copies.
Catalytic antibodies are of modest efficiency, as noted. Using a turnover number of 1 per second, 1014 saturated enzymes would yield 10 product molea~les in 1 second. In a 1,000 microliter volume the col,ce"l-alion of the 10'4 product molecules would be on the order of 0.1 micromolar. Even if solubility limits were 1 mg/ml of enzymes, then col,ce,)l,alions would decrease by only one order of magnitude. Thus, high diversities of substrates and candidate enzymes can be mixed under reaction conditions which yield a high diversity of products via a catalyzed web of reactions on practical time scales.
In an embodiment of this aspect of the present inven~ion, a group of enzymes representing a diversity of catalytic activity are separated in part or in entirety from one another and the subsl,ales contacted sequentially. For example, a group of enzymes are separated by membranes, such as dialysis bags, or by immobilizing dirrere"L
enzymes (represellling dirr~re~1l catalytic activities) on solid supports, ~ WO 94/24314 21~ 0 4 5 7 PCT/US94/04314 such as resins. Candidate enzymes can be localized on phage, using phage display libraries as is known in the art, or other means to ge"e,ale and display combinatorially diverse libraries of molecules, such as peptides or other molecules on beads, or surfaces, or polysome trapped peptide libraries. Additionally, candidate enzymes may be contained within or displayed upon one or more types of eukaryotic or prokaryotic cells, the cells and the substrates being brought into contact.
In any case, a group of subsl,ates is circulated (e.g., by peristaltic pump) through the separated enzymes. For example, substrates are circul~ted in and out of dialysis bags with pore sizes which prevent escape of the enzymes. Subslrates are bound by the first set of enzymes, modified, rele~sed and ciru~l~te~l to the next set of enzymes.
Aller"dli-/ely, a group of subslra!eS may be co"ri"ed and enzymes having one or more catalytic activities circl ~ te~l through sequentially. In genelal, the reactions are cond~cted over a period of several hours at temperatures of about 37C or below. Cofactors such as ATP, NADH, 2 and CoA are added where appropriate. Many of the cofactors may either be added directly or generated in situ. For example, 2 may be introduced by injecting the gas or air directly into the reaction mixture or by use of an electrode to ge"erale 2 An electrode need not directly contac;t a reaction mixture, but rather may be introduced into a compartment from which 2 may pass to the reaction mixture. For example, an electrode may be placed inside of a dialysis bag which in turn is surrounded by a dialysis bag containing a set of enzymes. It will be readily appreci~ted by those of ordinary skill in the art that a group of subsLrales may be subjected to the various separated enzymes in a variety of orders. Further, it will be evident that after subjecting a group of subsl-ales to the various separated enzymes, one or more steps may be repeated if desired. The repetition of steps need not be in the order initially performed and additional substrates may be intro~ ced at any step if desired.

WO 94124314 21~ 0 ~ 5 7 PCT/US94/04314 ~

In another embodiment of the present invention, a combinatorial library of organic molecules or other mo'~cu~es, which are similar to an initial molecule of inlelest, are generated by derivatization of the initial molecule in a very large number of possible ways to produce a high diversity library of "local" mimics of the initial molecule of i"~eresl.
Within the pr~sen~ invention, ~vo ways are provided for generating such a library, one which does not use enzymes, but uses a variety of possible ~d~ ~cts or other molecules which may undergo reactions with the initial mol~cl~e of inleles~, and also uses a variety of chemical reagents and physical conditions to drive the synthesis of a library of derivatized products of the initial molecule. Aller"dLi~ely, the core initial molecule plus a set of candidate adducts and other molecules which may react with the initial molecule are used, but also incll ~ded is a set of enzymes which may increase the rate of formation of the local high diversity library of derivatked forms of the initial compound. Based upon the prese"l d;sc~osllre provided herein, it will be readily appresi~ted by those of ordinary skill in the art that the methods ~or producing general high diversity libraries of product molec-~les and for producing local high diversity libraries of derivatized forms of an initial compound may be combined. For example, a new initial compound may be generated by the general procedure (e.g., subsllates with dir~ere,ll core structures).
Such a new compound is then used, with or without derivatives, to g~"~rale a local high diversity library of derivatized forms of the compound. Further, it will be evident to those of ordinary skill in the art that libraries may be generated using a combination of the methods herein without enzymes and the methods herein with enzymes.
Generation of a high diversity library of derivatized forms of a steroid hormone core, such as estrogen, is used as a non-limiting example. A set of reactants, including estrogen and a variety of other small molecules which are candid~tes to react with estrogen to form new product molecl~les partially or entirely containing the steroid core, are ~ WO 94/24314 216 0 4 5 7 PCT/US94/04314 7ed in a common reaction milieu. These are reacted in the p~esence of a set of enzymes to catalyze the reactions arro~ded by the system.
Enzymes can be chosen by a number of means, some known in the art, others specified herein. The formation of a library of derivatized molecu!es under these reaction conditions can be ~ssessed by a number of means known in the art. For example, the steroid core may be radioactively l~heled at a varietv of posiliol1s. Tl,erearler, the reaction mixture can be subjected to HPLC analysis, mass spectrograph analysis, or other modes of analysis to test for the diversity of molecl ~les which are labeled. All radio~ctively labeled moleuJles contain atoms derived from the steroid core, hence the new molecule species are at least partially comprised of the steroid core. If it is desirable to assure that a large part of the steroid core is contained in the novel species, then two or more di~lil ,cL radioactive labels can be used to label distinct and distant atoms in the core. Simultaneous presence of all labels suggests strongly that those pGI lions of the steroid core are intact. Aller"alives to radioactive labels include isotope labels and other means known in the art. The high diversity library is tested (e.g., by means described herein) to Jeler")i,le if it contains molecllles of inleresL. If such molec~les are detecteli~ they may then be isolated by a variety of means, including sib selection as described herein.
The detection of molecllles which are candidates to act as antagonists of estrogen is ~isc~ ~ssed first as a non-limiting example of detection of one or more molecl~es of i"le,esl in the library of this eslroge" example. Detection of molecules which have higher affinity than estrogen for the estrogen receptor (and hence which may be of use in hormone replacement therapy at lower concenL,aLions and thus lower side effects than estrogen itselfl is f~iscllssed as a second non-limiting example. Detection of candidate antagonists in the reaction mixture may be accomplished by use of very high specific activity radioactive estrogen bound to receptors by means known in the art. Unlabeled WO 94/24314 216 0 4 ~ 7 PCT/US94/04314 ~

competitors in the library will displace the labeled estrogen, and this competitive interaction can be ~ietected by loss of label.
Detection of candidate high affinity agonists for replacement therapy may be carried out by use of appropriate cell assays similar to the frog melanocyte assay or the use of pH changes described in detail herein. Presence of a high affinity agonist in the reaction mixture is demonstrated bec~l~se a very low co"ce,ltrdlion of the agonist compared to estrogen suffices to trigger the cell response.
Such assays may be carried out in the absence of estrogen, or in the prese"ce of increasing co"ce"l,aLions of estrogen. In the latter case, cell r~sponse at lower co"cent.alions of estrogen than would elicit a response with estrogen alone, detects the presence of an agonist in the high diversity library. If the agonist can act alone to trigger the cell response, then during the sib selection winnowing procedure, as its col,ce,-t-alion increases the tl,resl,old level of eslrogen required for triggering a cell response will dwindle.

The creation of a set of candidate enzymes able to catalyze reactions derivatizing the core molecule, e.g., estrogen, is carried out by selecting from a large set of enzymes ffor example the mouse or human immune repertoire), a subset of candidates which bind to the initial set of sul~lrates, the core molecule plus the candidate sub~lrates which are to react with the core and derivatize it. This set of enzymes may then advantageously be enlarged by generating a mutant variant spectrum of each. The purpose of this step is the following: The enzymes have been sele~ted bec~ ~se they bind to the s~ bslrales of the potential reactions, rather than selectively binding the transition states of the reaction. Generation of mu~ant spectra around each such initial enzyme which binds substrate(s) increases the probability that the mixture of enzymes will include candidates which bind the transition state of the reaction, hence are improved candidates to catalyze the reaction.

~ WO 94/24314 21 ~ O ~ ~ 7 PCTIUS94/04314 In a repetitive procedure, a sl ~ccession of candidate enzymes can advantageously be selecterl as candidates to catalyze the s~ccescio,~ of reactions steps leading away from core molecule for example the steroid core, and the initial ~ cts, to generate the high diversity library. At each reaction cycle with a given set of molecl~le, an enlarged set of molecules, many derivatized forms of the core molecule, will be generated. In order to find further enzymes to catalyze the next reactions a~rded by the enriched reaction system to create still further derivatized molecules of the core, it is advantageous to select from a high diversity library of candidate enzymes, new can~ tes which may act on the newly formed species of product moleu~les These new candidate enzymes plus their mutant spectra, as well as the previously identified candidate enzymes, may be used in the s~bse~luent reaction cycle to catalyze the fo~ dLion of still more kinds of derivatized forms of the core molecule. Given limited enzyme solubility, in order to keep the co"ce"lrdlions of critical enzymes as high as possible, it can be advantageous to utilize only the newly identified candidate enzymes, plus their mutant spectra identified from the high diversity library of candidate enzymes, plus the set of candidate enzymes from the last cycle or few cycles of the reaction sequence leading from the core molecule and initial ~-~duch. In CGIllrdSl, candidate enzymes leading from the initial core and initial ~ducts, can be advantageously eli.ninaled in later iterative steps, since they have already acted to catalyze formation of their prorlucts.
For example, one means to identify such further enzyme candidates at each iterative step cGI)si~ls in labeling the substrate and the product molecules in the reaction mixture, each at a variety of positions, with radioactive iodine. The purpose of labeling a variety of positions on each compound with iodine is to assure that the iodine labeling of at least some members of that species of compounds will not prevent binding of candidate enzyme moleulles at almost any WO 94/24314 216 0 ~ 5 7 PCT/US94/04314 ~

compound site ur~ ,deled by the iodine label. These labeled molecules are then reacted with the high diversity of candidate library enzymes, for example with human antibody moleu~les, to detect which antibody molecules bind the labeled molecules from the reaction mixture. This set incl~ Ides antibody molecules which bind the novel product molecules created in the reaction system. The antibody molecules plus their mutant variants are then used to enlarge the set of candidate enzymes.
A variety of means are known in the art to identify the antibody mo'eclJles which bind iodine labeled molecules in the reaction mixture. Among these, it is advantageous to use plaque assays or cell assays ex~,ressing the antibody library to test which plaques or cells bind iodine î~heled material. If a fluoresce,)l label is used instead of iodine, it is advantageous to make use of the natural display of antibody mo'Qc~les on cell surfaces of immortalized B cells, where each such monoclonal antibody producing cell displays its unique antibody. It is then advantageous to e~pose the population of cells to the fluoresce, labeled molecules in the reaction mixture, then sort the B cells. Those immortalized cells which are l~heled ge"eraLe antibody molecules which bind the labeled molecules from the reaction mixture. These immortalized cells can be grown to create a librar,v of monoclonal antibodies which are the candidate enzymes. In addition, it is possible to select antibodies, or other sequences which consLilute the further enzymes at each iterative step ~ lded to above by using the product molecules to create affinity columns, then using the columns to select s~lbset~ of libraries of phage displayed antibody molecules, polysome trapped antibodies, or libraries of DNA or RNA aptomers, or other sequences which bind the products on the column hence which may function as candidate enzymes. Thus, in addition to the use of a high diversity antibody library to find candidate enzymes, it is also possible to use other high diversity libraries. Among these, it is preferred to use ~ WO 94/24314 21 6 0 4 5 7 PCTIUS94/04314 high diversity RNA libraries, DNA libraries, and libraries of stochastic peptides alone or as fusion proteins with a variety of evolved proteins.
Another pre~er,ecl means to create a set of candidate enzymes which may help derivatize a core molecule with a set of ~dducts or other subsLrales, consists in using known enzymes involved in the normal biosynthetic pathway leading to the core, plus mutant varia, Its of those enzymes. Similarly, known enzymes utilizing any of the cts as subslrales, plus mutant variants of those enzymes, may be used. ,n order to catalyze a s~ ~ccession of reactions from the core molecl~le and further novel s~bslrales which may react with it, it is advanta~eo~ ~s to use the substrates and products present at each iteration of the reaction cycle to identify the enzymes which bind subsl.ales and/or products, then create further mutant spectra of these identified enzymes as candidates to catalyze the next reaction steps from the core molecule. Enzymes which bind subsl,ates and products can be identified by means known in the art, including binding assays to cloned enzymes via plaque or other assays. It is also advantageous to use a set of candidate enzymes formed by the union of a set of known enzymes and their mutant spectra, as just desc,ibed, plus a set of can~ tes derived from a high diversity library of candidates, such as the mouse antibody repertoire as described above.
In all the embodiments of this invention it can be advanl~geo~s to use procedures to select subsl,aLes at each of the stages of amplification of diversity which are good candidates to undergo reactions which yield a desired molecule of interest. A
procedure to do so consisl~ in creating sets of "shape-complements" to the "shape" of the desired target, then using the sets of shape-complements to bind and affinity select candidate substrates whose own "shapes" are similar to the target shape of the desired molecule. As a non-li",ili"g example, if the target molecule of interest is estrogen, it may be used to ge"era~e a set of monoclonal antibodies against estrogen, or WO 94124314 216 0 ~ ~ 7 PCTIUS94/04314 ~

a polyclonal serum against estrogen. These antibodies can be used to affinity purify candidate s~b~L~ s with shapes similar to estrogen.
Re~ions building upon these candidate substrates can be carried out, and the products searched for estrogen mimics.

6 In addition to antibodies, other shape diversity libraries ofDNA, RNA, or otherwise can be used to find shape-comple",e,lts to the target mol~cule~ here estrogen.
This "target shape" procedure can be advantageously extended in three ways. First, among the antibody molecules binding to estrogen ("rank one" antibodies), some will bind to the active site or the vicinity of the active site, and others will bind to other sites. These may be disc,i,l,inated by using the antibodies, each as a monoclonal, to ge"erale antiidiotype antibodies ("rank two" antibodies) by means known in the art. Any rank one antibody which generates a rank two antiidiot,vpe antibody that competes with estrogen for the binding site on the rank one antibody is likely to be a rank one antibody whose binding site actually binds the active site of estrogen. The set of each such rank one antibodies can be used to affinity select candidate subslrales with shapes similar to estrogen.
Second, the set of second rank antigens which compete with estrogen for binding sites on rank one antibodies can be used to affinity select candidate enzymes which will act on estrogen-like subslrales to yield estrogen mimics.
Third, this set of rank two antibodies can be used to generate "rank three" antibodies which can be used to affinity select a wide variety of estrogen-like subslrales. In addition to antibody mo'Qcu'~s and antiidiotype antibody molecllles, other sets of shape and shape-complement molecules, including DNA, RNA and other complex molecules can be used. These can, as one non-limiting example, be sele~ted from high diversity combinatorial libraries of molecules.

~ WO 94/24314 216 0 ~ ~ 7 PCT/US94/04314 In another embodiment of the present invention, a group of molecules are used which contain autocatalytic sets, e.g., ~lto~t~lytic sets of catalytic polymers. Reaction mixtures comprise such organic molecllles which are simultaneously substrates and catalysts. Re~ctions are carried out in a chemostat under flow conditions. For example, molecu'es A, B and C are present wherein molecule B catalyzes its own formation out of suL~l,ate molecule A, and molecule C catalyzes its own formation out of sut,~l,ale molecule A. This reaction is carried out in a chemostat where a receptor mo'ecuie, such as acetylcholine receptor is affixed to the walls of the chemostat and can bind any molecule that looks like acetylcholine. In this example, molecule B but not C looks sufficiently like acetylcholine to bind to the receptor for acetylcholine that is on the chemostat walls. Under flow conditions, the B molecule will tend to be selectively retained within the chemostat and the C molecule will not be retained. This provides selective conditions which leads to the selective amplification of the B autocatalytic set compared to the C
~ ~toc~lytic set. For example, if B, even when bound to the receptor acts as a catalyst leading to its own formation, then its retention within the system is selectively favored, and B is amplified with respect to C.
More generally, in a complex reaction mixture in which molecule B
funcLiG,)s as a catalyst in its own formation out of the complex reaction mixture, then retention of B is selectively favored bec~se it binds to the receptor for acetylcholine. Thus, in general, by taking a system under chemostat conditions in which one has a receptor for a molecule X, where fi~n~ing analogs of X is of i"lelest (X here is for example acetylcholine), then this is a general procedure to select among ~utoc~t~lytic sets for those sets synthesizing X-like mimics. Hence, this selective method enhances the capacity to use random complex reaction mixtures to s~,LI ,esi~e drug candidates able to mimic X.
In another aspect of the present invention, methods are provided for generation of new compounds without the use of enzymes.

21604~7 In one embodiment, the method comprises the steps of (a) reacting a group of cli~ere,)t subslrales, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehy.l~ ~ti"g agent to yield a first reaction mixture; (b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture; (c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture; (d) pe"or,)1ing a cG"der,salion reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture; (e) exposing the fourth reaction mixture to light of wavelength of about 220 nanometers to 600 nanometers, thereby producing one or more organic molecules di~erel1L from the substrates and agents; (f) screening the exposed fourth reaction mixture for the presence of an organic molecule having a desired property; and (g) isolating from the exposed fourth reaction mixture the organic molecule having the desired property.
In anolller embodiment, the method comprises the steps of: (a) reacting a group of di~erel ,l substrates, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehydrating agent to yield a first reaction mixture; (b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture; (c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture; (d) performing a cGndensalion reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture; (e) exposing the fourth reaction mixture to light of wavelength of about 220 nanometers to 600 nanometers, thereby producing one or more organic molecules different from the substrates and agents; (f) screening the exposed fourth reaction mixture for the ,uresence of an organic molecule having a desired property; and (9) ~ WO 94/24314 216 0 4 5 7 PCT/US94/04314 cleler,))ini"g the structure or functional properties characterizing the organic molecule having the desired property.
In this aspect of the present invention, a group of dirrerenl suL.~t,ales, such as those described above, are subjected to a series of reaction conditions from which one or more compounds having a .lesi~ed property are produced without the use of enzymes. More specifically, a group of dirrere,~t subslrales are reacted under suitable conditions with a dehyd~ling agent to yield a first reaction mixture.
Suitable dehydrating agents include carbodiimides, carbonyldiimidazole, sulfonyl halides, pl,osge~e equivalents and activated phosphoramides, as well as other agents in common use for solid phase peptide synthesis and nucleotide synthesis, etc. It will be evident to those of ordinary skill in the art that the most prefel,ed solvent(s) are dependent upon the particular group of suL,~l,a~es selecterl For example, if all the substrates are fairly polar in nature, a solvent such as methanol may be used.
CG"ce,lt,aled solutions of individual subsl,ales are made and then the group of substrates prepared by mixing aliquots of each conce~llrdled solution. Mixtures of solvents which are miscible with one another (i.e., do not form two phases) are appropriate where all the substrates are not soluble in a single solvent. Examples of solvent mixtures are acetone and water, dimethyl formamide and water, or ethanol and water.
Reaction conditions may be varied, but generally the reaction will be performed from about one hour to over, I.ghl at a temperature from about room temperature to the boiling point of the solvent.
The first reaction mixture, such as that described above, is reacted under suitable conditions with a reducing agent to yield a second reaction mixture. Suitable reducing agents include dissolving metals, hydride reagents, molecular hydrogen with suitable metal catalysts (e.g., platinum, palladium, nickel or rhodium), etc. Examples of reducing metals include sodium, lithium, potassium, various amalgams, calcium, iron, and tin. Examples of hydride reagents include sodium borohydride, lithium aluminum hydride, and borane. Reaction conditions may be varied, but generally the reaction will be performed from about one hour to overnight at a temperature from about room temperature or below (e.g., in an ice bath). It will be evident that certain reducing agents perform best in certain solvents. For example, where hydride reagents (such as sodium borohydride) are used, it will be evident that non-hydroxylic solvents (such as dimethylformamide) are ~.re:ter,ed.
The second reaction mixture is reacted under suitable conditions with an oxidizing agent to yield a third reaction mixture.
Suitable oxidizing agents include ozone, peroxides, chromate, perma"ga"ale, osmium tetroxide, chlorine, bromine, and air in the prese"ce of suitable metal catalysts (such as ruthenium tetroxide).
Re~tion conditions may be varied, but generally the reaction will be performed from about 1-2 hours to over, li~ht at a temperature from about room temperature or below (e.g., in an ice bath). It will be evident that certain oxidizing agents function best in certain solvents. For example, a mixture of water and alcohol may be used with hydrogen peroxide, but water only with permanganate, and l,exane (or petroleum ether) with halogens such as chlorine or bromine.
A CGI ,de"sation reaction is performed under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture. The third reaction mixture may be subjected to CGI ,dens&lion by dehydrating agents or heat. Suitable dehyd~li"g agents include molecl ~l~r sieves, carbodiimides, ~eoLropic distillation (to remove water), etc. For example, toluene may be added and then azeotropic distillation performed to remove water. It will be evident that reaction conditions vary depending upon the type of dehydration agent used.
The fourth reaction mixture is exposed to light. The light generally is within a range of about 220 nanometers to 600 nanometers, which ineh~es portions thereof or discrete wavelengths if desired.
Reaction conditions may be varied, but generally the irradiation of a ~ WO 94/24314 21 6 0 4 ~ 7 PCTtUS94/04314 reaction mixture will be performed from about 15 minutes to 2 hours at a temperature from about room temperature or below (e.g., in an ice bath).
All the above-described reactions are generally pei~or,ned at ambient pressure. Certain exceptions, such as reduction using molec~ r hydrogen, will be evident. It will be readily appreci~ted by those of o,dil,a,y skill in the art that a group of subsl,ales may be subjected to the various reaction steps in orders which differ from the order provided above. Further, it will be evident that after subjecting a group of subsl,ates to any one or a subset of the various reaction steps above in any order one or more of the steps may be repeated if desired.
Further, it will be clear that other reagents, used singly, or in mixtures, or used sequentially, in addition to the above examples, or with the above examples where pr~ctic~l can be utiiized. The repetition of steps need not be in the order initially performed and ~d~iitional substrates may be 16 introd~ced at any step if desired. In addition, one or more of the sul,slrdles used initially, or introduced at a subse~luent reaction step, may be gel,era~ed by any of the methods provided herein, i.e., by rando"~ chemistry with or without enzymes.
As described above, in an embodiment of this aspect of the present invention, the group of sul~sl,ales is provided by derivatization of an initial molecule or a class of molec~les Such a group of subslrales is subjected to the above-described reactions without enzymes to ge,)er~le a high product diversity which is cenlered around the initial molecule or a class of molecl~les.
A variety of means are available which allow detection of low concelllraLions of one or more species of a desired molecule in a mixture of molecules generated by the methods provided herein. For example, a variety of cell systems are well known to those of ordinary skill in the art which allow detection of low concentration ligands, e.g., ligands binding a hormone receptor. In this regard, for example, a system has been developed which clones human G peptide hormone WO 94124314 216 0 4 ~ 7 PCT/US94/04314 ~

receptors into frog melanocytes (Lerner, Proc. Natl. Acad Sci. USA).
The hormone receptors, typically located in the cell membrane, respond to binding of the cor,espo".l;ng hormone, but trigger a cell response releasing or reabsorbing melanophores. In a forty minute reversible cycle, cells darken dramatically, then can be induced to lighten in color again. Respo"se of the cell depends upon the affinity of the hormone for the receptor. Typical responses occur in the nanomolar to 100 picomolar hormone co,)cel,lrdliol- range. For some hormone receptor-I,Gr",GI,e pairs, where affinity is higher, response occurs in the picomolar I,GI,oone cGncenl~dliGn range. This cell system is an example of an assay system which allows detection, in a mixture of molecl ~les, of one or more species of ligands able to bind to the receptor. The set of mo'ecl~le ligands able to bind the receptor are then the ligands of interesl, for they are candicl~tes to act as drugs by antagonizing, agGni~ing, sl~bstituting for, or modifying the effects of the natural I ,Gr" ~one.
A second example of a cell assay is that available commercially from Molecular Devices (Palo Alto, CA). It col~sisls of an array of cl,er,lfels which respond to very small changes in local pH. In turn, these small pH changes reflect the altered metabolic activity of a population of cells upon receipt of some molecular signal, such as a hGr",o~e binding its receptor. For example, cell assays in which a hormone binds a receptor are known to those of ordinary skill in the art and allow nanomolar or subnanomolar concentrations of the hormone ligand to be letected A prefer,ed means of using the present invention cGnsisl~ in exposing such cells to a high diversity library of molecules generated by the methods provided herein, to detect the presence of one or more species of molecules able to trigger the cell response. That set of small molecules, each of which is highly likely to bind the I,ol",ol,e receptor, are the molecules of interest which may serve as drugs. Another example is to use blast B cells, which on their surface ex,~.ress antibodies directed to a molecule of interest, to detect in a high diversity library the presence of moleulles which sufficiently mimic the molecule of inlerest to be able to bind to its antibody on a B cell. Thus, an animal is immunized with a molecule of inleresl and the early B cells isol~ted A high diversity library of molecl~'~s generated by the methods provided herein is screened using the population of B cells. For example, binding may stimulate cell cycling or division by the last B cell bound. Cell cycling or division may be detected by means known in the art.
Allt:"~ali~/ely, a variety of assays to detect the presence of a ligand of intelesl exist which are based on direct binding assays. Thus, for example, a receptor for a hormone can be used directly to detect binding of a r~dic~ctivity labeled ligand. Other means, known in the art, to accomplish this include the following:
(i) The estrogen receptor is used as a non-limiting example. The cloned receptor can be affixed to a flat surface, for example, a filter. Very high specific activity estrogen is prepared, and bound to the receptor popul~tion. This set of bound ,ece~.tor~ is then used in a competitive assay. The bound rece~.lols are exposed to a library of compounds generated by the methods of the present invention. If the library contains ligands which also bind the estrogen receptor, those ligands will compete with the radioactively labeled estrogen itself for the receptors. Hence the r~d ~ctively labeled estrogen will be competitively displaced from the receptor, and can readily be ~etected by means known in the art. Thus, this assay allows detection of one or more species of ligands in the mixture which compete with estrogen for the estrogen receptor. This set of ligands is the set of interest, as they are candidates to be drugs mimicking or antagonizing estrogen.

WO 94/24314 21~ 0 ~ ~ 7 PCTIUS94/04314 ~

(ii) The estrogen receptor is again used as a non-g example. By means known in the art, one raises antibody mOIE~CI I'QS which are able to bind the receptor when the receptor is not bound by estrogen, but not bind the receptor when occupied by estrogen. Alle",ali~ely, one generates antibody molecules which bind the estrogen receptor only when the receptor itself does bind estrogen.
These antibody molecules can then be decorated with reporter groups by a variety of means known in the art, and used to detect the presence of one or more ligand species in a librarv of high diversity, which bind to the estrogen receptor. In the case of antibodies which only bind the receptor if the receptor is itself unbound by estrogens, one tests for loss of antibody binding in the ~.lesence of the library of compounds and in the simultaneous absence of esL,oge,1. In the case of antibodies which bind the receptor only if the receptor is bound by estrogen, one tests for an inc~ease in binding of the antibody in the plesence of the receptor and high diversity librarv.
(iii) In order to detect ligands in a high diversity library which are cand;~i~tes to mimic or antagonize the action of a given l,Gr",o"e or other molecule of interes~, it is advantageous to generate one or more monoclonal antibodies which bind the hormone or other molecule of inl~resl. This set of monoclonal antibodies can then be used, rather than a receptor, for the target molecule that is to be mimicked, in binding assays such as those noted above to detect the presence of one or more ligand species in the reaction mixture which are cand i~tes to mimic or antagonize the action of the target molecule.
An advantage of this procedure is that a receptor for the target molecule need not be available. Use of a set of monoclonal antibodies is advantageous because, a priori, it is not certain which molecular feature, or epitope, of the target molecule mediates its biological action. Use of a set of monoclonal antibodies, each responding to a different epitope on the target molecule, enhances the probability that the ligands ~ WO 94124314 216 0 4 5 7 PCT/US94/04314 detecter~ in the high diversity library will include those which mimic the biologically important epitope of the target. In some cases it may be possible to selectively use only those monoclonal antibody molecl~es which bind to the known important epitope of the target molecule.
(iv) Means are established in the art to detect protein-p,otei.) binding based on plasmon resonance and detection of a shift in refractive index. In a detection system developed by Pharmacia (Piscataway, NJ), a monoclonal antibody, or a l,or"~o"e receptor, is layered onto a gold chip. Binding of hormone, or other ligands to a receplor, is detected in very low conce"l-dlions (e.g., in the nanogram range or less). Thus, any receptor, or antibody, or other "shape complement" of a target molecule of inleresL can be placed on the gold chip, the latter can be exposed to a high diversity library, and the ,crese"ce of liganding species can be ~lelecte-l Another example of direct measL"e")ent of ligand-binding, which the applicant believe was developed by Evotech, can measure ligand billdi,l53 in the fe",tol"olar range. Rudolph Rigles of the Karolinska Institute in Stockholm has described a laser assay system in which a laser is focl~sed on an approximately 1 cubic micron volume of fluid, and can detect the presence of fluorescently labeled compounds at fel"lo")olar co"ce"lr~lions, 10-15 M, in tens of seconds. By fluorescent labeling of small "shape-complement" molecl lles of a desired target molecule, the bil ,di,)g of a target-mimic molecule to the shape-complement can be detected through alteration of the diffusion of the ligand-bound versus free shape-complement molecule. Thus, if estrogen is the target molecule, and a small RNA aptomer is the shape-complement which binds estrogen, then fluorescent labeled versions of that RNA aptomer can be used in Rigler's system. An estrogen-mimic which binds the fluorescently labeled RNA will slow its diffusion as detected in the laser system. Thus estrogen-mimics at very low, 10 15 M
or fel"~o",olar, col,cenlraLions can be detectef~.

WO 94/24314 216 0 4 S 7 PCT/US94/04314 ~

A further means to detect ligands of inlere~l at very low cGnce"lldlions consists in seeking ligands which block a DNA
polymerase. By blocking the DNA polymerase chain reaction (PCR) enzyme, amplification of the DNA can be blocked. Since PCR
amplification can yield billions or more copies of the initial DNA
sequence, blocking PCR amplification yields a readily detect~ble signal of a ligand which blocks the polymerase. Clearly, this method generalizes to other means to amplify DNA, RNA, or DNA- or RNA-like molecules such as ligation amplification, and extends to general means to block polymerases directly or indirectly with ligands of i"LeresL.
Given that the diversity of the library of molecules which must be tested for molecules of i"leresL is related inversely to conce,)l,~lio,)s and given that the requirement that the founding subsl,ales must be jointly soluble in the reaction mixture, then driving the ~etection level to very low co"cellt,alions permits the invention to be ili~ed to explore libraries of extremely high diversities. Diversi~ies of 10~5 can be generaled, and the presence of ligands of co"cenL-dLio"s of 10-15 to 10-16 M can be both detected and generated from initial millimolar mixtures of 1,000 to 100 subsL,dLes. Additionally, with a sufficiently high diversity of enzymes or reaction conditions, a high diversity library may be ~eneraled with a founder set of organic compounds with a diversity as small as 10.
As described above, compounds of inLeres~ in the high diversity library may act as catalysts for a desired reaction, or as cofRc~ors with other molecules to form an active catalyst. Other molecules may act as inhibitors of enzymes. In order to eYclude the possibility that the enzymes or catalysts are found among the candidate set of enzymes which may have been used to gel-eraLe the library, the latter set of enzymes can be quanLiL~ /ely removed from the high diversity library by aflinity columns bearing molecules directed to a constant part of each of the set of enzymes, or other means known in ~ WO 94/24314 216 0 4 5 7 PCTIUS94/04314 the art. The resulting high diversity library itself is then assayed for cand ~tes of i, Itel esl.
Detectionof molecules able to inhibit an enzyme may proceed by ~etecting ligands able to bind the enzyme, as described above. Identifying molecules which are candid~tes to catalyze a reaction alone or as a cofactor, may proceed by testing high diversity libraries alone, or in the prese,)ce of a helper molecule, say a protein, for which a desired molecule will be a cofactor. The system is tested for the presence of ligands able to bind a stable analogue of the transition state of the reaction. Such binding molecules are the candidate catalysts or cofactors sought, for they are candidates to catalyze the reaction itself.
All~r"ali~ely, a variety of means are known in the art which allow ~etection of the products of a catalyzed reaction itself. For example, cllro",ogenic or fluorogenic suL,slrates for a variety of reactions of i"lelest are available. Catalysis of the reaction increases the rate of forl "dliO, I of the colored or fluol esce, ll product. Aller"dLi./ely, assay systems are available or readily prepared which detect the presence of a product molecule bec~use that product molecule binds a receptor, an antibody mQlecule, or other shape complement. Thus, detection of higher rates of formation of that product molecule demo,l~lrales that the reaction itself was catalyzed.
Following the generation of high diversity libraries of compounds and the screening for the prese,1ce of compounds having properties of i"leresl, such compounds of i"lerest are characterized with 2~ or without isol~tion. A variety of means, including those known in the art, are available to characterize or isolate such compounds of interest.
Characteri,dlion and/or isolation, depend upon the inrolllldlion desired, and can be carried out at dirreren~ mole abundances of the target molecule of i,~lere~L. Thus, using modern mass spe~Lroglaph analysis, about 10 15 to 10-18 moles can be assayed for mass and charge, then fragmented in a variety of ways known in the art WO 94/24314 216 ~ 4 ~ 7 PCT/US94/04314 ~

and the fragments assayed for mass and charge. Using this data, it is possible to derive the structure of the molecllle of inleles~. For example, ligands of i,ltere~,l may be isolated by binding to a given hormone receptor, or monoclonal antibody, then ~he liganding molecu'es rele~sed by means known in the art, and finally characterized analytically. One means comprises attaching a target receptor or antibody to a solid support. A reaction mixture or subset thereof is contacted with the solid support. Those molecules that are bound will be retained, while the non-bound molecules are readily separated from the solid support. The molecllles of unknown structure which have been retained, are then eluted. The freed moleulles are characterized analytically, e.g., by mass spectroscopy, NMR, IR, UV, and may be sy"ll,esi~ed in batch quantities.
Examples of analytic techniques involving mass spectrometry include gas chromatography-mass spectrometry (GC-MS), HPLC-mass spectrometry (LC-MS), and field desorption mass spectrometry (FD-MS).
In other cases, ~he co"ce"l,dlions of molecules of i"le~esl in the high diversity library will allow detection of their presence, but may be too low for further isolation or charac;teri~lion. A prerel ,ed procedure called "sib selection" allows ready winnowing of the set of candidate enzymes, the set of founder subsl,ales, and the set of reaction conditions and chemical reagents, to smaller sets. This winnowing simultaneously reduces the side products generated in the high diversity Iibrary, increases the concentration of the target molecule of inleresl, and identifies the subset of candidate enzymes which catalyze the pathway leading to s~" Ill ,esis of the target molecule, and identifies the set of founder substrates required for synthesis of the desired target. Thus, this sib selection procedure is a means to generate a previously unknown molecule of il,lerest, as well as identify both that molecule and the substrates and enzymes needed to form that molecule.
A library, where the target of interest is a molecule which binds the estrogen receptor, is used as a non-limiting example. For ~ WO 94/24314 216 0 4 5 7 PCT/US94/04314 example, a high diversity library derived from D and L amino acids, including "G"nal.lral amino acids, and small peptides which may be composed thereof is provided by the methods described herein. Such a library will CGI lldil I linear, branched, cyclic and other singly or multiply col,~lrained forms due to formation of disulfide (S-S) intramolecular bonds.
An aspect of the present invention where sub:il,dles and candidate enzymes are used is discussed first. Further below, another aspect of the present invention where candidate enzymes are not used, but one or more reagents or reaction conditions are used, is disc~ssed The presence of one or more ligands for the estrogen receptor is rletecte~ in the high diversity library of this example by any of the means described above, or any other means. The set of candidate enzymes and set of founder subsl,-dles suffice to lead to reactions which g6"6rdle the desired ligands. As a non-limiting example, a set of four reaction steps, using seven of the initial sul)slrdles at ditterel,l reaction steps, may lead to the desired target molecule. By winnowing down the set of initial subsl-ales to the seven needed and the set of four enzymes needed the target molecule may be synthesized in high concentrations.
High cG"cel)L,dLions may be achieved bec~se, given the solubility limits, higher conce, llralioils of the seven critical sub lra~es may be attained than when 1,000 initial subslrales were used, and because only the four critical enzymes would be present.
Sib selection achieves this winnowing. One may start with the candidate set of enzymes, but could equally easily start winnowing the set of subsl~aLes. The set of candidate enzymes can be derived, for example, from a cloned polynucleotide library. Thirty- two aliquots are created, each of which contain a random half of the initial diversity of the candidate enzyme library. Thus, if the initial enzyme library diversity was 1,000,000, thirty-two aliquots are created, each containing a diversity of 500,000 candidate enzymes. The chance that any aliquot has the four WO 94/24314 216 0 4 5 7 PCT/US94/04314 ~

critical enzymes is theretore 1/16. Hence, on average, 2 of the 32 aliquots have the four critical enzymes. The full set of initial subslrales are added to each aliquot, the reactions run, then each aliquot tested for the prese"ce of the desired target molecule which binds the estrogen receptor. One or two of the aliquots are positive. Each of these aliquots has decreased the diversit,v of candidate enzymes by a factor of two, from 1,000,000 to 500,000. One of the aliquots which is positive is chosen. The other can be stored for later analysis. Again 32 aliquots are created, each again having a random half of the remaining candidate enzyme diversity. Hence each of the 32 aliquots now has a diversity of 250,000 candidate enzymes. Each is again tested for formation of the target molecule which binds the estrogen receptor. Therefore, in a logarithmic number of iterations, the set of candidate enzymes may be winnowed down to the four needed to catalyze the synthesis of the target molecule. In the presenl case about 18 ilelalio"s are required.
This winnowing procedure, ll,erefore, allows the isolation of a set of enzymes needed to sy"ll,esi~e a target molecule of i"leresl.
Thereafter, mutation, recombination and selection can be used on this set of enzymes to increase their efficiency and specificity in producing the target molecule. Thus, this procedure yields an efficient set of enzymes for later synthesis of the target molecule from its progenitor sul)sl,ales. In a further use of the present invention, mutant forms of these enzymes can be utilized to catalyze a related family of reaction steps leading to variant forms of the target molecules. Those variants may be more useful than the initial molecules.
In this example, the set of substrates may also be winnowed to the seven nee~ed This winnowing can occur either before or after the set of enzymes is winnowed. The process is the same.
Thirty-two aliquots are created, each containing a random 80% of the 1,000 initial s~lbslrdles. The chance that any aliquot cG"lai, ls the seven critical subslraLes is .87. Thus, on average one or more of the aliquots ~ WO 94/24314 216 0 4 5 7 PCT/US94/04314 conlains the requisite set of 7 substrates. Each aliquot is tested for the presence of the target molecule of inlelesL that binds the estrogen receptor. A positive aliquot is cl ,osen. Thirty-two aliquots are again generated each containing a random 80h of the remaining now redlJced subslrale diversity. The aliquots are again tested for those which contain the target molecule of inLeresl. In a loyar~ mic number of steps it is possible again to winnow to the seven critical initial subsl,~tes.
The number of steps is modest.
It is clear that the fraction of the candidate enzymes or initial subsl-ales used in each aliquot at the first winnowing step and each step thereafter can be chosen such that the expected number of aliquots which form the desired molecule is one or yrealer than one at each step of the winnowing process.
In modes of generating a high diversity library where no candidate enzymes are used but one or more reaction conditions and reagents are used the set of initial substrates may be winnowed using the sib selection procedure described above. This i"cleases the co"cer,l-dlion of the target molecule bec~se the diversity of molecules prese"l and resulting side reactions is sharply red~ced In addition in advantageous cases it may be possible to winnow out those reagents or physical conditions not needed to sy, lll ,eske the target molecule.
One aim of the sib selection procedure is to obtain a sufficient ab-" ,da"ce of the target molecule for its characie, i~liol, and sy"ll ,esis by independent means known in the art. Typically microgram or milligram quantities are sufficient for such analysis by sla"dard techniques. As noted it may often be possible to de~uce structure and composition from far smaller quantities by mass spectrographic analysis or other means known in the art.
It will be appreci~te-~ that it is not necess~ry to actually isolate a compound to homogeneity from a reaction mixture where sufficient information about the compound or its functional properties WO 94/24314 2 ~ 6 0 4 5 7 PCT/US94/04314 ~

can be accumulated in its less than purified state. For exampie, sufficient structural information may be obtainable using analytical techniques appropriate for mixtures of compounds. Allernali~/ely, a compound in a reaction mixture may be characterized functionally (e.g., 6 it is defined by the set of molecules with which it is capable of interacting). For example, a compound in a reaction mixture may interact with a particular amino acid or small sequence of a polypeptide, resulting in enha,)ced or dih,inisl,ed function of the polypeptide. For example, the compound might be a suicide substrate which covalently links to a polymer near the catalytic site. Such a bound suicide subslrale may be used to identify catalysts with a desired activity, or to characterize features of the active site of such a polymer. The site of interaction on the polypeptide may be detected by analytic techniques which are capable of ~letecting perturbations to individual amino acids or regions of the polypeptides. This information regarding the locus for allerdliG" of the polypeptide's function (i.e., information about the target) may be equally or more important than the structure of the compound in the reaction mixture which interacted with the polypeptide. It will be evident that, based on this type of information, one may modify a particular amino acid or region of a polypeptide in a variety of ways.
The following examples are offered by way of illustration and not by way of limitation.

~ WO 94/24314 216 0 4 5 7 PCT/US94104314 EXAMPLES

Preparation of Ubiquitin Fusion Libraries With Diversity of 1x107 The single-stranded DNA needed for 38, 71, and 104 amino acid polypeptide libraries is synthesi~e~l The total diversity is on the order 10'5. PCR amplification is carried out by routine methodology.
Ligation and l,a,)~or",ation efficiency, without attempts to o,cLi",i~e, ligates on the order of 107 random sequences into plasmid, and after Ira"~for",ation yields about 30,000 clones. An efficiency yielding of about 10,000,000 to 100,000,000 l,a"stor")ants per ug of plasmid DNA is attainable (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989). Using 50 ng per I,ansfor",alion, 500,000 to 5,000,000 clones per Irallstormation is achieved. T,dns~or"lation may be opli,ni ed by (i) purifying the insert DNA, (ii) o,~"i,oking ligation con~lilions, or (iii) ofjLil"i~i"g lransfor",ation technique and conditions. Even at unoptimized efficiency, a polypeptide diversity of 1,000,000 with thirty lrans~or",ations is at~ained.
On average, each sequence among the 107 ligated is unique. The diversit,v obtained is tested by counting total Irans~ormants created, sampling random ampicillin resistant clones, carrying out plasmid preparations, restriction mapping and screening for inserts. This allows calcu'~tion of the total number of transformed clones obtained, but, since any sequence might be present in multiple copies, the total alone does not yet specify the total diversity.
Clone redundancy in the library is tested using plasmid preparations of a pool of 5,000 plasmids. Redundancy among these distinct plasmids is tested via hybridization with the unique random DNA
region from each of several specific plasmids among the 5,000. To carry WO 94/24314 21~ 015 7 PCT/IJS94/04314 ~

this out, 5,000 Ira,)~tor",ed colonies are grown on a single plate, lifted onto nylon filters (GeneScreen Plus, DuPont), the cells Iysed, the DNA is W- crosslinked to the filter, washed, the DNA denatured with NaOH, and then neutralized~ Thereafter hybridization is carried out under slringe, ll conditions with r~d ol~heled unique DNA probes purified from each of several plasn,ids among the 5,000~ Probe DNA is cut from the ~ cent uhitllJitin sequences and gel purified prior to labeling~ Probe is l~heled by random primer labeling (Prime-lt, Stratagene Cloning Systems)~
Autoradiography of the resulting filters reveals if any insert DNA
sequence occurs in an expected one, or many among the 5,000 colony diversity on the plate. Given the distribution of numbers of colonies bound for each of 10 to 20 probe insert DNA sequences, the expected diversity of the library may be c~lcul~te~l based on maximum likelihood methods.

Ge,-erali"g A Diversity of Product Molecules The combi"alorics of the libraries described in Example 1 are tested for the onset of catalyzed reactions as libraries of polymers act on one anothen The number of possible interactions is enormous~
For example, for ligation reactions involving two DNA substrates and one polypeptide catalyst, the combinatorics admit of 1021 possibilities of interactions in the DNA and peptide libraries of a 10,000,000 diversity.
Even where the probability that an arbitrary polypeptide catalyzes a given ligation reaction is 104 (an estimate based on the ease of finding catalytic antibodies), a very large number of distinct reactions are catalyzed. Although the combinatorics favor the onset of catalyzed reactions, as the diversity of reactants increases, the conce"Lralion of any type of sequence decreases proportionally. For bimolecular 2~0457 reactions, the forward rate decreases as the square, for trimolecular reactions the rate decreases as the cube of the falling co"ce"l,dlions.
Using an estimate of the probability of catalysis of 10~ and seeking two sub~l,ale reactions Such as ligation, transesterification, or transamination to score, the desired product co, Icentr~liolls and catalyzed reactions may be achieved with diversities of 10~ in both the DNA and polypeptide libraries. For unimolQclJI~r reactions such as cleavage, or phosphorylation, diversities on the order of 105 to 10~ in both the substrate and catalyst library are needed.
A first set of experiments utilizes single stranded DNA
sequences as substrates. S~bse~uent experiments use polypeptides as sub~lrales. This choice is made for three reasons. First, prod~ction of novel DNA sequences, whose length differs from the initial set of sl,bslrales, all of identical length, is easy to detect on sequencing gels.
Second, single slrar,ded DNA, like RNA, is able to fold into complex structures (Lu et al., J. Mol. Biol. 223:781-789, 1991), hence afford a wider variety of sites for binding and catalysis than double stranded DNA
sequences of the same total diversity. Third, single stranded DNA is easier to obtain from the libraries than the corresponding RNA, and somewhat more stable against degradation. Nevertheless, RNA of high diversity specified by the libraries may be purified. Aller"dli~/ely, DNA
sequences may be modified to include RNA polymerase premier sites such as T7 to allow in vitro RNA transcription (Ellington and S~ost~k, Nature 355:850-852, 1992), and obtain high diversity RNA libraries for use as subsLrales. Thus, protocols are stated in terms of single stranded DNA subslrales, but single stranded RNA libraries may also be used.
The plastein reaction (Wang et al., Biochem. Biophys. Res.
Commun. 57:865, 1974; Silver and James, Biochemistry20:3177, 1981) is a general model for the experiments. In this reaction, protein subslrales are incub~ted with trypsin, which cleaves the subslrdles to smaller peptides. Since any enzyme catalyzes forward and reverse WO 94/W14 21 6 0 ~ 5 7 PCTIUS94/04314 ~

reactions, trypsin is capable of catalyzing ligation of larger polypeptides from the smaller peptide fragments. It has been found that dehydrating the reaction mixture, to shift the equilibrium in favor of synthesis, suflices for trypsin to catalyze ligation and transamination reactions leading to formation of high molecular weight polypeptides in the absence of ATP
hydrolysis (Levin et al., Biochem. J. 63:308, 1956; Neumann et al., Biochemistly 73:33, 1959). If the high molecular weight ~,a~ ial is removed and the reactants again cGncentraled, further high molecular weight polypeptides are formed. Absence of a requirement for ATP
hydrolysis is not too su".lising, since transamination reactions can ,~,,oceed without net formation of new peptide bonds.
In the first set of experiments, single stranded DNA
sequences of constant length from the libraries, end labeled after the reaction in one set of experiments, and uniformly labeled prior to the reaction in another set of experiments, are incub~ted with 32p nucleotides. The suL,sl,~tes are then incubated with affinity purified polypeptides from the libraries of Example 1 with length 38, 71, and 104 of tuned diversities in the ranges noted. Divalent cations, such as Mg++, Pb++, Mn++, as well as ATP as a potential energy source may be included In addition, c~nce,llralions of DNA sub~l,ales and polypeptides are tuned over a range sufliciently broad to include conditions under which biological polynucleotkles are cleaved or are ligated in vitro. In ligation reactions, typical DNA"ends" cGI~cenllalions are nanomolar. In a variety of reactions, typical enzyme concentrations are micromolar or higher. DNA subsl,a~e ranges in the nanomolar cGncer,lralions are easily created under the present experimental conditions. For polypeptides from the 71 amino acid library, a diversity of 10,000 polypeptides at 1.0 mg/ml yields about a 10.0 nanomolar concentration for each fusion protein. Therefore, reactions catalyzed efliciently by such novel enzymes, produce product 100 times slower than were their concentrations higher. In typical DNA cleavage or ~ WO 94/24314 216 0 4 S 7 PCT/US94/04314 ligation reactions, subst~ntial product can be ~etec~ed after times on the order of minutes. Thus, in general, detect~hle products are seen on the order of h~"~JIeds of minutes to thousand of minutes.
The polypeptides in the library catalyze, for example, cleavage, ligation or transesterification reactions among the single sl,a"ded DNA target molecllles Of these, cleavage is energetically favored in an aqueous medium, while tra~ses~eritication reactions, like L,ansa"-i.,dlion reactions among polypeptides in the plastein reaction, are nearly co"slanl energetically in ~q~eo~ls media. In ~ddition, a variety of c,osslinking reactions between two single stranded substrates may occur. Transesterification reactions between two substrate sequences of length L can yield two product molecules, one of which is larger than either of the two subsl,ate sequences. The beginning library of DNA molecules are all of the identical length. Thus, on a large 38 cm polyacrylamide gel (BRL Sequencing Apparatus) run under denaturing cGndilions, the entire library runs as a single band. However, where th polypeptides catalyze cleavage, ligation, Iral,sesleritication or crossli"l~ing reactions with DNA molecule subslrates, new shGI ler or longer DNA sequences appear on the gel. Using standard DNA
sequencingon long gels, bands which differ by a single nucleotide can be disc,i",i"dled over about a 400 base range. The gel is run to adjust the position of the random library full length single stranded DNA
sequences at a desired posilion on the gel. Using aliquots of the same reaction mixture sampled at the same moment, and running gels for dirterenl durations, a large range of molecular weights are scanned for novel bands. As noted, all products of reactions in one set of experiments are end labeled, since uniform labeling of substrate sequences prior to reaction with 32p may induce radiation breaks in single stranded subsl~ales. The end labeled material should be stable, but less label is present on the gel, rendering detection more difficult, and only one fragment of a cleavage reaction is visible. Uniform labeling WO 94/24314 21~ ~ ~ S 7 PCT/US94/04314 achieves higher specific activity and legitimately marks reactions yielding product molecules which are larger than our single stranded subslrales.
In order to assure that the new molecular size c~sses ~,rese"t de novo catalysis due to the polypeptide library, control reactions are carried out using a control Dbrary encoding ubiquitin alone.
If affinity purified ubiquitin alone, derived from the control library, catalyzes reactions among the DNA subsllates, then this can be controlled for in two ways. First, novel random peptides are cleared free from ubiquitin as noted above, the novel peptide fragments repurified by size under non-denaturing conditions, and retested for catalysis using these random peptides freed of ubiquitin. Second, the particular reaction subslrales acted on by ubiquitin or cell background material can be identified by a logarithmic dilution technique, as described below, and eliminated from the DNA subsllale library.
A number of features of this system may be ~ssessed First, the probability that a pr~tein catalyzes a delectable reaction on DNA subslrales may be estimated. At low diversities of the libraries, the appearance of a few distinct bands of lower or higher molecular weight than the initial DNA sul)sl,ate library may be seen. Where these are the only reactions catalyzed, then as the incubation period increases, no further bands appear. Each cleavage reaction involving a single DNA
sul~t,dle may give rise to two product sequences. Transesterification reactions between two subsllales again give rise to two product sequences per reaction. Crosslinking and ligation reactions yield one new product sequence. Single crosslinking and end ligation reactions yield one new product sequence. Single crosslinking and end ligation reactions among a uniform set of single stranded DNA sequences length L should all have a total length of 2L nucleotides. There~ore, for new bands corresponding to lengths less than 2L, the number of reactions is estimated as half the number of such new bands. Using this data, one may estimate the probability that an arbitrary polypeptide catalyzes a 21604~7 detect~hle reaction. (Some c~osslinked DNA sequences with 2L
nucleotides may have aberrant migration characteristics, perhaps leading to erroneously count them as products of transesterifc~tion reactions.
This could cause a two-fold error in the estimated probability.) Second, this estimated probability may be co"rir,l~ed by increasing the substrate and polypeptide diversity. Third, by tuning polymer length at co, Istant diversity, the effective number of sul)sL,ale sites and of catalytic sites may be measured as a function of polymer length.
In an additional set of experiments to test whether the set of polypeptides catalyze reactions, unlabeled single or doubled stranded DNA sequences of constant length derived from the libraries is incuh~ted with 32p labeled nucleotides or short oligonucleotides, acrylamide gels run, and the labeled material is tested for incorporation into large molecular weight DNA matelial.
A new ge"eral loyarill,r"ic dilution" procedures is carried out to isolate both the specific polypeptide(s) catalyzing any specific reaction, and the specific subsl-ales involved. The procedure introduced here also serves to isolate both the specific set of subsllales and the specific set of novel enzymes leading to the synthesis of a target molecule of inleresl.
To carry out this procedure, divide the total diversity of the initial cloned polypeptide library into four ditrerelll aliquots, each conlain;. ,9 a random half of the total diversity of the polypeptide library.
Aliquots may be created which reduce total diversity by random halves by knowing the diversity of the library, and the number of copies of each sequence by methods known to those skilled in the art.
For reactions with two suL,sllales and one enzyme, the probability that any random half of the diversity of the polypeptide librarv has the requisite enzymatic polypeptide is 0.5. Thus, two of the set of four random half-library aliquots contains the required polypeptide. If no random halved aliquot had the required polypeptide, a larger number of WO 94/24314 21~ 0 4~ ~ PCT/US94/04314 ~

haived aliquots is tested. Each new diminished library is incubated with the full set of single stranded DNA substrates, and the products analyzed on a long sequencing-type gel. On average, for two such gels, the desired product of the reaction continues to be presenl. Thus, the corresponding half polypeptide library contains the polypeptide which catalyzes the reaction. That now di"~i~ ,ished library is again divided into four random halves in four ~liquot~ Each is incl Ib~ted with the full set of DNA substrates, the gel run and the product identified if formed in at least one of the four aliquots. By a logarithmic number of halvings of the initial polypeptide library, the single polypeptide catalyzing a specific reaction is isolated. Simultaneously, the fusion gene encoding this polypeptide is isol?ted. Thus, if the polypeptide diversity is on the order of 10,000, then about 13 halvings suffice.
In the same way, the specific substrates for the reaction in ~luestion are ol,taine.l. For two sul,sl~ate reactions, eight random halves of the DNA su~sl,ate library are prog,essively formed. The probability that any aliquot cGntaills the two sul~sl,dtes is 0.25, hence on average two of the eight have the two subsl,ales. These aliquots with the now known catalytic polypeptide are incuh~ted, gels run, which aliquot exhibits the desired reaction product CGI l~inlled, thereby concluding that the cor,esponding half of the subslr~le diversity co"lains the desired two suL,s1,a1es. Over a logarithmic number of successive rounds, the two subs1,ales are thus isolated.
As noted, a main virtue of this approach is that it is possible to carry it out for any set of molecule subs1,a1es, and any set of polypeptide, RNA, or other potential catalysts. In short, where a diversity of new products are formed under these experimental conditions, and where one such product is of inLeresl and can be reliably found in the product mixture after reaction, then a modest number of halving steps isol~tes both the subsL,ates for and enzymes for the reaction leading to the product. This approach generalizes to cases in which several ~ WO 94/24314 21~ 0 4 ~ 7 PCT/US94/04314 enzymes carry out a sl~Gcession of reactions from an initial set of su6~lrales. It is merely "ecess~y to alter the ra,)cJo,n fraction of the diversity in each aliquot, and number of aliquots at each step, to assure that at least one such aliquot colllaills the requisite set of subslraLes or enzymes. At any diversity, a logarithmic number of steps is required to isolate both the set of substrates and the set of enzymes leading to sy,lthesis of a desired novel target compound.
The polypeptide libraries of tuned diversity may be permitted to act on themselves as substrates. Many of the same considerations apply to polypeptide and DNA sequences as substrates for reactions. Cleavage is energetically favored in aqueous medium, while Ird,)sdl,lination reactions are energetically neutral. Thus, as noted, in the plastein lea~Lion, i"cleasi"g the col,cellLrdlion of the peptide fragments by dehyd~dliol1 shifts the llansal,lination reactions in favor of syl,lhesis of large mo'ec~ r weight polypeptides, and the reactions proceed without ATP hydrolysis (Neumann et al., Biochemistry 73:33, 1959). Thus, after incubation of a set of labeled polypeptides of a COI)S~dlll length and mean molecular weight, formation of novel lower and higher mo'~cu~r weight sequences may be seen. A variety of endoprote~ses, exoproteases and other enzymes may be used to drive the efficient sy, lll ,esis of larger polypeptides from smaller peptide substrates. Enzymes used include subtilisin, papain, thermolysin, chemotrypsin, and carboxypeptidase Y, in enzyme conce-,lr~lions ranging from micromolar to millimolar, and substrate concenlldLions .anging from millimolar to molar (Wong and Wang, E:xperientia 47:1123-1129, 1991).
Based upon a solubility of 1.0 mg/ml for the polypeptide fusion library, then at a diversity of 100, each 71 amino acid fusion peptide is present at approximately 0.6 micromolar concel,L,dLion. With a diversity of 1,000,000, each is present at 0.06 "a"ol"olar co"ce,lLrdLion. In a volume of 10 ml, a diversity of 1,000,000 WO 94/24314 216 0 ~ ~ 7 PCT/US94/04314 cor,es~onds to 10 nanograms of each. These conce"LrdLions are ~letect~hle. For example, gold stained blots on Immobilon P filters can detect spots with 3.5 nanograms, and polyacrylamide gel staining can detect bands or spots of 2.0 nanograms (Pluskal et al, Bio/Techniques 4(3):272-282, 1986; Ausubel et al., eds., Current Protocols in Molecular Biolo~y, Greene Publishing and Wiley^ln~r~cience, New York, 198n-R~d el~heling increases tlete~t~hility by more than an order of magnitude (~iarrells, Methods Enzymol. 254:7961-7977, 1979). In order to maxil,lke subsL,ale, hence product conce"L,dLions, the diversity and conce,lt,dLion of the polypeptide library may be tuned to find that minimum diversity and maximum concelltrdLion at which preferred new prominent bands appear. In addition ~o running one-dimensional SDS
polyacr,vlamide gels, reaction mixtures are analyzed on two-dimensional gels, running first an isoelectric dimension, followed by SDS page analysis (O'Farrel, J. Biol. Chem. 2~0:4007~021, 1975; Garrells, Methods Enymol. 254:7961-7977, 1979; Summers and Kauffman, Developmental Biology 113:49-63, 1986). Automated facilities for .liyiLi~ed gel data analysis are available. Two-dimensional gels may be used to collfirll, that unique bands on one-climensional gels cor,espond to unique spots in two dimension, hence a single product polypeptide. This allows one to count the number of reaction products.
For subcritical reaction systems of minimal diversity, only a few novel products are formed, and no further catalyzed reactions occur due to these new polymers. Thus, as incl~b~tion increases, no new bands or spots are generated. From the number of novel polypeptides produced, the probability that an arbitrary polypeptide catalyzes a reaction may be quantified. As above, cleavage and transamination reactions among polypeptide substrates length L t,vpically yield two products of length less than 2L. Ligation and crosslinking reactions yield one product with a total of 2L amino acids. Using two-dimensional gels, the number of distinct products of molecular weights corresponding to a WO 94/24314 216 0 ~ 5 ~ PCT/US94104314 total of 2L amino acids are discli~"inated, since one knows an expected mean molecular weight and a c~lc~ hle variance. Thus, for a modest number of novel bands and spots, the total number of reactions catalyzed may be estimated. From this, the probability that a polypeptide catalyzes a reaction can be salcu'~ted As the lellyllls of the polypeptides are altered, one may obtain measures of the scaling relation for numbers of types of reactions catalyzed as a function of polymer length of substrates and enzymes.
As noted above, phase transitions afford the ability to catalyze an explosion of molecule diversity from a diverse founder set of organic molecules acted upon by a sufficient diversity of potential catalytic polymers. Where target small moleul4s of i"leres~ are .Jetecie-J among the products of the catalyzed reactions, the logarithmic partitioning procedures above should allow the recovery of the specific sul)sl,ales and novel enzymes leading to the molecule of interest.
In supracritical reaction systems, by definition, new products become substrates for yet further reactions engendering still further new products which again are candidate sul~sllales. Three signatures are mol,ilored to establish supracritical behavior. First, over time, the diversity of substrate and product species increases. This is the major criterion. Second, over time, the maximum molecular weight product inc~eases. Third, the mean and variance in the molecular weight distribution among the products increases in a c~iclJ~hle way.
The second and third signatures require elaboration. In a suprac,ilical reaction system where the initial substrate single stranded DNA, polymers are all of length L, the maximum length polymer which can be formed by a single ligation reaction is of length 2L. The maximum length which can be formed by use of two such newly formed polymers in a new ligation reaction where they are the substrates is 4L, then 8L and so forth. Thus, visu~ tion of an increasing maximum molecular weight among the product molecules is evidence favoring WO 94/24.314 ~ 1 fi ~o ~ ~ ~ PCT/US94/04314 supracritical behavior of the reaction system. More generally, in model reaction systems whose founder subslrale sets are only a few monomers in length, the mean and variance in molecular weights among the product polymers increase over time and gives rise to a characteristic unimodal distribution. The diversity of polymers of a given length presenl in the system can be plotted on the ordi"ale and the lengllls of those polymers on the absc;ssa. As reactions proceed creating a diversity of small and large products, the resulting curve may rise steeply to a peak as length increases, then fall off wi~h an exponential tail.
In the first set of experiments, the diversity of new bands which appear on sequencing gels are analyzed as a function of time and as a function of the diversity of the polypeptide library catalyzing the reactions. In minimally diverse DNA su,`Jsl,dle systems a modest number of new products may appear early, then not increase over time.
In systems with a subs~nlially higher diversity of single stranded DNA
subslrdle sequences, detection of a sustained increase in total diversity over time (as limited by the product co"cerlt,alions required for detection) and detection of a sustained increase in the highest molecular weight cl~sses seen, are strong evidence for supracritical behavior of the reaction system.
In a second set of experiments, forward reaction velocities are driven, and the reaction system maintained in non-equilibrium conditions, by suslai,1i,)9 the concenLralion of the founder set of single stranded DNA sequences through periodic or continuous addition of labeled single stranded DNA sequences for",i"sa that set. Sustained non-equilibrium conditions through "driving" by addition of founder SUbSllale molec~lles may be important to achieve high conce"lralions of high molecular weight polymers. The catalyzed reactions funnel monomers to specific large polymers.
Addition of founder substrate DNA polymers is carried out in two ways. In the first way, slJL)slrales are added to an otherwise ~ WO 94/24314 ` 216 015 7 PCTIUS94/04314 closed stirred reactor. In the second way, substrates are added to a flow chemostat. The two environments are quite dir~erenl. In a closed stirred reactor, product molecules are not removed from the system except by back reactions or further reactions in which they are subslrates. In a flow chemostat, product molecl ~les are removed. As shown in detail by Eigen and Schuster (The Hypercycle: A Principle of Natural Self-O~a~ dlion, Springer-Verlag, New York, 1979), the chemostat system driven by continuous addition of subsl,ale molecllles is an environment which carries out selection on the reaction products:
The total mass of subsl,ate nucleotides ultimately becomes constant.
The fraction of these which are organized into product molecules of dirrere,)t sizes may change. Those product molecules which are pro~uce~ faster than they are diluted by the outflow actually accumulate in cGncelllrdliG", the re")ai"der are gradually eliminated. Thus, the closed reaction system allows one to test for the total increase in product diversity over time. The flow chemostat environment allows one to test, as a function of flow and driving rates, whether the reaction system settles down to a sustained set of founder polymers and their direct and indirect reaction prod~ ~ctC.
Parallel experiments are carried out in which both the subsl,ales and the catalysts are polypeptides. To do so, one may again begin with the minimal diversity 71 or 104 amino acid polypeptide libraries required to see the onset of catalysis of new molecular size prod~cts then tune diversity upward several orders of magnitude.
Minimally complex polypeptide systems can form a small number of novel product polymers which does not increase further over incubation time. A supracritical system shows an increasing diversity over time.
One-dimensional and two-dimensional gel electrophoresis are used to analyze the total increase in diversity over time. Unlike analysis of DNA sequences, however, use of two-dimensional gels may allow one to discriminate several novel product molecules with the same WO 94/24314 21~ 7 PCT/US94/04314 molecular weight on SDS page analysis. A sustained increase in total diversity over time (as limited by the product concenlraliGns ~letect~hle), and a sustained increase in the highest molecular weight cl~ses seen, is strong evidence for supracritical behavior of the reaction system.
In a second set of experiments, labeled amino acids and short peptides, up to hexamers, are incllb~ted with libraries of increasing diversity from the larger amino acid library plus the polypeptide library.
By one- and two-dimensional gel analysis, the labeled amino acids and small peptides are tested for incorporation into high molecul~r weight material. Control experiments use affinity purified ubiquitin alone with the labeled amino acids and small peptides, and the labeled amino acids and small peptides incub~terl by themselves.
Supracritical behavior may be dem~"slrated in a particularly clean way: Theolelical work shows that a sufficiently low diversity founder set of amino acids and small peptides will be subc,ilical. However, if the c~nce"l,dlions of members of that founder set are mainlai. ,ed by exogenous addition, and the set is inc~ ~b~ted with a high diversity of larger polypeptides added once only at the outset of the experiment, then the larger polypeptides can catalyze the formation of many polypeptides built up out of the founder set. Those novel polypeptides themselves come to play catalytic roles in sustaining the ~r",dlion of themselves and yet further novel polypeptides. Indeed, such a system might include collectively ~ ~oc~t~lytic sets of polypeplides. In short, the small peptides alone, in sustained cG,-ce"l,alions, are subcritical, but transient exposure to a high diversity of larger polypeptides triggers supracritical behavior which is thereafter sustained without further addition of the larger polypeptides.
To carry out this experiment, the above flow chemostat experiments are extended using labeled amino acids and small peptides, incl~ ted with an initial set of diverse 71 or 104 amino acid polypeptides. The conce, Itr~lions of the founder set of labeled amino WO 94124314 2 ~ 6 0 4 ~ 7 PCT/US94/04314 acids and small peptides is sustained. At a critical diversity of 71 or 104 amino acid polypeptides not only incorporation of amino acids and small peptides into high molecular weight material is seen but p~rsistence of that incorporation under the che",osLal conditions which leads to the exponential dilution and ultimate loss of all initial 71 or 104 amino acid polypeptides. Such sustained synthesis of large polymers from the sustained founder set demG"~t,ales that transient incuh~tion with the high diversity library of 71 or 104 amino acid polypeptides triggers a phase transition in the system of amino acids and small 1 0 peptides.
In order to conti"n that exposure of a collection of organic mo!eclJ!es to a diversity of polypeptides leads to synthesis of an i"c,easinJ diversity of organic molecules a reliable means of letecting and d;sc,i"~i"aLin~ small quantities of organic molecules is required.
HPLC analysis appears to fulfill the requirements. With UV absorbance detection HPLC can detect conc~nL,aLions down to the nanomolar range. For example, tryptophan can be ~letected down to about 10 ~anomolar. It may be possible to increase the range of small molecllles which are r~etect~ble using IR rather than UV spectra (Kemp and Vellaccio Organic Chemistry, Worth Publishers Inc. 1980). A chosen set of fifty to a few hundred organic moleu ~les gives rise to a discrete set of peaks which can be disc,i",i"aLt:d from a far more complex mixture co"taining a number of additional peaks due to the p,esence of new product molecules Evidence of reactions include both the appearance of new peaks and the disappearance of the initial subsL,dLe peaks.
In these experiments sets of founder organic molecules are first assembled with well-displaced peaks on HPLC analysis followed by sequential addition of trial substrate compounds to solutions containing previously accepted members of the founder set. Founder sets are WO 94124314 216 0 ~ ~ 7 PCT/US94/04314 ~

created which optimize both founder conce"l,dlions and diversity, such that novel product molec~ ~es yield easily detect~hle peaks.
As in the other experiments described above, experiments are carried out with a fixed input of founder organic molecl ~les, and under conditions which drive forward synthesis and hold the system displaced from equilibrium by continuous addition of the founder set of organic molecules to otherwise closed stirred reaction systems. In a subset of experiments, radioactively labeled founder set molecl ~les are used to establish that radioactive atoms are incorporated into new product molec~'e~. The conce"l,alions of product molecl~les ultimately depends upon the ratio of the diversity of founder set to product set, the number of reaction steps from the founder set to a given product molecule, and the detailed forward and reverse kinetics along the reaction pathway(s) leading to and from the product species. On average, however, if the founder set diversity is 100 and the set members are present in millimolar CGI ,ce"lralion initially, if the system were otherwise closed and if the final diversi~y were about ten million, then the terminal product concenlralions might be about 10 nanomolar.
Once having established the conditions under which only a few reactions are catalyzed and thus in which product peaks are easily Jetected the foundation is provided by which to increase the diversity of the polypeptides to which the same founder set is exposed. For a sufficient diversity of polypeptides, a very large increase in the diversity of small organic product molecl ~es, hence peaks, is seen in the system.
As in our analysis of systems using DNA or polypeptides of fixed initial length, here too, as reactions proceed, ever larger molecular weight products can be formed. Thus, in supracritical systems, both diversity and maximum molecular weight increase with time and with the diversity of the polypeptide library.
These experiments demon~lrale that a large diversity of organic compounds can be formed by catalyzing reactions from a ~ WO 94/24314 216 0 ~ 5 7 PCT/US94/04314 sustained founder set of small organic molecules. Thus, these experi",e,)l~ lead to the application of these new technologies to the generation of high diversity libraries of small molecules as drug cand ~tes Once a diversity of novel organic products is gei,eraled, the logarithmically iterative procedure defined above may be utilized to isolate both the set of novel enzymes leading to a specific product molecule, and the set of founder organic molecules which are the initial sub:,l.ales needed for the chain of reactions leading to the product molecule. This procedure is a minor modification of that described above and reflects the fact that several, e.g., 4, enzymes might be needed to catalyze a chain of reactions, and reflects the fact that several, e.g., 7, initial subsl,ates may be required in those reactions. The four enzymes may be IGgariLl"nically isolated as follows. At each step, the current polypeptide library diversity is lando",ly partitioned into ten aliquots each col)Laini"~a a rancJo", 0.7 of the total diversity. The probability that any aliquot contains the four reql~isitive polypeptides is .24, hence on average two of the aliquots have the four enzymes.
Reactions with the full diversity of initial substrates are carried out and the target of i"leres~ identified in one or two aliquots, thereby reducing the polypeptide library diversity by a factor 0.7. Successive cycles will, again in a logarithmic number of steps, isolate the four enzymes needed.
To cut the subsL-ate diversity down to the seven substrates needed the subslrale diversity is randomly assigned to 10 aliquots each collLaill;,)g a random 0.8 of the initial diversity. The probability that any aliquot has the seven critical subslrales is .21, thus on average two aliquots are s~ ~Gcessf~
This analysis is of considerable interest for two reasons.
First, it establishes that a sequence of reactions, not just a single reaction, is catalyzed by a set of novel enzymes, leading from a set of initial substrates in the founder collection to a target molecule many WO 94/24314 216 0 ~ 5 7 PCT/US94/04314 ~

synthetic steps away. Second, such a procedure co"slilutes a radically new approach to the problems of organic sy, Ill ,esis. Here diversity and sc.eenir,L~ procedures are used to identify simultaneously not only de novo enzymes, but also the set of subslrdles leading via a sequence of catalyzed reactions to a target organic compound. The second eresl, of course, relates to drug discovery.
There are several all~r"ali./e approaches to finding such drug can~id~tes. In a first, a receptor for the normal ago, lisl is already in hand and is used to screen for small molecule mimics of the agonist.
In a second, no receptor is yet available, but only the agonist itself. In a third, inhibitors of an enzyme are sought. As an example of the first approach, one might wish to detect the presence of an organic molecule of i"tele~ ,resellt in nanomolar co"cel,l-alion, bec~use it binds to a specific cloned cell receptor. Such detection is attainable by a competition assay with the normal ligand for the cloned receptor.
Labeled normal ligand would not bind or would show redl ~ced binding in the presence of the entirely unknown small molecule present in the reaction mixture. As ~isu~ssed below, nanomolar conce"~rations suffice for detection. Where a binding event is detected when the unknown product is in the nanomolar range, then the above described logarithmic dilution process may be used to find both the enzymes and substrates leading to synll ,esis of a new organic molecule able to bind a cell receptor. Note that neither the target molecule, nor the spe~cific initial s~bstrdles, nor the enzymes required for sy,ltl,esis of the target from the founder set of subsL,ales, need to be known in advance. Any such molecule is a drug candidate to bind to the receptor, hence modify or mimic or antagonize the activity of the normal agonist.
In the second approach, the receptor for the agonist is not known, but the agonist is known. Here a set of random polypeptides which bind to the agonist, hence are its shape complements, is sought.
This set of polypeptides then can be used, in place of the unknown ~ WO 94124314 216 0 4 5 7 PCT/US94/04314 receptor, to screen for novel organic molecules which compete with the agonist for binding to members of the set of shape complement polypeptides. While one would not yet know which polypeptides bound the agonist by groups of atoms which reflected the function of the agGnisl, some among the polypeptides presumably do bind the impG,~nt agonist epitopes. Thus, the set of organic molecl~ies binding to the polypeptide set is a set of candidate drugs to mimic or mod~ te the activity of the agonist.
A third approach seeks a novel small molecule inhibitor of an enzyme such as HIV plotease by slowing cleavage of the peptide sul,sl,ale.
To seek agonist mimetics of estrogen, for example, the cloned estrogen l~eceptor which is immobilized on Immobilon P filters as dot blot arrays is utili~ed. Competition assays are carried out with r~r~ c~ctively l~beler~ estrogen and the molecules formed in the reaction mixtures. Dot blot filters are incub~ted with decreasing CGI Icel ltrdLions of labeled estrogen and constant cGncenlralions of the mixture of organic mclecu'es Control filters have no organic molecules ~ e-l As estrogen concentlaliG,) decreases, tests are condlJcted to cJeLer,nine whether competitive displacement of the labeled estrogen occurs.
Tritium labeled estrogen and its analogues are available as 150 Ci per millimole. Thus, a picomole of this probe is 0.15 microcuries. '2~'1 labeled estroye" and its analogues labeled at over 2200 Ci per millimole are available. A picomole is 2.2 microcuries. Thus, even less than picomole quantities of organic molecule competitors which displace such bound labeled estrogen are ~etect~ble. Since novel products in the 100 to 1000 picomolar range are generated, even estrogen mimics with modest affinity for the receptor displace labeled estrogen present in picomole cG"celltlalion, and thus are detect~hle.
From the foregoing, it will be appreci~te~i that, although specific embodiments of the invention have been described herein for WO 94/24314 216 0 4 ~ 7 PCTtUS94tO4314 ~

purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims (50)

Claims

1. A method for the production of an organic molecule having a desired property, comprising the steps of:
(a) providing a starting group of different organic molecules;
(b) causing at least one chemical reaction to take place with at least some of the different organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecules different from the organic molecules in the starting group;
(c) repeating step (b) at least once by substituting the intermediate reaction mixture as the starting group to thereby produce a final reaction mixture as a result of the last repetition;
and (d) screening the final reaction mixture resulting from step (c) for the presence of the organic molecule having the desired property.

2. The method of claim 1 further comprising the step of isolating from the final reaction mixture the organic molecule having the desired property.

3. The method of claim 1 further comprising the step of determining the structure or functional properties characterizing the organic molecule having the desired property.

4. The method of claim 3 further comprising the step of synthesizing the organic molecule having the desired property.

5. The method of claim 1 further comprising the step of adding more of the starting group of different organic molecules to the intermediate reaction mixture after at least one repetition of step (b).

6. The method of claim 1 wherein the different organic molecules of the starting group all share a common core structure.

7. The method of claim 1 wherein the different organic molecules of the starting group are selected from the group consisting of alkanes, alkenes, alkynes, arenes, alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds, organometallic compounds, hetero-atom bearing compounds, amino acids, nucleotides, and mixtures thereof.

8. The method of claim 7 wherein the different organic molecules of the starting group are selected from the group consisting of acids, amines, alcohols, amino acids, nucleotides, and unsaturated compounds.

9. The method of claim 8 wherein the different organic molecules of the starting group are selected from the group consisting of amino acids and nucleotides.

10. The method of claim 1 wherein the at least one chemical reaction for each repetition of step (b) is independently selected from the group consisting of substitution, addition, elimination, rearrangement, dehydration, reduction, oxidation, condensation, hydrogenation, dehydrogenation, dimerization, epoxidation, isomerization, cyclization, decyclization, halogenation, sulfonation, alkylation, acylation, nitration, hydrolysis, esterification, transesterification, carboxylation, decarboxylation, amination, and deamination.

11. The method of claim 1 wherein the chemical reaction is caused by changing the conditions of the intermediate reaction mixture, by taking a step selected from the group consisting of adding water, removing water, adding air, adding oxygen, adding ammonia, changing temperature, changing pressure, adding an oxidizing agent, adding a reducing agent, adding a source of radiation, adding a hydroxylating agent, adding a hydrogenating agent, adding a dehydrogenating agent, adding an epoxidizing agent, adding a halogenating agent, adding a sulfonating agent, adding an alkylating agent, adding an acylating agent, adding a nitrating agent, adding a hydrolytic agent, adding a carboxylating agent, adding a decarboxylating agent, changing concentration, adding a new solvent, changing pH, and adding a catalyst.

12. The method of claim 1 wherein the at least one chemical reaction is caused by adding a set of different enzymes.

13. The method of claim 12 wherein at least 10,000 different enzymes are added.

14. The method of claim 13 wherein at least 1,000,000 different enzymes are added

15. The method of claim 14 wherein at least 100,000,000 different enzymes are added.

16. The method of claim 1 wherein the conditions causing the chemical reactions of steps (b) and (c) are the same.

17. The method of claim 1 further comprising the step of using a selection method on the intermediate reaction mixture to produce a subset of organic molecules with a higher likelihood of producing the organic molecule having the desired property.

18. The method of claim 17 wherein the selection method comprises using a chemostat.

19. The method of claim 1 wherein at least one agent, selected from the group consisting of oxidizing agents, reducing agents, hydrogenating agents, dehydrogenating agents, hydroxylating agents, hydrogenating agents, dehydrogenating agents, epoxidizing agents, halogenating agents, sulfonating agents, alkylating agents, acylating agents, nitrating agents, hydrolytic agents, carboxylating agents, and decarboxylating agents, is added during at least one repetition of step (b).

20. The method of either claim 1 or claim 7 wherein the starting group contains at least 10 different organic molecules.

21. The method of claim 20 wherein the starting group contains at least 100 different organic molecules.

22. The method of claim 21 wherein the starting group contains at least 1,000 different organic molecules.

23. A method for the production of an organic molecule having a desired property comprising the steps of:
(a) providing a starting group of different organic molecules;
(b) causing at least one chemical reaction to take place with at least some of the different organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecules different from the organic molecules in the starting group;
(c) repeating step (b) at least once by substituting the intermediate reaction mixture as the starting group to thereby produce a final reaction mixture as a result of the last repetition;
(d) screening the final reaction mixture resulting from step (c) for the presence of the organic molecule having the desired property; and (e) if the organic molecule is found in the final reaction mixture then performing the following additional steps:
(1) dividing the starting group of different organic molecules into at least two subgroups each containing less than all of the different organic molecules in the starting group;
(2) performing steps (b) and (c) on each of the subgroups in the same way as performed with the starting group to produce a final reaction submixture corresponding to each of the subgroups;
(3) screening each of the final reaction submixtures resulting from step (2) for the presence of the organic molecule having the desired property; and (4) repeating at least once steps (1) through (3) for at least one of the successful subgroups from which the organic molecule having the desired property is produced by substituting the successful subgroup as the subgroup in step (1) to thereby identify a narrowed group of different organic molecules from which the compound having the desired property can be produced.

24. A method for the production of an organic molecule having a desired property, comprising the steps of:
(a) providing a starting group of different organic molecules;
(b) causing at least one chemical reaction to take place with at least some of the different organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecules different from the organic molecules in the starting group;
(c) repeating step (b) at least once by substituting the intermediate reaction mixture as the starting group to thereby produce a final reaction mixture as a result of the last repetition;
(d) screening the final reaction mixture resulting from step (c) for the presence of the organic molecule having the desired property; and (e) if the organic molecule having the desired property is found in the final reaction mixture, then performing the following additional steps:
(1) providing at least two additional starting groups of different organic molecules, each additional starting group corresponding to the starting group of step (a);
(2) performing steps (b) and (c) on each of the additional starting groups in the same way as performed with the starting group of step (a) with the exception that, for each of the additional starting groups, at least one of the chemical reactions is eliminated to thereby produce an additional final reaction mixture from each of the additional starting groups;
(3) screening each of the additional final reaction mixtures resulting from step (2) for the presence of the organic molecule having the desired property;
(4) repeating, at least once, steps (1) through (3) for at least one of the successful additional starting groups from which the organic molecule having the desired property is produced by substituting the successful additional starting group as the additional starting group in step (1) to thereby identify a narrowed group of chemical reactions from which the compound having the desired property can be produced

25. A method for the production of an organic molecule having a desired property, comprising the steps of:
(a) providing a starting group of at least 100 different organic molecules selected from the group consisting of alkanes, alkenes, alkynes, arenes, alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds, organometallic compounds, hetero-atom bearing compounds, amino acids, nucleotides, and mixtures thereof;
(b) causing at least one chemical reaction selected from the group consisting of substitution, addition, elimination, rearrangement, dehydration, reduction, oxidation, condensation, hydrogenation, dehydrogenation, dimerization, epoxidation, isomerization, cyclization, decyclization, halogenation, sulfonation, alkylation, acylation, nitration, hydrolysis, esterification, transesterification, carboxylation, decarboxylation, amination, and deamination to take place with at least some of the different organic molecules in the starting group to create an intermediate reaction mixture having one or more organic molecules different from the organic molecules in the starting group;
(c) repeating step (b) at least once by substituting the intermediate reaction mixture as the starting group to thereby produce a final reaction mixture as a result of the last repetition;
(d) screening the final reaction mixture resulting from step (c) for the presence of the organic molecule having the desired property;
(e) isolating from the final reaction mixture the organic molecule having the desired property; and (f) determining the structure or functional properties characterizing the organic molecule having the desired property.

26. The method of claim 25 wherein the different organic molecules of the starting group all share a common core structure.

27. The method of claim 26 further comprising the step of using a selection method on the intermediate reaction mixture to produce a subset of organic molecules with a higher likelihood of producing the organic molecule having the desired property.

28. The method of claim 27 wherein the at least one chemical reaction is caused by adding a set of different enzymes.

29. A method for the production of an organic molecule having a desired property, comprising the steps of:
(a) reacting a group of different substrates, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehydrating agent to yield a first reaction mixture;

(b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture;
(c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture;
(d) performing a condensation reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture;
(e) exposing the fourth reaction mixture to light with a wavelength of about 220 nanometers to 600 nanometers, thereby producing one or more organic molecules different from the substrates and agents;
(f) screening the exposed fourth reaction mixture for the presence of the organic molecule having the desired property;
and (g) isolating from the exposed fourth reaction mixture the organic molecule having the desired property.

30. A method of generating for characterization an organic molecule having a desired property, comprising the steps of:
(a) reacting a group of different substrates, the group comprising acids, amines, alcohols, and unsaturated compounds, under suitable conditions with a dehydrating agent to yield a first reaction mixture;
(b) reacting the first reaction mixture with a reducing agent under suitable conditions to yield a second reaction mixture;
(c) reacting the second reaction mixture with an oxidizing agent under suitable conditions to yield a third reaction mixture;

(d) performing a condensation reaction under suitable conditions upon the third reaction mixture to yield a fourth reaction mixture;
(e) exposing the fourth reaction mixture to light with a wavelength of about 220 nanometers to 600 nanometers, thereby producing one or more organic molecules different from the substrates and agents;
(f) screening the exposed fourth reaction mixture for the presence of the organic molecule having the desired property;
and (g) determining the structure or functional properties characterizing the organic molecule having the desired property.

31. The method of claim 30, additionally including, prior to step (9), isolating from the reaction mixture the organic molecule having the desired property.

32. The method of either claim 29 or claim 30, additionally including, prior to step (f), repeating steps (a)-(e) with or without introducing additional substrates.

33. The method of either claim 29 or claim 30 wherein the order in which the substrates are subjected to the reactions of steps (a)-(e) is varied.

34. The method of either claim 29 or claim 30, further including after step (9), producing the organic molecule having the desired property.

35. The method of either claim 29 or claim 30 wherein the desired property is the ability to function as a drug, a vaccine, a ligand, a catalyst, a catalytic cofactor, a structure of use, a detector molecule, or a building block for another compound.

36. A method for the production of an organic molecule having a desired property, comprising the steps of:
(a) reacting a group of different enzymes representing a diversity of catalytic activities under suitable conditions with a group of different substrates to create a reaction mixture, thereby producing one or more organic molecules different from the enzymes and substrates in the reaction mixture;
(b) screening the reaction mixture for the presence of the organic molecule having the desired property; and (c) isolating from the reaction mixture the organic molecule having the desired property.

37. A method of generating for characterizing an organic molecule having a desired property, comprising the steps of:
(a) reacting a group of different enzymes representing a diversity of catalytic activities under suitable conditions with a group of different substrates to create a reaction mixture, thereby producing one or more organic molecules different from the enzymes and substrates in the reaction mixture;
(b) screening the reaction mixture for the presence of the organic molecule having the desired property; and (c) determining the structure or functional properties characterizing the organic molecule having the desired property.

38. The method of claim 37, additionally including, prior to step (c), isolating from the reaction mixture the organic molecule having the desired property.

39. The method of either claim 36 or claim 37 wherein the group of different substrates is selected from the group consisting of alkanes, alkenes, alkynes, arenes, alcohols, ethers, amides, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds, organometallic compounds, hetero-atom bearing compounds, amino acids, nucleotides, and mixtures thereof.

40. The method of claim 39 wherein the group of different substrates is selected from the group consisting of acids, amines, alcohols, amino acids, nucleotides, and unsaturated compounds.

41. The method of claim 40 wherein the group of different substrates is selected from the group consisting of amino acids and nucleotides.

42. The method of claim 39 wherein the group of different substrates contains at least 100 different organic molecules.

43. The method of claim 42 wherein the group of different substrates contains at least 1,000 different organic molecules.

44. The method of either claim 36 or claim 37, further comprising after step (c), producing the organic molecule having the desired property.

45. The method of either claim 36 or claim 37, wherein the desired property is the ability to function as a drug, a vaccine, a ligand, a catalyst, a catalytic cofactor, a structure of use, a detector molecule, or a building block for another compound.

46. The method of either claim 36 or claim 37 wherein the group of different enzymes comprises at least 10,000 different enzymes.

47. The method of claim 46 wherein the group of different enzymes comprises at least 1,000,000 different enzymes.

48. The method of claim 47 wherein the group of different enzymes comprises at least 100,000,000 different enzymes.

49. The method of either claim 36 or claim 37, wherein the substrates of the group of different substrates all share a common core structure.

50. The method of either claim 36 or claim 37, further comprising the step of using a selection method on the reaction mixture to produce a subset of organic molecules with a higher likelihood of producing the organic molecule having the desired property.