WO2003009940A2 - Method and device for directed sort combinational synthesis - Google Patents

Abstract

A sorting apparatus and method for directed sort combinatorial chemical synthesis is provided. This invention relates to a directed flexible, sorting apparatus and method for combinatorial chemical synthesis using a directed split and recombine process. The process utilizes one algorithm that is universal in directing and sorting a library of any size. This invention further provides a method of sorting solid supports for combinatorial chemical synthesis comprising delivering one or more supports which are organized in a one dimensional linear array in one or more reaction vessels, to an isolation and transfer chamber, transferring one or more support from each of said reaction vessels to one or more subsequent reaction vessels, in a patterned distribution; wherein the patterned distribution of supports in each subsequent transfer is identical and the position of each support in a reaction vessel codes for its previous synthesis history. In another aspect, the invention provides a directed sort apparatus wherein all members of a set at a given point in time are reacted with the same chemical building blocks throughout synthesis.

Description

METHOD AND DEVICE FOR DIRECTED SORT COMBINATIONAL SYNTHESIS

This application claims the benefit of the filing date of U.S. Provisional Application Serial No. 60/ 307,186 ;Titled: Method and Device for Directed Sorting in Combinational Synthesis filed 7/24/2001.

BACKGROUND

Recently, protocols have been developed for combinatorial synthesis in which a large number of chemical compounds are individually synthesized. These methods proceed by a sequence of steps, each step adding a particular, selected one of a plurality of building blocks, i.e. small organic molecules, to a growing, intermediate compound. Thereby, the number of potential final compounds is a product of mathematical terms, one term for each synthesis step representing the number of possible building blocks that can be added at that step.

In combinatorial synthesis, addition step reactions typically proceed by combining the partially-

synthesized, intermediate compound with the building block having an attached activating residue.

(Hereinafter, building blocks are assumed to have necessary activating residues attached.) Also

added to an addition step reaction are activating reagents and other reagents and solvents. The

building blocks are often added in a molar excess to the partially synthesized compound present so

that the thermodynamically favorable building block addition proceeds substantially to completion.

After addition of one building block, the intermediate compound is separated from the spent

reaction solution and prepared for the addition of a further building block. Often, the intermediate

compound is attached to a solid-phase support by, e.g., a cleaveable linking residue, in order to simplify separation of intermediate compound from spent addition reaction solutions. In such

solid-phase protocols, a final step of cleaving the linking residue frees the final compound.

Building blocks (activated as necessary), activating and other reagents, and reaction conditions

have been recently perfected for a wide variety of classes of final compounds. Exemplary of such

reactions and protocols are the following for addition of natural and artificial amino acids to form

peptides: Lam et al., 1991, A new type of synthetic peptide library for identifying ligand-binding

activity, Nature 354: 82-84.; U.S. Pat. 5,510,240 to Lam et al. for Method of screening a peptide

library; Lam et al., 1994, Selectide technology: Bead-binding screening. Methods: A Companion to

Methods in Enzymoloqy 6: 372-380. For protocols for the synthesis of benzodiazepine moieties,

see, e.g.: Bunin et al., 1992, A general and expedient method for the solid phase synthesis of 1,4-

benzodiazepine derivatives, J. Amer. Chem. Soc, 114: 10997-10998.; U.S. Pat. 288,514 to EUman

for Solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support.

Also, for protocols for the addition of N-substituted glycines to form peptoids, see, e.g., Simon, et

al., 1992, Peptoids: A modular approach to drug discovery. Proc. Natl. Acad. Sci. USA, 89: 9367-

9371; Zuckermann et al, 1992, Efficient method for the preparation of peptoids [oligo(N-

substituted glycines)] by submonomer solid-phase synthesis. J. Amer. Chem. Soc, 114: 10646-

10647; WO PCT94/06,451 to Moos et al. for Synthesis of N-substituted polyamide monomers,

useful as solvents, additives for food, enzyme inhibitors etc. Approaches for synthesis of small

molecular libraries were recently reviewed by, e.g., Krchnak et al., 1996, Synthetic library

techniques: Subjective (biased and generic) thoughts and views, Molecular Diversity, 1 : 193-216;

Ellman, 1996, Design, synthesis, and evaluation of small-molecule libraries, Account. Chem. Res., 29: 132-143; Armstrong et al., 1996, Multiple-component condensation strategies for

combinatorial library synthesis, Account. Chem. Res., 29: 123-131.; Fruchtel et al., 1996, Organic

chemistry on solid supports, Angew. Chem. Int. Ed., 35: 17-42; Thompson et al., 1996, Synthesis

and application of small molecule libraries, Chem. Rev., 96 :555-600; Rinnova et al., 1996,

Molecular diversity and libraries of structures: Synthesis and screening, Collect. Czech. Chem.

Commun., 61: 171-231; Hermkens et al., 1996, Solid-phase organic reactions: A review of the

recent literature, Tetrahedron, 52: 4527-4554.

Predictable synthesis of a large number of individual compounds, which are subsequently collected

into a library of compounds, is of interest and utility in several fields, in particular libraries for drug

discovery, agricultural, health, veterinarian, nutritional sciences, and materials sciences. It is of

particular interest to identify active compounds which provide structural information for use in the

development of therapeutic licensed or marketed compounds and compound series, to establish

structure activity relationship data, to validate the function of molecules, to identify

pharmacophores (active molecular structural elements), and to identify active compounds in

particular in the field of pharmaceutical lead-compound selection. A pharmaceutical lead-

compound for a drug is a compound which exhibits a particular biologic activity of pharmaceutical

interest and which can serve as a starting point for the selection and synthesis of a drug compound,

which in addition to the particular biological activity has pharmacologic and toxicologic properties

suitable for administration to humans or animals. It is apparent that synthesis of large numbers of

compounds and screening for their biological activities in a controlled biological system can be of

assistance in lead compound selection. Instead of turning to botanical or other natural sources, the pharmaceutical chemist can use combinatorial protocols to generate in the laboratory compounds

to screen for desired activities. See, e.g., Borman, 1996, Combinatorial Chemists Focus on Small

Molecules, Molecular Recognition, and Automation, Chemical & Engineering News, Feb. 12,

1996, 29-54.

To achieve the benefits of these developed combinatorial protocols, automated synthesis apparatus

is advantageous. Dealing manually with hundreds, thousands, or perhaps tens of thousands of

separate compounds is expensive, time consuming, and prone to error. Therefore, synthesis robots

for automating one or more steps of combinatorial protocols have been recently developed.

Examples of such recently developed robots are described in: Cargill et al., 1996, Automated

Combinatorial Chemistry on Solid-Phase, Laboratory Robotics and Automation, 8: 139-148; U.S.

Pat. 5,503,805 to Sugarman et al; U.S. Pat. 5,252,296 to Zuckermann et al. for Method and

apparatus for biopolymer synthesis; WO PCT 93/12,427 to Zuckermann et al., for Automated

apparatus for use in peptide synthesis; Krchnak et al., 1996, MARS— Multiple Automatic Robot

Synthesizer. Continuous Flow of Peptide, Peptide Res. 9: 45-49.

The most efficient, and as far as the instrumentation the least demanding, combinatorial chemistry

method for the synthesis of large compound arrays is the split and mix (split and recombine)

method introduced by Arpad Furka (Furka et al. 1988b; Furka et al. 1988a; Furka et al. 1991) and

later independently used by Kit Lam (Lam et al. 1991) and Richard Houghten (Houghten et al.

1991). The method suffers from limitations, e.g., the amount of compound synthesized is restricted

by the loading of a solid support particle, and the chemical history of each particle has to be

recorded or determined by analytical methods (Lam et al. 1997).

The split and mix method was developed on resin beads and those typically accommodate

nanomolar amounts of material at best. In order to increase the amount of compound, two

different routes were followed. Richard Houghten invented T-bags (Houghten, 1985), where the

resin beads were enclosed into a permeable container. IRORI MicroKans are based on the same

idea. Another option is to apply a macroscopic modular support, introduced by M.H. Geysen

(Geysen et al. 1984; Geysen et al. 1986; Geysen et al. 1987) and Ronald Frank (Frank and Dδring,

1988). In order to increase loading and to improve reaction kinetics, the polypropylene mold was

grafted with polystyrene to produce SynPhase Crowns and soon after SynPhase Lanterns with a

larger surface area and a very convenient shape . The reaction kinetics and product yield from

Lanterns are comparable with resin beads; however, handling Lanterns provides distinct

advantages, particularly for the synthesis of sizable libraries containing mg amounts of each

compound.

Different methods were used to identify each compound after synthesis. The lost chemical history due to mixing of individual particles after each combinatorial step was addressed by chemical

coding on beads (Kerr et al. 1993; Nikolaev et al. 1993; Ohlmeyer et al. 1993), T-bags were

manually labeling (Houghten, 1985), attaching radio-frequency tags (Moran et al. 1995; Nicolaou

et al. 1995) to MicroKans or Lanterns, etc. Recently (Smith et al. 1999) and later others (Furka et

al. 2000; Furka, 2000) described positional tracking of the chemical history by arranging the solid

phase particles into a one-dimensional string referred to as necklace coding.

In general solid state synthesis is a well-know procedure, reaction vessels are conventional and

typically are inexpensive commercially available vessels, microtitre plates, syringes, and so forth,

capable of resisting the solvents and reaction conditions used in synthesis protocols, where reaction

temperatures may range from -100 to +200 ° C and reactions may take minutes or sometimes days

to complete. In a preferred embodiment the reaction vessel is a threaded Teflon tube enclosed on

both sides by luer fittings. Reaction vessel arrays are sealed with various sealing means. However,

reaction vessels can be tailored to withstand any rigorous reaction conditions (i.e. high or low

temperatures; high or low pressures; or closed systems).

Solid-phase combinatorial synthesis typically proceeds according to the following steps. In a first

step, reaction vessels are charged with one or more solid-phase supports, and the first of the

plurality of building blocks or a linker moiety is covalently linked to the solid support.

Subsequently, a plurality of building block addition steps are performed, all of which involve

repetitive execution of the following substeps, and in a sequence chosen to synthesize the desired

compound. First, a sufficient quantity of a solution containing the building block moiety selected for addition is accurately added to the reaction vessels so that the building block moiety is present in a molar excess to the intermediate compound. The reaction is triggered and promoted by activating reagents and other reagents and solvents, which are also added to the reaction vessel. The reaction vessel is then incubated at a controlled temperature for a time, typically between

minutes and 24 hours, sometimes as long as several days, sufficient for the building block addition reaction to go to substantial completion. Optionally, during this incubation, the reaction vessel can be intermittently agitated or stirred. Finally, in a last substep of building block addition, the reaction vessel containing the solid-phase support with attached intermediate compound is

prepared for addition of the next building block by removing the spent reaction fluid and thorough washing and reconditioning the solid-phase support. Washing often typically involves cycles of adding and removing a wash solvent. Optionally, during the addition steps, multiple building blocks can be added to one reaction vessel in order to carry out more than one synthetic step on one solid-phase support. After the desired number of building block addition steps, the final compound is present in the reaction vessel attached to the solid-phase support. The final compounds can be utilized either directly attached to their synthetic supports, or alternatively, can be cleaved from their supports. In the latter case, the linker moiety attaching the compound to the solid-phase support is cleaved, and the library compound is extracted.

The advent of combinatorial chemistry techniques has brought about the need to handle numerous different solid-phase (support) bound intermediates concurrently. The modular solid phase support has become popular for the synthesis of combinatorial arrays of compounds, particularly due to the advantageous physico-chemical properties and the possibility to apply a directed sorting approach to distribute supports between combinatorial synthesis steps. These methods can also be applied to sort containers holding resin or solution phase, where the container is treated as a single "support".

In order to preserve the identity of each support during the combinatorial synthesis on Lanterns others have described a necklace coding principle. A necklace may be a string, wire, rod, or device inserted through aligned holes in each object set. In order to sort Lanterns before a subsequent combinatorial step applicants have found that Lanterns packed into a tube provided handling advantages when compared to a string of Lanterns, particularly as a result of the Directed Sort Apparatus described in this disclosure.

This invention relates to a sorting apparatus and method for directed sort combinatorial chemical synthesis; more particularly it relates to a directed flexible, sorting apparatus and method for combinatorial chemical synthesis using a directed split and recombine process. The process utilizes one algorithm that is universal in directing and sorting a library of any size.

In one aspect, this invention provides a method sorting solid supports for combinatorial chemical synthesis comprising delivering one or more supports which are organized in a one dimensional linear array in one or more reaction vessels, to an isolation and transfer chamber, transferring one or more support from each of said reaction vessels to one or more subsequent reaction vessels, in a patterned distribution; wherein the patterned distribution of supports in each subsequent transfer is identical and the position of each support in a reaction vessel codes for its previous synthesis history.

In another aspect, this invention provides a directed sort apparatus a directed sort apparatus for performing combinatorial solid phase synthesis of compounds comprising two or more first

reaction vessels for holding one or more object sets organized in a first ordered pattern; an isolation and transfer chamber for directed redistribution of object sets from each of said reaction vessels to one or more subsequent reaction vessels for receiving object sets from the isolation and transfer chamber; and repeating steps for each building block added.

In another aspect, the invention provides a directed sort apparatus for performing combinatorial solid phase synthesis of compounds comprising performing a reaction in each of one or more first reaction vessels for holding one or more object sets organized in a one dimensional linear array, and delivering to an isolated and transfer chamber for directed redistribution of object sets from each of said reaction vessels to one or more subsequent reaction vessels for receiving object sets from the isolation and transfer chamber; and repeating the steps for each building block added.

In another aspect, the invention provides a directed sort apparatus wherein all members of an object set at a given point in time are reacted with the same chemical building blocks throughout synthesis. Directed sort combinatorial synthesis of this invention is a method for split and mix chemical synthesis. One compound per support or many compounds per support may be used to produce large amounts of each compound with great efficiency, simplicity, and speed. The solid supports can be delivered either for reaction in flow through tubes or for handling in microplates. The sort apparatus follows an algorithm that is universal in directing the sorting of any library regardless of size. A single support can be sorted at a time or multiple supports, "object sets", can be used to increase the scale/yield of each compound synthesized. The problem of lost chemical history due to mixing of individual supports after each combinatorial step is addressed by preserving the order of supports, e.g., as they are placed into the reaction vessels.

Throughout the reshuffling process, in addition to solid phase synthesis, the algorithm can be used to sort any object, including vessels for solution phase synthesis or even for sorting the control of valves for directing the addition of reagents to certain reaction vessels. Use of the single universal algorithm only requires the chemist to enter a library design (i.e. a number of steps and the number of building blocks in each step). A computer following the algorithm can then design the synthesis and the synthesis batch size. All building blocks can be validated allowing for increased quality control.

Combinatorial chemistry synthesis protocols prescribe the stepwise, sequential addition of building blocks to intermediate partially-synthesized intermediate compounds in order to synthesize a final compound. These protocols are, generally, divided into liquid-phase protocols and solid-phase protocols. In liquid-phase protocols, final compounds are synthesized in solution. In solid-phase synthesis, final compounds are synthesized attached to solid-phase supports that permit the use of

simple mechanical means to separate intermediate or partially-synthesized intermediate compounds

between synthetic steps. A preferred solid-phase support for the present invention includes

synphase lanterns which may optionally be functionalized in order to covalently attach intermediate

compounds. However any support such as those made of various glasses, plastics, or resins can

also be used.

The directed sort combinatorial synthesis method provides a low cost alternative to radio tagging.

The method combines the efficiency of the split mix approach with the simplicity of recording the

chemical history of individual solid phase particles by tracking, or directing, the location of each,

e.g., support throughout each step of synthesis and final distribution into vessels for cleavage and

compound extraction/storage.

In further embodiments, this invention comprises other combination and sub-combinations of the

previously described elements, functioning either in conjunction with other apparatus or independently of any apparatus. Directed sort combinatorial synthesis can be accomplished

I through a directed sort of supports or reaction vessels where the latter are physically moved, or in

yet another embodiment , through the directed sort of supports or reaction vessels in which they

are not moved, but rather addition of reagents is directed, or a combination of both. One skilled in

the art can envision numerous ways of controlling the delivery of reagents to locations (e.g. control of valves directing the addition of reagents to certain reaction vessels) described by the algorithm,

rather than directly sorting the supports, to achieve the same directed synthesis. In a further

embodiment , an array of reaction vessels, e.g. a 3-D array, e.g., (Figure 8), is separated from the

nearest neighbor reaction vessels by valves. It should be recognized that a similarly functional

array can be constructed where, physically, the reaction vessels are not in an array, but are linked

by tubing and valves to produce the same functional relationship of connectivity between sets of

reaction vessels.

One skilled in the art can see that, rather than indexing the delivery vessels and keeping the

receiving vessels fixed, the receiving vessels can be indexed and the delivery vessels remain fixed,

or other mechanical, valve control, or support transport means can be used to achieve the same

directed sort outcome, and the isolation and transfer chamber can be replaced with valves or other

support transport means such as the electromotive transport of supports in a microfluidic system.

Similarly, one skilled in the art can see that rather than using supports,

actual reaction vessels can be used instead, within which the reactions are carried out, such as

solution phase reactions, or reaction vessels containing multiple solid phase resin particles, such as

a microcan or closed reaction vessel, accessed, for instance, through a septum(s) contained in the reaction vessel. Throughout this disclosure, the following terms have the following conventional meanings:

"Chemical library" or "combinatorial library" or "compound library" or "array" is an intentionally

created collection of different compounds, usually prepared in parallel, and screened for biological

activity in a variety of different formats.

"Building block" refers to any molecule that can be covalently attached to other molecules to

generate structurally different compounds, generally small organic molecules. The addition of a

chemical building block is often referred to as subunit addition or substituent addition.

"Combinatorial chemistry" or "combinatorial synthesis" refers to an ordered strategy for the

parallel synthesis of diverse molecular entities which leads to the generation of chemical libraries.

The strategy consists of the systematic and repetitive covalent connection of structurally different

building blocks to each other to yield large arrays of compounds.

"Linker" refers to a molecule or group of molecules covalently attached to the solid support on

one end and to the first building block on the other end. Linkers have different molecular structures

and, therefore, different lengths, shapes, sizes, degree of hydrophobicity and hydrophilicity, steric

bulk, and chemical reactivity. The selection of a linker in solid phase synthesis is dependent on both

the synthetic scheme and the biological screening format.

"Solid support" refers to a material or group of materials having a rigid or semi-rigid surface, appropriate size, shape, and porosity, and high chemical resistance. Examples of solid supports are

glass, silica, cellulose, polystyrene cross-linked with divinylbenzene, polystyrene-

polyethyleneglycol copolymer, silica gel, alumina gel, plastic, polyamides, polyimides,

polytetraflouroethylene, phenol-formaldehyde polymers, poly(chlorotrifluoroethylene),

halogenated polyolefins and other support materials commonly used in peptide, polymer, and

small-molecule solid phase synthesis.

"Resin" refers to a solid support material which has been grafted with a linker for attachment of the

first building block. Examples of preferred resins are IRORI MicroKans , Wang resin (a

polystyrene-based resin with a 4-alkoxybenzyl alcohol linker), Rink amide resin (a polystyrene-

based resin with a 4-(2',4'-dimethoxyphenylaminomethyl)phenoxymethyl linker), and Sasrin resin (a

polystyrene-based resin with a 2-methoxy4-alkoxybenzyl alcohol linker). Other preferred resins are

described in the Combinatorial Chemistry & Solid Phase Organic Chemistry Handbook published

by NovaBiochem, La Jolla, Calif; the Solid Phase Sciences catalog published by Solid Phase

Sciences, San Rafael, Calif., or the Rapp Polymere catalog published by Rapp Polymere GmbH,

Tubingen, Germany.

"Reaction vessel" refers to the vessel in which reactions take place. In some cases it may act as a

holder for "solid supports". The reaction vessel is capable of resisting the solvents and reaction

conditions used in synthesis protocols.

Also, "object set" refers to one or more solid support, resin, or in the case of liquid phase synthesis a reaction vessel that shares the same chemical history. Members of an object set move

together throughout the combinatorial chemical synthesis process. An object set may be only one

support, vessel or resin can or if scale up is desired in order to increase the yield of each given

compound produced than multiple supports, vessels or resin cans can be used. Each member of the

object set will have the same number and order of building block reactions resulting in the same

final compound.

These and other features, aspects, and advantages of the present invention will become better

understood by reference to the accompanying drawings and the following description, where:

FIG. la TEFLON tube as reaction vessel, dispensing vessel and receiving vessel

FIG. lb TEFLON tube as reaction vessel, dispensing vessel and receiving vessel

FIG. 2 illustrates sorting algorithm; each string represents a different reaction vessel; each letter

represents the addition of a building block; the numbers on each object set corresponds to the

order of object sets before each reshuffle

FIG. 2 A further illustrates the algorithm of figure 2; each necklace represents a different reaction

vessel; each letter represents the addition of a building block. The relationship of the building

blocks after each reshuffle step and after each building block addition is shown. FIG. 3a Depicts the details of a dispensor which can be used to transfer object sets such as lanterns

one-by-one from a delivery tube to selected receiving tubes.

FIG. 3b Depicts arrangements using a plurality of dispensers in a conversion manifold for

distributing object sets one-by-one from a set of delivery tubes to a set of receiving tubes using a

2-D rotary arrangement.

FIG. 3 c Depicts arrangements using a plurality of dispensers in a conversion manifold for

distributing object sets one-by-one from a set of delivery tubes to a set of receiving tubes using a

2-D planar arrangement

FIG. 3d Depicts arrangements using a plurality of dispensers in a conversion manifold for

distributing object sets one-by-one from a set of delivery tubes to a set of receiving tubes using a

linear arrangement.

FIG . 4 illustrate a 12 channel solid support reshuffler

FIG . 5 Traceless synthesis of benzimidazoles

FIG. 6 Analytical gradient HPLC profile of crude benzimidazole FIG. 7a Depicts the circular 2-D linear arrangement of delivery tubes, conversion manifold, and receiving tubes used for the simple sorting of object sets between synthesis steps.

FIG. 7b Details of the device shown in Figure 7a.

FIG. 8 3-D array in which each reaction vessel is separated from the nearest neighbor reaction vessels by valves.

FIG. 9 Diagram of a 3 building block X 4 building block library ;

FIG 10. Diagram of a 4 building block X 3 building block library

FIG 11. Depicts a three step process with three building blocks in each step. Each string is a separate reaction vessel with nine supports in each.

FIG 12 Depicts the three step process of figure 11. The relationship of the building blocks

FIG. 13 Depicts the use of multiple short tubes in place of one long tube, forming a "virtual tube

For clarity of disclosure, and not by way of limitation, the detailed description of this invention is presented herein with respect to figures that illustrate preferred embodiments of elements of this invention. However, this invention includes those alternative embodiments of these elements performing similar functions in similar manners that will be apparent to one skilled in the art from the disclosure provided. Additionally, this invention is disclosed with respect to its preferred application to solid-phase, combinatorial chemistry synthesis. The invention is not so limited, and

includes application of the various elements disclosed to other chemical protocols having similar functional steps, as also will be apparent to one skilled in the art. For example, components of this invention can be applied to appropriate liquid-phase, combinatorial chemistry synthesis protocols, to other solid- or liquid-phase chemical protocols, or to any combination thereof.

Sorting algorithm

This invention can employ a general sorting protocol that (i) applies the same algorithm before any combinatorial step of the synthesis, and (ii) which is independent of the number of building blocks in individual steps, and (iii) which permits multiples of standard capacity (or length) dispensing and receiving tubes to be used to synthesize any size library. For example, Lanterns can be organized in one-dimensional, or linear, array in each dispensing tube (Figure 2). The first receiving tube is

filled with Lanterns from the dispensing tube(s) (Fig. 3) by transferring one Lantern at a time from

each of dispensing tubes until the receiving tube contains n_x = n_t0t/BB_x Lanterns, where n_x is the

number of Lanterns in a tube for the x-th combinatorial step, n_tot is the total number of compounds

(which number is equal to the number of Lantems unless multiple supports per compound are used

to increase the amount of compound produced), and BB_X is the number of building blocks in the x-

th step. Then, the next receiving tube is filled the same way. The algorithm is briefly described

using an example of a library synthesized in three combinatorial steps using 2, 3, and 4 building

blocks in the 1^st, 2^nd, and 3^rd steps, respectively (Figure 2). Rather than directly transferring

between reaction vessels, columns of object sets can be transferred onto a necklace (i.e. string,

wire, rod, or device inserted through aligned holes in each object set) and then to the next set of

reaction vessels, or a necklace can be used to transfer object sets from a reaction vessel to a

distribution device.

This algorithm is applicable to the directed sort by any process, including the Directed Sort

Apparatus described in this disclosure. In the example of the algorithm in Fig. 2 two tubes (or

strung necklaces) are depicted, each containing 12 Lanterns which are reacted with representative

"A" [light] or "B" [dark] building blocks's, i.e., reagents or building blocks. Then all the Lanterns

are reshuffled into three new tubes (or onto three new necklaces). One Lantern was taken from

each dispensing tube and put into the receiving tube (the last Lantern from the first tube ends up at

the bottom of the first receiving tube, the last Lantern from the second tube ends up as the second

Lantern from the bottom on the first receiving tube, etc.). Altogether 8 Lanterns are transferred to the first receiving tube. The same process is repeated 2 more times. As a result, three new tubes

containing identical arrays of Lantems were formed.

These three tubes are again reacted, respectively, with the appropriate building block (e.g., "C" "D" or "E" ) and then the reshuffling is repeated using exactly the same algorithm as previously.

The result is four new tubes, each containing identical arrays of Lanterns formed that are then reacted, respectively, with one of four possible building blocks, (e.g., representative "F", "G", "H", and "I") in the last combinatorial step. As a result of this process, all 24 "letter" combinations are made and the position of any Lantern within the final four tubes (or on a necklace, had that method been used) unambiguously detem ines the chemical history (kind of building block reacted with the

Lantern in each step of the process) or putative structure synthesized onto each Lantern. Figure 2a further depicts the relationship of the building blocks (e.g., letters) as the Lanterns move through the synthesis steps.

In the example of the algorithm in Fig. 12 three tubes (or strung necklaces) are depicted, each containing 9 Lantems which are reacted with representative "A" , "B" or "C" building blocks's, i.e., reagents or building blocks. Then all the Lantems are reshuffled into three new tubes (or onto three new necklaces). Altogether 9 Lantems are transferred to the first receiving tube. The same process is repeated 2 more times. As a result, three new tubes containing identical arrays of Lantems were formed. These three tubes are again reacted, respectively, with the appropriate building block (e.g., "D"

"E" or "F" ) and then the reshuffling is repeated using exactly the same algorithm as previously.

The result is three new tubes, each containing identical arrays of Lanterns (object sets) that are

then reacted, respectively, with one of three possible building blocks, (e.g., representative "G",

"H", and "I") in the last combinatorial step. As a result of this process, all 27 "letter"

combinations are made and the position of any Lantern within the final three tubes (or on a

necklace, had that method been used) unambiguously determines the chemical history (kind of

building block reacted with the Lantern in each step of the process) or putative structure

synthesized onto each Lantern. The Lantems (object sets) of figure 11 are numbered to show the

correlation between the position of the Lantern after each reshuffle. Figure 12 further depicts the

relationship of the building blocks (e.g., letters) as the Lantems move through the synthesis steps.

Sorting from tubes into tubes can provide handling advantages when compared to sorting onto

strings or necklaces. Thus, one can use sorting tubes as reaction vessels. Besides being compatible

with the Directed Sort Apparatus described in this disclosure, the tubes provide convenient

reaction vessels with the advantage of using a continuous flow method for washing resins beads,

which has been recognized for a long time to be very efficient (Lukas et al. 1981; Dryland and

Sheppard, 1986; Krchnak et al. 1987; Frank and Dδring, 1988) and has been used in commercial

synthesizers.

Apparatus Directed Sort Apparatus can be used to sort Lanterns between each consecutive combinatorial step. Such apparatus can simply permit the Lanterns to be sorted one by one from one dispensing tube at a time and transferred to the each appropriate receiving tube (Figure 3). Figure 3a Depicts the details of a dispenser which can be used to transfer object sets such as lanterns one-by-one from a delivery tube to selected receiving tubes. A plurality of dispensers may be used in a

conversion manifold for distributing object sets one-by-one from a set of delivery tubes to a set of receiving tubes using a 2-D rotary arrangement (Figure 3b), a 2-D planar arrangement (Figure 3c), or a linear arrangement ( Figure 3d).

For efficiency, however, a sorting device, such as the Directed Sort Apparatus can handle a multiplicity of dispensing and receiving tubes concurrently (Figure 4). The receiving tubes can be used as reactors, and in turn become dispensing tubes for distribution before the next round of synthesis. Alternatively, the Lanterns from each receiving tube can be transferred into reaction vessels, so long as the order is maintained. Finally, at the end of the synthesis, the Lanterns can be transferred into cleavage or storage vessels, such as 96-well plates, one Lantem per well, using the Directed Sort Apparatus. Therefore, for purely practical reasons to conveniently accommodate the 96 well plate format, the apparatus can be built to accommodate twelve tube reactors at a time and

allow sorting Lantems during synthesis and distribution of Lanterns into 96-well plates at the end of synthesis. The algorithm of this invention enables the synthesis of any reasonable size of combinatorial library to be synthesized by the 12 tube Directed Sort Apparatus in a batch- wise manner. Chemistry

A chemical route has been developed to a traceless synthesis of tri-substituted benzimidazoles on SynPhase Lantems according to Figure 5. Aminomethylated Lanterns are acylated with an Fmoc- 4-methoxy-4 -(gamma-carboxypropyloxy)benzhydrylamine. Aromatic nucleophihc substitution of

fluorine in o-fluoronitrobenzenes by the immobilized amino group provides o-nitroaniline 1. The nitro group is reduced by tin(II) chloride dihydrate in N-methylpyrrolidone (NMP) and then reacted with isothiocyanates to form polymer supported thioureas 2. Cyclization to 2- arylaminobenzimidazoles 3 is accomplished by diisopropylcarbodiimide (DIC). In order to introduce a third combinatorial step, the resin-bound 2-arylaminobenzimidazoles is reacted with

isocyanate to yield Lantern-bound product 4. The target compounds 5 are obtained by cleavage from the Lantern using TFA. Figure 6 documents purity of one crude product.

Directed sort combinatorial synthesis can be accomplished through a directed sort of supports or reaction vessels where they are physically moved, or in yet another embodiment , through the directed sort of supports or reaction vessels in which they are not moved, but rather addition of reagents is directed, or a combination of both. One skilled in the art can envision numerous ways of controlling the delivery of reagents to locations (e.g. control of valves directing the addition of reagents to certain reaction vessels) described by the algorithm of this invention, rather than directly sorting the supports, to achieve the same directed synthesis. One can also envision use of an array of reaction vessels, e.g. a 3-D array (Fig 8) , in which each reaction vessel is separated from the nearest neighbor reaction vessels by valves, recognizing that a similarly functional array

can be constructed where, physically, the reaction vessels are not in an array, but are linked by

tubing and valves to produce the same functional relationship of connectivity between sets of

reaction vessels. For example, the reaction vessel need only have a single pair of valves or ports

(for entry and exit of reagent, gas, etc) but there is a heirachal assembly of valves or a switching

mechanism of valves to selectively access combinations of reaction vessels, for instance as

described by the algorithm.

For demonstration purposes, one can depict an actual regular 3-D array of interconnected reaction

vessels, as shown for the synthesis of a 3 x 3 x 3 library in fig. 8. Each reaction vessel has 6 valves

for flow-through of reagents in the x-axis, y-axis, and z-axis directions, valve a to valve c, valve b

to valve d, and valve f to valve e, respectively. For explanation purposes, each horizontal layer of

the 3-D array of reaction vessels is labeled x, y, and z, and each vertical layer of the 3-D array of

reaction vessels is labeled 1, 2, and 3. Thus, i) opening valves b and d wifl permit reagents to flow

in the z-axis direction, from reaction vessels in horizontal layer x to vessels in horizontal layer y to

vessels in horizontal layer z, essentially causing the x, y, and z reaction vessels immediately above

one another to become a single reaction vessel; ii) opening valves a and c will permit reagents to

flow in the y-axis direction, reconfiguring the effective reaction vessels; and iii) opening valves f

and e will permit reagents to flow in the x-axis direction, reconfiguring yet again the effective

reaction vessels. Thus an example of a synthetic reaction scheme for a three step reaction, 3

building blocks in each reaction (BB 1 ,2, 3 in reaction 1, BB 4, 5, 6 in reaction 2, and BB 7, 8, 9 in reaction 3), would be to: i) add BB1 and carry out reaction 1 entering through open valve a and exiting (with recycling if desired) through open valve c of all the reaction vessels in layer x, add BB2 and carry out reaction 1 entering through open valve a and exiting (with recycling if desired) through open valve c of all the reaction vessels in layer y, and add BB3 and carry out reaction 1 entering through open valve a and exiting (with recycling if desired) through open valve c of all the

reaction vessels in layer z; ii) add BB4 and carry out reaction 2 entering through open valve f and exiting (with recycling if desired) through open valve e of all reaction vessels in layer x, through add BB5 and carry out reaction 2 entering through open valve f and exiting (with recycling if desired) through open valve e of all reaction vessels in layer y, and add BB6 and carry out reaction 2 entering through open valve f and exiting (with recycling if desired) through open valve e of all reaction vessels in layer z; iii) add BB7 and carry out reaction 3 entering through open valve b and exiting (with recycling if desired) through open valve d of all reaction vessels in vertical layer 1, add BB8 and carry out reaction 3 entering through open valve b and exiting (with recycling if desired) through open valve d of all reaction vessels in vertical layer 2, and add BB9 and carry out reaction 3 entering through open valve b and exiting (with recycling if desired) through open valve d of all reaction vessels in vertical layer 3. In this way every compound is synthesized in a specific location through valve opening and shutting, defining and re-defining the effective reaction vessels and effectively "sorting" the individual reaction vessels and supports contained therein (in the case of solid phase synthesis).

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated.

Examples

Directed Sort Teflon tube reaction vessel

A -28 UNF thread is cut on both ends of Teflon tube (Cole-Palmer, Vemon Hill, IL; OD 8 mm, ID 5.6 mm). The length of the tube is cut to allow 5.3 mm for each Lantern plus 2 times 5 mm for both threads. The tube is filled with Lanterns and enclosed from both sides using two female Luer fittings with -28 UNF thread (Figure la). Figure la depicts the use of a teflon tube as a reaction vessel which can be capped at both ends after object sets have been loaded in. The diameter of the tube constrains the object sets to remain in an ordered, linear array, preventing mixing. Reagents can be flowed into or through the tube, and in the case of lanterns, the hollow aligned cores of each permit insertion of a necklace for manipulating the object sets, or of a necklace containing or serving as a catalyst, etc.

Figure lb Depicts the basic concept of the lantern, depicting its hollow core, various types of reaction or delivery and receiving vessels including a simple tube, tubes contained in a microplate, or a syringe, and the ability to deliver object sets into, or remove object sets from such a tube using a necklace. The ability to add or remove reagents is depicted, as is the ability to use caped or minimally occluded tubes sufficient only to retain the object sets unless pressure is applied.

Reactions on SynPhase Lanterns Two Lantem sizes are available, L-Series Lantem (5 x 5 mm in size) with a loading of 15 mmol and D-Series Lantem (5 x 12.5 mm in size) with 35 mmol. L-Series Lanterns are used though the described concept is applicable to either series of Lanterns, since the diameter of both Lanterns is identical such that they can fit into the same tube and each has a hole in the center convenient for mnning a wire through and "stringing" the Lantems.

One L-Series Lantem weights 38 mg, giving a nominal substitution of ca 0.4 mmol/g. However, since Lantems do not swell, the solvent usage is substantially lower when compared to resin beads. 67 Lantems represent 1 mmol and fill a volume of 12 mL. Since Lantems do not swell under standard conditions, 5 mL of solvent is sufficient to wash all Lanterns. The same volume is used to perform each reaction. In most solid phase reactions an excess of reagent is used and 5 mL of reagent for Lantern synthesis at a 0.5 M concentration make 2.5 mmol of reagent (2.5 molar excess). Sufficient for synthesis to maintain the same concentration with resin, the volume of reagent would have to be doubled because one gram of resin (usually loaded at 1 mmol/g) typically requires 10 mL of solvent per solvent exchange.

Washing Lanterns

The tube reactor filled with Lantems is attached using 1/8 Teflon tube to two Teflon distribution valves used for operating the Domino Blocks. Four ports of the solvent selection valve are connected using a 1/8 Teflon tube to four reservoirs with solvents. The common port was connected to the tube reactor. The second end of the tube reactor is connected to the tube reactor selection valve. The common port of this valve is connected to the evacuated waste container. In order to wash Lanterns the appropriate solvent is chosen by the solvent selection valve. The flow through the tube reactor is adjusted by the tube reactor selection valve. The typical volume of washing solvent is 200 mL per tube reactor of 50 Lantems.

Support (Lantem) Directed Sort Apparatus

Two sorters are tested. The first uses a three chamber principle, the support delivery chamber (a

Teflon tube), the support isolation and transfer chamber, and the support receiving chamber (a Teflon tube). The delivery and receiving chambers are offset so that a support in the delivery chamber could not directly pass through into the receiving chamber. The support isolation and transfer chamber separates the delivery and receiving chambers in such a way that only one, or the desired number of supports, could pass from the delivery chamber into the isolation and transfer chamber, and then these supports are either transferred within this chamber or the isolation and transfer chamber itself is re-positioned to allow the supports to be transferred to the receiving chamber. In the first apparatus the isolation and transfer chamber is incorporated into a push-rod, as shown in Figure 3. The pushrod configuration is a simple method to shuttle one or a specified number of supports (regulated by the dimensions of the isolation and transfer chamber) from the delivery chamber(s) to the receiving chamber(s) in any desired series of steps. Reliable transfer without jamming or failure to transfer is important for directed sort to be used for library synthesis where the identification of each synthesized compound is based on knowing the position of each support throughout the synthesis process rather then based on the analytical interrogation of some tag. This shuttle mechanism provides this reliability. A plurality of reaction vessels and sorters can be used to simultaneously yet individually transfer object sets from one set of reaction vessels to another. The plurality of sorters can be arranged as a 2-D or 3-D conversion manifold. In a preferred embodiment one such arrangement utilizes a 2-D circular set of sorters as a conversion manifold and a circular and rotatable set of reaction or delivery vessels above a second circular set of reaction or receiving vessels. In the case reaction vessels are used, they can be inserted. In the case the delivery vessels are different from the reaction vessels, a method, such as the use of a necklace (rod with a gripper/stop, simple wire, string, or post) can be used to transfer the column of object sets from each reaction vessel to a respective delivery vessel.

A second apparatus is suitable for large library synthesis (Figure 4). This "Directed Sort Apparatus" is designed to simultaneously move Lanterns from twelve (or any desired number) dispensing tubes (chambers) into twelve (or same desired number of) receiving tubes (chambers). Twelve dispensing tubes are attached to the upper part of the circular dispensing stainless steel manifold. The receiving tubes are connected in a linear fashion to the bottom of the conversion manifold. The conversion manifold, which contains twelve (or the same desired number of) isolation and transfer chambers, is connected with the circular dispensing manifold by twelve tubes. The conversion manifold serves the function of converting the circular arrangement of dispensing tubes into a linear arrangement of receiving tubes. Inside the circular dispensing manifold, a spring-loaded moving circular part with twelve openings of a size of a Lantem moves a single Lantern from below each of the dispensing tubes to a position above each of the receiving conversion tubes. Once each Lantern reaches the top of the respective receiving conversion tube, it passes through the conversion manifold into the receiving tube. The circular array of isolation and transfer chambers is designed with one isolation and transfer chamber for each dispensing tube, and only rotates a half step, from below each respective delivery tube to above each respective receiving tube. Thus, each isolation and transfer chamber directs the sort of supports from one delivery tube to a specific receiving tube. The delivery tubes rotate above the conversion manifold, so that supports from each delivery tube can be delivered to all 12 receiving tubes according to any desired sort pattern. To assure reliable repositioning of Lantems, a stainless steel 2 g weight is placed on the top of Lantems in all dispensing tubes. Though not necessary, because the transfer path that lanterns follow from the isolation and transfer chamber to the receiving tubes is curved to translate the circular array of delivery tubes into a linear array of receiving tubes, the receiving tubes are connected to an evacuated reservoir via a solenoid valve to apply negative pressure or air flow to increase the reliability of lantern transfer through the curved tubes. The valve is opened for a fraction of second at the same time when the moving Lantern is positioned above the receiving tube. The negative pressure gradient /air flow assists in the movement of lanterns into the receiving tube.

Those skilled in the art can see that there are a variety of ways to apply positive pressure to the top of the dispensing stack besides a simple weight, and that positive pressure (rod or air) can be used to expel object sets from the sorter.

Figure 7a depicts the circular 2-D linear arrangement of delivery tubes, conversion manifold, and receiving tubes used for the simple sorting of object sets between synthesis steps. In this example the delivery tubes rotate in a circular manner such that the tubes change position, while the conversion manifold and receiving tubes do not. The Conversion manifold instead rotates back and forth a portion (e.g. half) of a step (where a step is the distance between two delivery or receiving tubes), and consists of the number of chambers as there are delivery tubes, each capable of receiving a single object set in the first position, and when rotated, delivering said object set to a receiving tube.

Figure 7b details the device shown in Figure 7a. The first step depicts the conversion manifold in its first position aligned with the delivery tubes. In each delivery tube there is a weight which assures that the object sets enter the conversion manifold. The circular array of delivery tubes and conversion manifold is mated to a linear array of receiving tubes (arranged in the x direction) by flexible tubing, the connecting transport tubes, and there is vacuum at the bottom of each receiving tube. There is also a hole above the second positions for the conversion manifold to which positive pressure can be applied (gas or physical device) to force object sets from the conversion manifold into the receiving tubes.

Operation: i) In the first position object sets for each delivery tube (from each building block reaction labeled 1, 2 and 3) enter the conversion manifold but cannot pass through since it is not aligned with the receiving tubes; the next operation is depicted between the left panel and the right panel ii) the conversion manifold is rotated a partial (~half) step counter-clockwise (looking down from the top) to its second position, now aligned over the respective connecting transport tube for each receiving tube, a vacuum is transiently applied to .the bottom of each receiving tube, and the object sets drop into their respective receiving tubes; iii) the conversion manifold is rotated back a partial step to its first position (not shown), an object set from each reaction delivery tube is delivered into the respective conversion manifold position, iv) the delivery tubes and the conversion manifold are rotated synchronously clockwise one step, repositioning both so that they are indexed one receiving tube away from their original position in i); and v) the process loops back to ii) and the process is repeated until all the object sets have been delivered to receiving tubes. At the end, the object sets can be pushed out of each of their respective tubes into a reaction tube (flipped to maintain the orientation of top object set to top object set), or retrieved on a necklace and either reacted on the necklace or transferred from the necklace into a reaction vessel. In the case of transfer from delivery tubes to a microplate or other sets of receiving tubes, one object per well or tube (as at the end of a synthesis when it is desired to release one compound per well), after each ii) the receiving plate (consisting of tubes in the x linear direction , y rows deep) can be indexed one tube over in the y direction, or another set of empty tubes can be placed under the connecting transport tubes.

Distribution of Supports into 96-well Plates

Instead of twelve receiving tubes, a 96-well plate is placed below the conversion manifold for final distribution of Lanterns into wells after finishing the synthesis. This distribution from columns of object sets in tubes to single object sets in each well of a microplate would be frequently common for any number of reaction vessel configurations, whether circular or in another 2-D arrangement, and would be facilitated by a different configuration for the plurality of sorters (conversion manifold) used .

Chemistry

Acylation of Lanterns with a linker

A 50-rnL syringe is loaded with 50 Lanterns, Lanterns are neutralized with 50% piperidine in DMF, and washed 5 times with DMF. Fmoc-4-methoxy-4 -(ga ma- carboxyprophyloxy)benzhydrylamine (5mmol, 2.69 g) and HOBt.H₂0 (5mmol, 0.765 g) are dissolved in 15 mL NMP, and DIG (5 mmol, 0.782 mL) is added. The solution is added to the syringe with Lanterns and kept on a tumbler overnight (16 h).

Lanterns are washed 5 times with DMF, THF, and DCM, and dried by a stream of nitrogen. To quantify the amount of linker available on each Lantern (linker substitution), a half mL of 50 % piperidine/DMF solution is added to one Lantern in a 2.5 mL syringe and kept on a tumbler for 10 min. The Lantem was washed 5 times with DMF and all washes are collected, diluted, and the absorbance is measured at 302 nm against DMF. Fmoc release indicates a linker substitution of 37 umol/Lantern.

Nitroaniline: Step 1

A 20-mL syringe is was loaded with ten Lanterns (acylated with linker as described above) and 50 % piperidine/DMF solution is added. After 10 min the Lantems are washed 5 times with DMF, 3 times with dry DMSO, 5 mL of 1 M solution of o- fluoronitrobenzene and DIE A (0.17 mL) in DMSO is added to the syringe. The syringe is left shaking in an incubator at 75 C overnight (16 h). Lanterns are washed 5 x with DMSO, DMF, DCM, and dried by nitrogen. One ring from a Lantem is cut, the product cleaved by TFA for 1 h, and analyzed by analytical gradient HPLC at 280 nm.

o-Phenylenediamine: Step 2

A 20-mL syringe with ten Lanterns from step 1 is charged with 5 mL of 2 M solution of tin(II) chloride dihydrate in NMP, bubbled with argon for 15 min. The syringe is left on a tumbler overnight, washed 3 times NMP, DMF, DMF/water, DMF, THF, DCM, dried by nitrogen. One ring from a Lantern is cut, the product is cleaved by TFA for 1 h, and analyzed by analytical gradient HPLC at 220 nm.

Thiourea: Step 3

A 20-mL syringe with ten Lanterns from step 2 is charged with 5 mL of 1 M isothiocyanate solution in NMP. The syringe is left on a tumbler overnight, washed 3 times DMF, THF, and DCM, dried by nitrogen. One ring from a Lantern is cut, the product cleaved by TFA for 1 h, and analyzed by analytical gradient HPLC at 280 nm.

Benzimidazole: Step 4

A 20-mL syringe with ten Lanterns from step 3 is charged with 5 mL of 1 M DIC solution in DMF. The syringe is left on a tumbler overnight, washed 3 times DMF, THF, and DCM, dried by nitrogen. One ring from a Lantern is cut, the product cleaved by TFA for 1 h, and analyzed by analytical gradient HPLC at 280 nm.

Urea: Step 5

A 20-mL syringe with ten Lanterns from step 4 is charged with 5 mL of 1 M isocyanate solution in DMF. The syringe is left on a tumbler overnight, washed 3 times DMF, THF, and DCM, dried by nitrogen. One ring from a Lantem is cut, the product was cleaved by TFA for 1 h, and analyzed by analytical gradient HPLC at 280 nm.

Virtual Tube Sets

Multiple short tubes can be used in place of one long tube as depicted in figure 13, each set of short tubes forms a "virtual tube" when aligned head-to-tail as shown to the left of the vertical line. To the right of the vertical line is a comparison of the first two steps of the synthesis of a 3 x 3 x 3 library using short tubes (on the left) containing three object sets each and comprising "virtual tube" sets, versus on the right a long tube containing nine object sets each. The building blocks are indicated as BB1, BB2, BB3 for the first reaction and BB4, BB5, BB6 for the second reaction. The object sets are numbered so that their location within tubes and between sorts can be followed. Focusing first on the Virtual Short Tube synthesis, three tubes each are reacted with each building block. Each 3 tube set when ordered head-to-tail represents a "Virtual Tube" containing 9 object sets, as can be seen by comparing the numbers for BBl short tubes to the long tube corresponding to BBl to the right (see dotted arrow). Three 3-tube sorts are then carried out, in preparation for which one tube of each reaction set BB reaction of tubes is combined (as shown) with one of each of the other reaction set tubes to form the 3-tube delivery tube set. The "virtual tube" head-to-tail layout dictates how these tubes are recombined before the sorts. Three individual sorts are carried out with these different sets of tubes as shown, and the resulting receiving tubes re-sorted into head-to-tail "virtual tubes" for reaction with the next three building blocks (BB4, BB5, BB6). On the right side is shown the use of three long tubes for the same synthesis steps, with a single 3-tube sort. Comparing the head-to-tail "virtual tubes" in each reaction BB4, BB5 and BB6 to the long tubes in BB4, BB5, and BB6 reactions (dotted arrow) demonstrates an identical composition and order, verifying the use of the "virtual tube" concept with the sorter as a means to keep the tube lengths to a convenient length (e.g., breaking a 200 object set tube up into four 50 object set tubes).

The entire disclosure[s] of all applications, patents and publications, cited herein and of corresponding U.S. Provisional Application Serial No. 60/ 307,186, Titled: Method and Device for Directed Sorting in Combinational Synthesis, filed 24 July 2002, are incorporated by reference herein.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

What is claimed is:

1. A method of sorting object sets into groups for combinatorial compound synthesis by sequential addition of molecular building blocks comprising:

a) redistributing object sets from a first group of object sets, in which the individual object sets are in a certain first order, to a second group of object sets wherein the object sets are redistributed into a different second order such that after redistribution there are no adjacent object sets from the first ordered group of object sets which remain adjacent in the second ordered group of object sets, b) adding chemical building blocks to each group of object sets after said redistribution, and c) repeating steps a) and b) for each desired building block addition.

2. A method of Claim 1 wherein the object sets are contained within at least one holder to maintain their order during handling.

3. A method of Claim 2 wherein the number of holders equals the number of building block reactions for that step.

4. A method of Claim 2 wherein the holders are clustered in a linear set, one set for each building block reaction, so that the linear set forms a virtual holder.

5. A method of Claim 2, wherein the object sets are inserted from a necklace.

6. A method of Claim 2, wherein the object sets are removed using a necklace.

7 . A method of Claim 1, wherein the object sets are contained on a necklace to maintain their order during handling.

8. A method of Claim 2 /wherein the holder is open at both ends.

9. A method of Claim 8 ,wherein the holder can be constrained to retain the object sets.

10. A method of Claim 8, wherein the object sets can be inserted through an end of a holder.

11. A method of Claim8, wherein the object sets can be removed from an end of a holder.

12. A method of Claim 2, wherein the holder is open at one end.

13. A method of Claim 1, wherein the identical order of redistribution is repeated for each step of synthesis.

14. A method of Claim 1, wherein the identical order of redistribution is repeated in all but one step of the synthesis.

15. A method of Claim 14, wherein the order of redistribution for the step before the last synthetic reaction is different.

16. A method of Claim 14, wherein the order of distribution before the second reaction step is different.

17. A method of claim 1, wherein the number of object sets at every step is equal to the total number of compounds to be synthesized.

18. A method of claim 1 , wherein each object set is individually locatable and the history of building block additions are identifiable at any time.

19. The method of claim 1, wherein each object set contains more than a single member.

20. The method of claim 4, wherein all members of an obj ect set at a given point in time are reacted with the same chemical building blocks throughout synthesis.

21. The method of claim 1, wherein an object set comprises one or more reaction vessels or solid phase supports.

22. The method of claim 1 , wherein the object sets are themselves physically moved between groups.

23. The method of claim 1, wherein the object sets are redistributed without actually themselves being moved to each object set within any group reflects its prior and planned chemical building block additions and the combination of said reagent accessibility to each object set is specified and described by the location of the object set at any time, and wherein the distribution of object sets follow the equation

Nx=Ntot/BBx object sets where

N tot= total number of compounds to be synthesized

Nx=number of object sets in a reaction vessel in the x-th combinatorial step

BBx =number of building blocks in the X-th reaction steps.

24. The method of claim 23, wherein the order of redistribution of the objects sets is effected by changing the access of each building block to each object set, in such a manner that said access to each object set within any group reflects its prior and planned chemical building block additions and the combination of said reagent accessibility to each object set is specified and described by the location of the object set at any time.

25. A directed sort apparatus for performing combinatorial solid phase synthesis of compounds comprising:

a) one or more first reaction vessels for holding one or more object sets organized in a first ordered pattern, b) one or more isolation and transfer chambers for directed redistribution of object sets from each of said reaction vessels to c) one or more subsequent reaction vessels for receiving object sets from the isolation and transfer chamber;

wherein the object set redistribution pattern in each subsequent transfer is identical and the position of each object set in a reaction vessel codes for its previous synthesis history, and the object sets are transferred using the repetitive process of one object from each reaction vessel into subsequent reaction vessels until each subsequent reaction vessel contains Nx object sets where , Nx=Ntot/BBx object sets where

N tot= total number of compounds to be synthesized

Nx=number of object sets in a reaction vessel in the x-th combinatorial step

BBx =number of building blocks in the X-th reaction steps.

26. The directed sort apparatus of claim 25 wherein; an object set may be an individual object or multiples thereof and each set remains adjacent throughout all redistributions and building block additions.

27. The directed sort apparatus of claim 25 wherein; all members of an object set are reacted with the same chemical building blocks throughout synthesis.

28. The directed sort apparatus of claim 25 wherein an object set is one or more reaction vessel or solid phase support.

29. A directed sort apparatus of claim 25, for performing combinatorial solid phase synthesis of compounds comprising: a) performing a reaction in each of one or more first reaction vessels for holding one or more object sets organized in a one dimensional linear array, and delivering to, b) an isolation and transfer chamber for directed redistribution of object sets from each of said reaction vessels to c) one or more subsequent reaction vessels for receiving object sets from the isolation and transfer chamber; d) repeating steps a-c for each building blocks added, such that after each redistribution there is no adjacent object set from the first group of object sets which remains adjacent in the second group of object sets.

30. The directed sort apparatus of claim 29 ,wherein an object set may be an individual object or multiples thereof; and each set remains adjacent throughout all redistributions and building block additions.

31. The directed sort apparatus of claim 29, wherein all members of an object set are reacted with the same chemical building blocks throughout synthesis.

32. The directed sort apparatus of claim 29, wherein an object set is one or more reaction vessel or solid phase support.

33. A method of Claim 2 wherein each object set is individually sorted from a delivery holder into a receiving holder by an isolation and transfer chamber.

34. A method of Claim 33 wherein the delivery holder and receiving holder are offset such that object sets cannot pass between them without a distribution device.

35. A directed sort apparatus for performing combinatorial solid phase synthesis of compounds comprising: a) one or more first reaction vessels for holding one or more object sets organized in a first ordered pattern, wherein each object set is contained within a holder b) one or more isolation and transfer chambers for directed redistribution of object sets from each of said reaction vessels to c) one or more subsequent reaction vessels for receiving object sets from the isolation and transfer chamber; d) repeating steps a-c for each building blocks added, wherein the object set redistribution pattern in each subsequent transfer is identical and the position of each object set in a reaction vessel codes for its previous synthesis history, and the object sets are transferred using the repetitive process of one object from each reaction vessel into subsequent reaction vessels until each subsequent reaction vessel contains Nx object sets where , Nx=Ntot/BBx object sets where

N tot= total number of compounds to be synthesized

Nx=number of object sets in a reaction vessel in the x-th combinatorial step

BBx =number of building blocks in the X-th reaction steps.

36. A apparatus of Claim 35 wherein the delivery holder and receiving holder are offset such that object sets cannot pass between them without a distribution device.

37. An apparatus of Claim 35wherein the isolation and transfer chamber accommodates a single object set from the delivery holder at a time, and moves from the delivery holder position to the receiving holder position and delivers that object set into the receiving holder.

38. A method of Claim 33 wherein a plurality of delivery holders and receiving holders are used.

39. A method of Claim 33 wherein a plurality of isolation and transfer chambers are used to transfer object sets from a plurality of delivery vessels to a plurality of receiving vessels.

40. A method of Claim 1 wherein each delivery vessel contain more than one object set.

41. A method of Claim 1 wherein each receiving vessel contains more than one object set.

42. A method of Claim 1 wherein each receiving vessel contains a single object set.

43. A method of Claim 2, wherein each object set is individually sorted from a necklace into a receiving holder.

44. A method of Claim 1 wherein the object redistribution pattern in at least one (also two and every) transfers is identical and the position of each object set in a reaction vessel codes for its previous synthesis history, and the object sets are transferred one at a time from each reaction vessel into the next reaction vessels until each next reaction vessel contains Nx object sets where, Nx=Ntot/BBx object sets where

Ntot = total number of compounds to be synthesized

Nx = number of object sets in a reaction vessel in the xth combinatorial step BBx = number of building blocks in the xth reaction step.

45. A method of Claim 44 wherein after each redistribution there is no adjacent object set from the first group of object sets in the delivery vessel which remains adjacent to the second group of object sets in the receiving vessel.

46. A method of Claim 1, where a plurality of reaction vessels is in the form of a microplate.

47. A method of Claim 1, where a reaction vessel is a syringe barrel, Teflon tube, closed ended tube, or open ended tube.

48. A method of Claim 1, wherein the object sets are ordered in such a manner that a hole in each object set is aligned to form a hollow passage through all the object sets within the holder.

49. A method of Claim 2, wherein solutions flow through and around all the object sets within the holder.

50. A method of Claim 1, wherein the transferred object set is from one end of the stack of object sets.

51. A method of Claim 1, wherein object sets move through a dispensing tube and into a transfer device.

52.A method of Claim 39, wherein positive pressure or weight on top of the column of object sets is used to assure positive movement.

53. A method of Claim 42, were a plurality of positive pressure devices are joined together with a plurality of dispensing tubes.

54. A method of Claim 41, where negative pressure is used to move object sets into the translocation device

55.A method claim 1, where object sets move into the receiving tubes from the transfer device

56. A method of Claim 44, where positive pressure is used to move object sets from the translocation device into the receiving tube.

57. A method of claim 44, where negative pressure applied to the receiving tube is used to move the object set from the translocation device into the receiving tube.