JPH09508353A - Synthesis and screening of molecular diversity - Google Patents

Abstract

(57) Summary An apparatus and method for efficiently synthesizing diverse molecular products on a substrate. The parent container (200) contains a suspension of the substrate. The suspension is pressured with argon and sent to multiple reaction vessels (201-209) in one or more reaction vessel banks where the monomer addition reaction occurs. Optionally, the substrate can be tagged with tag monomers. A vortex motor (300) agitates the contents of the reaction vessel during the monomer addition reaction to enhance synthesis. After the addition reaction of the desired monomers and / or tag monomers, the suspension is pressured with argon and returned to the parent vessel (200) for mixing. The suspension can then be pressured with argon and redistributed into the reaction vessels (201-209) for further synthesis.

Description

Detailed Description of the Invention           Synthesis and screening of molecular diversity Related application   This application is related to U.S. Patent Application Serial Nos. 08/146886 and 08/149675. It is a part-pending application of the specification (both of which were filed on November 2, 1993). , Which are incorporated by reference for all purposes. Copyright notice   A part of the disclosure of this specification and claims is protected by copyright. including. The copyright holder is the owner of the patent documents or files appearing in the Patent and Trademark Office files and records. Makes no objection to reproduction by any of the patent disclosures, but otherwise All rights reserved. Field of the invention   The present invention is generally used to synthesize very large collections of various molecules. , And identify compounds with useful and desired activities from such collections And a method and device for isolation. The present invention also has the desired properties Introduce identification tags into such collections to facilitate compound identification. About things. BACKGROUND OF THE INVENTION   Ligands for macromolecular receptors are not available in the techniques of molecular biology or synthetic chemistry. Screening a diverse collection of peptides produced through the galleries Can be identified by Recombinant peptide library Reotide, a filamentous bacteriophage capsid protein and DNA Binding protein Lac I. Manufactured by inserting into the gene encoding (Cwirla et al., 1990,Proc. Natl. Acad. Sci. USA 87: 6378-6382 ； with Scott Smith, 1990,Science 249: 386-390; Devlin et al., 1990,Science 249: 404-406; C ull et al., 1992,Proc. Natl. Acad. Sci. USA 89: 1865-1869; and PCT public number  WO 91/17271, WO 91/19818, WO 93/08278, each of which is to be cited. Form part of this specification. These random libraries have 10⁹More Can contain various peptides, each of which is physically linked to the genetic material that encodes it. Fused to a sequence of large proteins that are linked together. A library like this Is more efficient at interacting with the receptor through several rounds of affinity purification. Screened and selected exposition or disposition Prey vector amplified in E. coli, and peptides involved in receptor binding The DNA of individual clones is sequenced to reveal the identity of the response. See also PCT Publication Nos. WO 91/05058 and WO 92/02536.   Chemical approaches for producing peptides or other molecular libraries have been It is not limited to synthesis using 20 genetically encoded amino acids. Natural Building block set to contain amino acids and other molecular building blocks not found in Expanding the array dramatically increases the diversity of available sequences and structures. . In some of the strategies described for creating synthetic molecular libraries, The reaction products are spatially separated, and the identity of individual library members is , Explicitly defined by the nature of the synthesis. Geysen et al., 1984,Proc. Natl. Acad. Sci.  USA 81: 3998-4002; Geysen et al., 1986,Synthetic Peptides as Antigens; C iba Foundation Symposium 119, Porter, R. And Wheelan, J. Hen (Wiley, New Y ork) pp.131-146; Fodor et al., 1991,Science 251: 767-773; US Patent No. 5143854 Description; and PCT publication WO 84/03564; 86/00991; 86/06487; 90/15070 and 92 / See 10092 publication (each of which is incorporated by reference) .   A library of more than 30 million soluble peptides is available for multi-peptide synthesis. It is prepared by the "tea bag" method. Houghten, 1985,Proc. Natl. Acad. Sci.  USA 82: 5131-5135; and U.S. Pat.No. 4,631,211 (these are each incorporated by reference). Thereby forming a part of this specification). Each library was synthesized, and A mixture of degenerate peptides in which the individual amino acids in the sequence are clearly defined To be screened. Screening (eg competition Iterative binding assays) and resynthesis will fractionate these mixtures and Used to define the most active peptide within the ibary. Houghten et al. 1991,Nature 354: 84-86; Pinilla et al., 1992,Peptide Research 5: 351-358; Bl ake, J. And Litzi-Davis, 1992,Bioconjugate Chem. 3: 510-513; and PCT See WO 92/09300 publication (each of which is incorporated herein by reference) Is).   Furka et al., 1988,Abstr. 14th Int. Congr. Biochem., Prague, Czech.Five: 47 ( Furka et al., 1991,Int J. Peptide Protein Res. 37: 487-493; and Sebestye n et al., 1993,Bioorg. Med. Chem. Lett. Three: 413-418) Spirit synthesis method Ram and co-workers attached to resin beads with a diameter of 100-200 μm About 10⁶A library containing the above peptides was prepared. Lam et al., 1991,Nature 354 : 82-84; Lam et al., 1993,Bioorg. Med. Chem. Lett. Three: 419-424; and PCT See WO92 / 00091 publication (these are part of the specification by reference) Form). The bead library should be incubated with labeled receptors Beads bound to the receptor screened by Selected and selected using a micromanipulator. Each bead is 50 ~ It contains 200 pmol of a single peptide sequence which is Edman degraded. Alternatively, it can be determined directly by either mass spectrometric analysis. In principle , By reducing the bead volume, a large diversity of labels can be obtained using this approach. Ibrary can be made. However, the sensitivity of peptide sequencing techniques is about Limited to 1 pmol, which is evident in the range of direct peptide sequencing analysis. It sets a limit. In addition, the library construction set , D- or other non-natural amino acid, or other chemical building block When expanded, direct and unambiguous sequence analysis cannot be provided.   Chemically synthesized molecules and natural products such as fermented broth of microorganisms High-throughput screening of collections has traditionally It has played a central role in the search for compounds that lead the development. Molecular diversity The combination of chemistry and associated technology to create and evaluate sex A huge wave of interest in drug discovery is a meaningful test in the development of this paradigm of drug discovery. It is a gold stone. Pavia et al., 1993,Bioorg. Med. Chem. Lett. ThreeSee: 387-396 (this Are incorporated herein by reference). To date, peptide chemistry has A fundamental tool for developing the use of combined methods in Gand identification Met. Jung & Beck-Sickinger, 1992,Angew. Chem. Int. Ed. Engl. 31: 36 See 7-383, which is incorporated herein by reference. This is High yield solid phase coupling chemistry and recombination of amino acid monomers, related genera Create a peptide library A large and structurally diverse range of collaborative work involving biological approaches to It may be attributed to the usefulness of the fence. In addition, the potency of many low molecular weight peptides and Specific biological activity makes these molecules attractive as starting points for drug discovery Make things. Hirschmann, 1991,Angew. Chem. Int. Ed. Engl. 30: 1278-1301 and And Wiley and Rich, 1993,Med. Res. Rev. 13: 327-384 (each quoted Which forms a part of this specification). However, unfavorable pharmacodynamic characteristics Sex, eg, poor oral bioavailability andIn vivoClearance in Limits the broader development of peptidic compounds as drugs. This understanding Recently introduced the concept of combinatorial organic synthesis beyond peptide chemistry to benzodiazetes. Known pharmacophores such as Pin (Bunin & Ellman, 1992,J. Amer. Chem.  Soc. 11410997-10998, which is incorporated by reference herein. ) As well as polymer molecules such as oligomeric N-substituted glycines ("peptides"). Id) ”and the development of oligocarbamate libraries Urged to. Simon et al., 1992,Proc. Natl. Acad. Sci. USA 89: 9367-9371; Zucker mann et al., 1992,J. Amer. Chem. Soc. 11410646-10647; and Cho et al., 1993,Scie nce  261: 1303-1305 (each of which is incorporated by reference. ).   For identifying useful compounds or improving the properties of lead compounds Has a huge library of molecules Despite its great value, such libraries, especially large ones Screening difficulties, for example, may reduce drug discovery and development costs. It restricts shocking access to large libraries. As a result, live Each member of the rally has a unique identification tag to facilitate compound identification (See PCT Publication WO 93/06121, which is hereby incorporated by reference. And US Patent Serial No. 946239 (filed Sep. 16, 1992) and And No. 762522 (filed September 18, 1991), which are incorporated by reference. Forms and screens a library of molecules). The development of a method for this is highly desired. In that method, a chemical synthesis method, The product of the conjugation synthesis, typically on resin beads, is responsible for each coupling in the synthesis. Or by linking the identification tag to the beads at the same time as other product manufacturing reaction steps. Is specified in. Each tag describes what happened during the desired reaction step, such as which Was the mino acid monomer coupled at a particular step in the peptide synthesis reaction? To identify. The structure or identity of the compound on any bead, such as the peptide Rows can also be inferred by reading the set of tags on the beads. Ideally, Such tags have a large amount of information and are very sensitive to detection and decoding. And is stable to the reagents used in the synthesis. The idea of oligonucleotide-encoded chemical synthesis is also described by Brenner and Lerne. r, 1992,Proc. Natl. Acad. Sci. USA 89: 5181-5183 (This is quoted Which form part of this specification).   The encoding method begins with orthogonally differentiated diamine linkers and Soluble chimeric peptides in which the combinatorial synthesis of rows comprises a "binding" chain and a "coding" chain Can be used to create a library of Kerr et al., 1993,J. Amer. Chem.  Soc. 115: 2529-2531, which is incorporated herein by reference. . Coupling of natural or non-natural amino acid monomers to the tether is described in “Coating”. By constructing an amino acid code consisting of four L-amino acids on the "" chain. Will be recorded. Compounds were prepared in equimolar amounts of peptides by affinity purification on the receptor. Selected from the tide mixture and analyzed by HPLC. Then each purified The sequence of the coding strand of the molecule was determined by Edman degradation to reveal the structure of the binding strand. It will be ridiculous. A similar peptidic code summary is also given by Nikolaiev et al., 1993,Pepti deResearch  6: 161-170 Recently reported.   Edman's sensitivity and throughput constraints limit the library of limited diversity. This would severely limit the scope of this aspect of the encoding method for analysis. Orient Although the use of gonucleotide nucleotides offers greater promise, it Improved methods for synthesizing tide-tagged molecular libraries are needed. Is required. In addition, the synthesis of very large tagged molecular libraries as well as There is still a need for other methodologies for screening.   It is possible to synthesize a diverse collection of molecules on multiple solid supports, such as beads. If this is desired, further issues can be raised. Various molecular manufacturing Examples of the use of beads to be synthesized are disclosed, for example, in the following applications: , Which are incorporated by reference for all purposes. US patent application serial number 07/876792 (filed April 29, 1992), US patent Serial number 07/762522 (filed Sep. 18, 1991) and US patent Application Serial No. 07/946239 (filed Sep. 16, 1992).   While achieving substantial success, the above techniques also suffer from certain limitations. . For example, if the synthesis of various products was performed on beads, such beads Many manual operations are required. For example, US patent application serial number 07/876792 Specification (filed April 29, 1992) (this is for all purposes only) In more detail), a collection of beads is suspended in a carrier and Of the beads, and the monomer addition reaction is performed on the separated set of beads. Beads thus synthesized are sometimes subdivided and selectively recombined , Mix the recombined beads, and repeat this procedure. A large number of If the monomer is included and the reaction involves many monomer addition steps, manual manipulation is extreme. Be monotonous and boring at the end You. In addition, "aggregation" of many synthesized products is a daunting task.   From the above, we synthesized and screened a very large tagged molecular library. It can be seen that an improved method and apparatus for training is desired. The present invention Meets these and other needs. Summary of the Invention   The present invention provides an easily readable identification tag unique to each molecule in the library. To create and screen a molecular library, marked with Provide an improved method for It also combines a wide variety of molecular products quickly and efficiently. An apparatus and method are provided.   In one embodiment, the invention provides an encoded synthetic chemical library. For tagging the products of a combinatorial chemical process for producing And a reagent. In an important embodiment, the present invention is modifiable and compatible Peptides and oligonucleotides on fine beads through a synthetic method Provide a method for performing the production. The large size produced by this combinatorial synthesis Oligonucleotide-encoded synthetic peptide libraries are available for many Each of the beads having a single peptide (having a defined sequence). , And its sequence artificially and uniquely encodes the structure of related peptides. It contains many copies of the single-stranded DNA tag that it contains. live The rally was identified by flow cytometry as a fluorescently labeled biological receptor. Interactions can be efficiently investigated, and individual beads are Selected by utilizing the ability of the FACS machine to sort beads. Can be The DNA tags on the sorted beads were amplified by PCR. And sequenced to determine the structure of the encoded and encoded peptide ligand Is done. Libraries include, for example, receptors such as anti-peptide monochrome To find high affinity (nanomol level) ligands for null antibodies Can be used.   The synthetic molecular library of the present invention combines compounds on each of a plurality of solid supports. Can be made by forming (the compound is different on different solid supports) it can. The compound is synthesized in a process that includes the following steps. (A) Support Distributing by stochastic method among multiple reaction vessels, (b) first chemical construction Exposing the support in each reaction vessel to a block, (c) pooling the support , (D) partitioning the support among a plurality of reaction vessels by a stochastic method, (e) Exposing the support in each reaction vessel to a chemical building block, and (f) step (a )-(E) are repeated at least 1-20 times. Typically, a substantially equal number of solid supports A carrier is distributed in each reaction vessel. In one embodiment of this method, the chemistry The building block was chosen from a set of amino acids and the resulting compound was a peptide. It is a do-oligomer.   In particular, the invention resides in the coupling chemistry associated with such methods. Regarding seed improvement. Such improvements are due to beads, phosphorus, etc. in such synthesis. Used to remove the Fmoc protecting group from the α-amino group of the car or the growing peptide chain Regarding chemistry that can be done. Preferably, such removal is 5-15%, preferably Is a treatment with 10% piperidine for 5 to 60 minutes, preferably 5 to 10 minutes Other conditions, such as 5 with 15-30% piperidine. ~ 30 minutes can also be used. Another improvement is activation of the peptide coupling reaction. Regarding science, there is an automated device tagged with an oligonucleotide. When used to perform the synthesis of a truncated peptide library, the present invention provides Provide a simple mixture of HOBt / HBTU to reduce reagent supply bottles You.   In another aspect, the invention synthesizes a tagged molecular library. In the method, each molecule in the library is covalently attached to a solid support. , And it is tagged with one or more different chemically inert hydrocarbon tags Where the tag comprises various hydrocarbon regions and molecular hooks. , A method for synthesizing a molecular library. Preferably such tags are Cleavable linker attaching a tag to the solid support, a molecular hook, and the cleavage It includes hydrocarbon chains of various lengths connecting the molecular hooks to possible linkers. The tag has the formula: (Where n is 1 to 10 or more, X is a cleavable linker, and And R is a molecular hook) Is more preferred. Such molecular hooks are preferably biotin. Groups, complements of highly associated peptide pairs, and protected activatable groups such as It is selected from the group consisting of photoactivatable groups. The preferred cleavable linker is light It is a linker cleavable by.   Moreover, a method for detecting the presence of such a chemically inactive hydrocarbon tag is available. Provided. The method comprises cleaving one or more different tags from a solid support, And subsequently immobilizing the tag on a second solid support. Then immobilized The processed tag is treated with an oligonucleotide sequence, thereby The reotide sequence selectively binds to the immobilized tag. After that, oligonucl The otide sequence is amplified and its presence detected, where the oligonucleotide The presence or absence of the tag sequence serves as an indicator of the presence or absence of the tag.   Another embodiment of the invention is tagged with a chemically inert hydrocarbon tag. Synthesized Molecules Attached to Solid Support in Selected Tagged Molecular Library A method for determining the order of synthetic steps for is provided. The method is based on the solid support Individually detecting the presence of one or more various tags of Or the absence thereof is an indication of the appearance of a particular synthetic step in the synthesis of the molecule.   In another aspect, the present invention provides a conventional flow cytometh. Encoded synthesis on beads that are too small to be separated by the Li system. Methods and apparatus for synthesizing academic libraries. Such a small bee The size of the resulting library was determined for beads-based libraries. Is a typical range of 10⁹~Ten¹³From a library without beads 10¹⁸It is possible to increase to the size of member. The present invention also The present invention relates to a screening method for an una library.   The present invention also provides encoded synthetic live sequences to identify useful compounds. A method of screening a rally. In one important aspect, the present invention Are used to characterize and identify compounds with useful activity. To create, tag, and screen product libraries It provides an important advance in the field of natural product screening in relation.   In another aspect, the invention provides a pool of compounds from the molecular libraries of the invention. To an improved method for the rapid and efficient identification of Smell this way A compound tagged with a desired property (eg, binding to a receptor) Oligonucleotide tags from a pool of concatemerized and single Closing to facilitate sequencing of many tags in a sequencing reaction Be converted. If the tagged compound is a peptide and the genomic co- If a code-based encoding mechanism was used, then the peptide Of analysis For this purpose, the individual tags from the concatamers were added to other selection and expression systems such as Subcloning into the plasmid and phage-based system described in the Background section. Can be converted into   In another embodiment, the present invention provides for the rapid and efficient synthesis of diverse molecular products. An apparatus and method for doing so are provided. In accordance with certain aspects of the invention, a wide variety of poly The mer is synthesized on a substrate such as glass beads. Optionally, the beads are synthetic During the reaction, molecular tags can be tagged at the same time. Just as an example, on beads Molecules synthesized in can contain peptides, while the molecular tags are oligonucleotides. It may include octides. Of course, a molecule is a basic "structure" common to other related molecules. As long as it has a "building block", other molecular products also can be produced by the techniques described herein. Can be synthesized using Examples are benzodiazapine, prostaglandins and beta Includes Turn Minetics.   According to one embodiment of the invention, a parent container is provided for mixing the bead suspension. Used for Mixed beads can be separated into multiple Are distributed to the reaction vessels. In the reaction vessel, the beads are various and selected. Exposed to the monomer and the monomer is Or react covalently. Optionally, the beads are exposed to a chemical “tag” and tagged. The beads are also covalently or otherwise linked to the beads. Then bee The chips pass through the manifold back to the parent container where they are recombined and mixed. Mixed The bead suspension was then redistributed between multiple reaction vessels and loaded with monomer. The process of addition, bead mixing and redistribution is continued. The process is on its surface Formation of a collection of beads or other substrates with a diverse set of formed molecules Results in   According to one aspect of the invention, the present invention is for synthesizing diverse molecules on a substrate. Apparatus and method. Substrate is distributed from the parent vessel to the selected reaction vessel You. Then, the reagent is introduced into the reaction vessel and a part of the molecule is synthesized. Then The substrate is transferred to the parent container for mixing. The substrate is then further synthesized. Are redistributed into the reaction vessel for. Cycle is the synthesis of the desired set of molecules Continue until During synthesis, the entire synthesizer is sealed from the outside atmosphere. You.   In general, the present invention provides that each molecule in a library is uniquely Create and screen molecular libraries that are tagged with constant tags An apparatus and an improved method therefor are provided.   A further understanding of the features and advantages of the present invention will be made with reference to the following description and drawings. . Description of the drawings FIG. 1 shows a schematic diagram of the synthesizer of the present invention. FIG. 2 shows a schematic diagram of the reagent reservoir. Figure 3 shows a three-way valve used in a synthesizer. You. FIG. 4 shows a two-way valve used in a synthesizer. FIG. 6A shows a rotatable disc controller holding multiple groups of reagent reservoirs. Figure 7 illustrates another arrangement of reaction vessel storage locations with bayers. Figure 7 shows lower manifold valve, injection valve, reagent reservoir And the interconnection between the reaction vessels. FIG. 8 shows a non-concentric stirrer. FIG. 9 shows the upper portion of the reaction container storage location. 10A and 10B show the lower reaction vessel bracket. 11A-11D show a substantially translucent tube according to one aspect of the present invention. Optical array block for using an optical sensor to detect the presence of Is shown. FIG. 12 shows a reaction vessel according to one aspect of the invention. FIG. 12A shows a temperature control jacket around the reaction vessel of FIG. FIG. 12B shows a cross-sectional view of the jacket and container of FIG. 12A. FIG. 12C shows another embodiment of the reaction vessel having a temperature control jacket. . Figure 12D shows a cross-sectional view of the jacket and container of Figure 12C. ing. FIG. 13 shows a parent container. FIG. 14 shows a simplified diagram of the electronic hardware for controlling the synthesizer. doing. FIG. 15 shows a simplified diagram of the controller circuit. FIG. 16 shows a control computer for discharging all liquid from the reaction vessel. The process performed is shown. Figure 17 shows the control computer to remove material from the bottom manifold. The steps performed are shown. FIG. 18 shows the control computer for stirring the contents in the parent container. The process shown is shown. 19A-19D show a method for redistributing bead suspension from a parent vessel to a reaction vessel. 3 illustrates the steps performed by the control computer. FIG. 20 shows control of placing reagents from a pressurized delivery system into a reaction vessel. It shows the steps performed by the computer. 21A-21D illustrate placing a reagent from a pressurized delivery system into a reaction vessel. A schematic diagram illustrates the steps performed by the control computer for this purpose. FIG. 22 shows a control computer for introducing the amino acid monomer into the reaction vessel. Shows the steps performed by. 23A-23C show control sequences for introducing amino acid monomer into the reaction vessel. The process done by a pewter This is illustrated in the schematic diagram. Figure 24 shows a control for transferring the bead suspension from the reaction vessel to the parent vessel for mixing. It shows the steps performed by your computer. Figure 25 shows a schematic diagram of the data flow between the main modules of the control software. Clear. FIG. 26 schematically illustrates the command interpreter structure. FIG. 27 schematically illustrates the mechanism used to access valve data. ing. FIG. 28 schematically illustrates the mechanism used to access sensor data. doing. FIG. 29 shows an apparatus for synthesizing combinatorial chemical libraries on fine beads. Is shown. The device is a synthetic having a sequence of reactive sites where compounds can be synthesized. From a vacuum manifold or porcelain plate attached to a solid substrate with a surface Become. Partition blocks are reaction wells lined up in a row corresponding to reaction sites. And used to separate library members after each mixing step. It is. The instrument is also used to aid in the synthesis of tagged chemical libraries. Can be put. FIG. 30 shows a method for monitoring the stability of thiazolidinone.¹³Use of C NMR Explaining. Panel C shows the carbo of the ring position 2 and the linker, attached to a support. At the α-position to the nyl group¹³Thiazolidine double labeled with C atom Non¹³C NMR spectrum is shown (labeled position is indicated by “*”) ). Panel B shows support bound duplex after treatment with 95% TFA for 1 hour. Labeled thiazolidinone¹³3 shows a C NMR spectrum. Panel A , 40 cycles of DNA synthesis followed by support bound double labeled thiazol Zinon's¹³3 shows a C NMR spectrum. Figure 31 further illustrates the stability of thiazolidinone for monitoring stability.¹³Use of C NMR Explaining. Panel C shows the ring position and the linker moiety attached to the support. At the α-position to the rubonyl group¹³Thiazolidinone double labeled with C atom of¹³C NMR spectrum is shown (labeled position is indicated by "*") . Panel B shows support-bound dinucleotides after 90 minutes photolysis in PBS buffer. Double labeled thiazolidinone¹³3 shows a C NMR spectrum. Panel A Was support bound double labeled after photolysis in PBS buffer for 3 hours. Of thiazolidinone¹³3 shows a C NMR spectrum. FIG. 32 shows support bound thiazolidinone for 40 cycles of DNA synthesis and Of the reaction mixture prepared by subjecting it to photolysis in PBS buffer for 3 hours 8 shows HPLC microanalysis. Figure 33 shows the graphic user interface provided on the control computer. Interface (“GUI”). As shown, the GUI is Between 1303 It includes a companion rectangular window 1301. User commands at the top of the window There is a menu bar 1305 with choices 1306-1313. These Each of the Mand Choices has an additional submeme to control the operation of the synthesizer. Including new. User selects the appropriate command choice with the mouse This allows the synthesizer to be programmed. Figure 34 depicts the GUI and shows the submenu options in the macro menu. Is shown. FIG. 35 depicts a dialog box for executing a macro. Figure 36 depicts a GUI, with submenu options in the group menu. Is shown. FIG. 37 illustrates a GUI, which is a submenu in the variable (variable) menu. Indicates options. FIG. 38 depicts a GUI, in the diagnostics (diagnosis) menu. Shows submenu options. FIG. 39 depicts a valve diagnostic screen. FIG. 40 depicts a sensor diagnostics screen. Figure 41 depicts a GUI with submenu options in the file menu. Is shown. 42-44 show dialects related to the synthetic setup. Shows a user box and allows the user to select the reaction vessel to be used in the synthesis. What to do (Figure 42), select start, loop and end macros (Figure 4) 3), and put the amino acid symbol or oligonucleotide code in each reaction vessel (Fig. 44). FIG. 45 shows a GUI, which is a submenu in the synthesis menu. Indicates options. FIG. 46 depicts a status display during composition or macro execution. FIG. 47 depicts the User Abort dialog box. FIG. 48 shows a GUI, and the submenu option in the edit menu is displayed. It shows that. Figure 49 shows the main modules of the control software under Windows (trademark) environment. A schematic diagram illustrates the flow of data between the two. Description of a specific embodiment   The present invention generally produces and screens tagged chemical libraries. To an improved method for playing. The present invention also includes a collection of diverse molecules. Useful in the synthesis of the tagged chemical libraries described above, for example. Related to the device.   Library tagged to correctly recognize the value of the improved method Basics for making and using As well as how the various synthesis and screening steps interact. Application and how the choice of reagents affects the results achieved. I have to. Tagged chemical libraries are often on solid supports. , And the choice of support and linker is critical to success. Rin Kerr attaches the support to the tag and attaches the support to the molecules of the library, and In embodiments without a solid support, tags are attached to the molecules of the library. Can be used for Choices regarding chemical building blocks, tags and synthetic methods are similar Is important and is influenced by the nature of the solid support and linkers available. You. Assays and uses where a tagged library is intended Also affects these choices, as well as the installation of equipment and reagents available.   The apparatus and method of the present invention is mainly used for the synthesis of oligonucleotides and peptides. However, the present invention is not limited thereto. The present invention is a substance , For example, polysaccharides, phospholipids, polyurethanes, benzodiazapines, pros Finding application in the synthesis of tagglandins and beta-turn analogs and other substances Let's. The cyclic material may be as disclosed in US Pat. No. 5,242,974 (Holmes). (Which is incorporated herein by reference).   The use and synthesis of a wide variety of materials, such as oligonucleotides and peptides, is described below. More details will be opened during the pending application below. Are shown and are incorporated herein by reference for all purposes. Part of. US Patent Application Serial No. 07/876792 (April 29, 1992) Application), US Patent Application Serial No. 07/762522 (filed Sep. 18, 1991) ) And US patent application serial number 07/946239 (filed September 16, 1992) .   The description of the invention is provided as illustrated by the following summary. Compendium I. Overview of Tagged Chemical Library Synthesis II. Solid support           A. type           B. Linker           C. Molecular support III. Chemical building blocks           A. Oligomer and monomer           B. Other building blocks IV. tag V. Synthesis method           A. Peptide library with oligonucleotide tags           B. Oligonucleotide-tagged peptide library synthesis           Improved method for           C. Small molecule synthesis           D. Methods for making soluble libraries VI. Assay method           A. Screening assay for bead-based libraries           B. Screening for soluble molecules           C. Screening a library of natural products VII. Instrument installation and reagents VIII. Equipment for parallel coupling synthesis reactions Example End of compendium   In addition to the above summary, the following glossary is provided to facilitate the description of the present invention. And many abbreviations and terms used herein to describe the invention. Is defined to have the stated general meaning as given.   Abbreviation: HBTU, O- (benzotriazol-1-yl) -1,1,3,3- Tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxy Cibenzotriazole; HATU, [O- (7-azabenzotriazole-1- Yl) -1,1,3,3, -tetramethyluronium hexafluorophosphate TFA, trifluoroacetic acid; TCA, trichloroacetic acid; DIEA, diisop Ropylethylamine; DMF, dimethylformamide; Fmoc, 9-fluorene Nylmethyloxycarbonyl; DMT, dimethoxytrityl; Trt, trityl Bz, Ben Zoyl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl ;^tBoc, tertiary-butyloxycarbonyl; PBS, phosphate buffered Saline; BSA, bovine serum albumin; mAb, monoclonal antibody.   Complementary or substantially complementary: These terms are the bindings of one compound to another. Competence, for example, the ability of a ligand to bind its complementary receptor. Scripture Formally, these terms refer to internucleotide nucleotides, such as double-stranded DNA moieties. Between the two strands of the offspring, or sequenced with an oligonucleotide primer, or Is a depiction of the base pairing between the primer binding site on the common strand nucleic acid to be amplified. Used in conjunction with. "Complementary" nucleotides generally refer to A and T (or A and U) and C and G, but with a wide variety of syntheses with binding properties known to those skilled in the art. Or there are modified nucleotides. "Substantial complementarity" means that RNA or DNA strands are Present when hybridizing to complementary nucleic acids under selective hybridization conditions. Exist. Typically, hybridization will be at least 14-25 nucleotides. Occurs when there is at least about 55% complementarity over the range of For more selective hybridization, complementarity is 65%, 75%, 90% and And 100% increase. Kanehisa, 1984,Nucl. AcidsRes. 12 : 203, which is incorporated herein by reference. High selectivity Hybridization conditions Is known as "stringent hybridization conditions" and It is defined below.   Epitope: This term corresponds to a subclass of receptors known as antibodies. Used to describe the part of the antigen molecule that is outlined by the interacting regions Can be   Identification tag: In its most general sense, this term allows a chemical reaction, eg , The individual solid supports experienced in the synthesis of oligomers on the solid supports. Used to indicate physical attributes that provide a means by which a nomer addition reaction can be identified . This identification tag is used in a series of reactions used in the synthesis of chemical libraries. Useful for recording a process. The identification tag can be any recognizable property (this Are, for example, shapes, sizes, weights, colors, optical densities, etc. that can be distinguished with a microscope or the like; Differential absorbance or luminescence; chemical reaction; magnetic or electrical properties; Can include any other unique mark that can be coded, and , Can be deciphered at the level of one (or several) molecules. Such an identification tag A preferred example of is an oligonucleotide. Because of the oligonucleotide The nucleotide sequence is the information encoded in a robust form. You. An "identification tag" refers to whether a solid support is used in a synthesis or not. In any case, it can be directly coupled to the synthesized oligomer. This latter state In general, the identification tag is conceptually a “support” for oligomer synthesis. Seeing it works again Can be.   Ligand: This term refers to a particular receptor, typically a particular receptor. Used to indicate the molecule that is recognized by its binding to the molecule. By the receptor A reagent that is bound to or reacts with a receptor is called a "ligand" and its term Has a defined meaning only in relation to its paired receptor . The term "ligand" means that the substance in question binds to the receptor or Specific molecular size or other structure or composition other than interacting Does not imply the characteristics of. A “ligand” is a natural ligand that a receptor binds to. Of a functional analogue capable of acting as an agonist or antagonist. It can function as either. Ligands that can be investigated by the present invention are cell membrane receptors. Scepter agonists and antagonists, toxins and venoms, virus Pitopes, hormones, sugars, cofactors, peptides, enzyme substrates, co Including factors, drugs (eg opiates, steroids, etc.) and proteins It is not limited to these.   monomer: This term refers to another molecule or set of molecules attached to each other, such as Indicates any member of a set of molecules that can form a set of gomers or polymers Used for. The set of monomers useful in the present invention is an example of peptide synthesis. For example, a set of L-amino acids, D-amino acids or synthetic amino acids is included. Not limited. In this specification The "monomer" used in is any of the basic set for the synthesis of oligomers. Means some member. For example, an L-amino acid dimer is a polypeptide A basic set of 400 "monomers" is formed for the synthesis of Variety of monomers The basic set can be used in a series of steps in the synthesis of polymers. Those skilled in the art , "Monomers" are simply one type of "chemical building block," and The other types of chemical building blocks can also be oligomers or small organic molecules or other It can be used in the method of the present invention regardless of which of the molecules is synthesized. You will understand that.   Oligomer or polymer: These terms refer to the chemical or Used to indicate a molecule formed by a method involving enzymatic addition. This Such oligomers include, for example, nucleic acids, polysaccharides, phospholipids and alpha, beta or Is a linear, cyclic or branched polyhedron of a peptide having any of omega amino acids. Mer, heteropolymer, polyurethane, polyester, polycarbonate, poly Urea, polyamide, polyethyleneimine, polyarylene sulfide, polysilo Xanthines, polyimides, polyacetates or other polymers, which are Those of ordinary skill in the art will readily appreciate upon reading the disclosure.   peptide: This term refers to monomers in which the monomers are linked together through amide bonds. Used to indicate an oligomer that is a rufa amino acid. "Pep Chido It may also mean "polypeptide". In the context of the present invention, the amino acid L-light It should be understood that it can be a physical isomer or a D-optical isomer. Pe Ptides are more than 2 amino acid monomers long, but more often than 5 Amino acid monomer length up to 10 and longer than 20 amino acids Are possible. However, peptides longer than 20 amino acids are more commonly referred to as "polypeptides. It is called "Petit". Use the standard one-letter abbreviations for amino acids (eg, pro P for phosphorus). These abbreviations are Stryer,Biochemistry, 3rd edition (1988) (this is Incorporated herein by reference).   Oligonucleotide: This term is typically prepared by synthetic means, Used to indicate a double-stranded DNA or RNA molecule. Oligo used in the present invention Nucleotides may be present in environments with oligonucleotides of different lengths from May be suitable, but is usually 50-150 nucleotides in length, It is preferably 80 to 120 nucleotides. For example, oligonucleotide tags Is the monomer-by-monomer addition step used to synthesize the oligomer. Can be constructed nucleotide-by-nucleotide, which is equivalent to Furthermore, Very short, ie 2-10 nucleotide oligonucleotides, To extend existing oligonucleotide tags to identify Can be used. Suitable oligonucleotides are described by Beaucage and Carruthers, 1981,Tetr.  Lett. twenty two: 1859-1862 by the phosphoramidite method or Ma tteucci et al., 1981,J. Am. Chem. Soc. 103: 3185 according to the triester method (this Are incorporated herein by reference) or by other methods. Therefore, for example, when using a commercially available automated oligonucleotide synthesizer Therefore, it can be prepared.   Operably linked: This term refers to one segment of nucleic acid and another segment Means a functional relationship between. For example, if the promoter drives transcription of the coding sequence A promoter (or enhancer -) Is "operably linked" to the coding sequence. Generally operably linked Means that the linked nucleic acid segments or sequences are contiguous, and If the two protein coding regions must be linked, they are adjacent and Means that you are in the reading frame.   Parallel coupling: This phrase uses two to separate distinct points on the substrate. Means to simultaneously couple the building block compounds of. Such groups The body has a solid support with distinct groups to which these building blocks are attached. It can be a carrier or two distinct if each building block is attached independently. Another chemical compound having a pendant group can also be constructed. Simultaneous coupling Attaches the new building block compound to the first coupled building block. Such a distinction before adding Means the coupling of two compounds to. Therefore, it is used in this context The term "simultaneous" means that two building blocks are exactly The meaning is not limited.   Receptor: This term refers to a molecule that has a specific affinity for a given ligand. Means Receptors can be naturally occurring or synthetic molecules. Receptor In their unaltered natural or separated state, or with other species Can be used as a collection of. Receptor can be covalently or non-covalently May be associated with other substances. Examples of receptors that can be used in the methods of the invention include anti- The body, cell membrane receptors, monoclonal antibodies, specific antigenic determinants (eg, Antiserum, polynucleotides, nucleic acids, rhease, cells, or other substances) It includes but is not limited to cutin, polysaccharides, cells, cell membranes and organelles. Receptors are also known as "anti-ligands". "Ligand-Rese "Pter-pair" is two molecules, typically two macromolecules, It is formed when it binds and forms a complex. Other examples of receptors are antibiotics A specific transport protein or enzyme that is essential for the survival of the microorganism in which Enzyme binding sites, ligand binding sites on antibody molecules, nucleic acids, Lerner et al., 1991,Sc ience  252: 659, which is incorporated by reference herein. Various catalytically active polypeptides, and hormone receptors such as insulin Phosphorus and Including but not limited to long hormone receptors.   Substrate or solid support: These terms refer to materials that have hard or semi-rigid surfaces. It is used to Such materials are preferably small beads, spherules, particles. It takes the form of a disk or other conventional form, although other forms may be used. A few In some embodiments, at least one surface of the substrate can be substantially flat . A roughly spherical shape is preferred.   Stringent hybridization conditions: This phrase is highly selective Hybridization condition, in which the nucleic acid is such that the bound sequences are completely Or, as long as it is largely (ie, greater than 80%) complementary, other nucleic acids (or Remain in stable association with other segments of the same nucleic acid). This Such conditions typically result in salt concentrations below about 1M, eg below 500 mM. And often contains salt concentrations below 200 mM. Ha for oligomers Hybridization temperatures are typically above 22 ° C, eg about 30 ° C. Higher, and often above about 37 ° C. Longer fragments are specific high Requires higher hybridization temperature for hybridization. Other factors also dramatically affect the stringency of hybridization (see Such factors include base composition, complementary strand length, the presence of organic solvents and base (Including the degree of match), the combination of factors is one factor -Only absolute hand More important than a dan.   Synthesis: The compound isIn vitroWhen manufactured by chemical or enzymatic synthesis in It is said to be "synthetic". The synthetic library of the present invention contains virus or plasmid. Vector, such as a bacterial, yeast or other biological host. Can be contrasted with a library in a vector capable of I. Overview of Tagged Chemical Library Synthesis   The present invention generally synthesizes and screens tagged chemical libraries. A method for performing In essence, each “of the chemical libraries of the invention is A book is a chemical substance or molecule of interest, a chemical substance or molecule of interest or its important It consists of a tag that identifies the perspective and the link between the tag and the chemical or molecule of interest. You. In one important embodiment, the chemical or molecule of interest is an oligomer, For example, a peptide, a tag is an oligomer, such as a nucleic acid, and The linkage is a solid support or particle from which oligomers and tags, for example Optionally to facilitate detection or to provide a soluble library. Can be refused. Such libraries may bind to the receptor or some Screened to isolate individual oligomers with other desired properties sell. General methods for producing tagged chemical libraries are Explained by manufacturing a huge, highly diverse collection of Gomer, Here, (Libra Lee typically includes duplicate "books," but each different library Lee members have a unique monomer sequence to other library members Oligomer. Such libraries or collections are, for example, Xⁿ Monomers assembled into oligomers of length n yielding different compounds Can include all combinations of X different monomers. Collecti Can also have different monomer units in, for example, only one or a few sites. Whilst containing the same sequence at all other sites.   General methods for synthesizing such collections of oligomers are typically Includes a random combinatorial (“stochastic”) approach and monomer unitization Includes biological and / or enzymatic assembly. One process consists of multiple steps (a) Distributing a plurality of solid supports in reaction vessels, (b) in different reaction vessels Of the support in each reaction vessel using different combinations of the first monomer and tag. The step of coupling the first monomer and the first tag to (c) collecting the support Step, (d) distributing the support in a plurality of reaction vessels, (e) different for each Using different combinations of second monomer and second tag in the reaction vessel, the first module The second monomer is coupled to the nomer and the solid support or first tag Coupling the second tag in tandem, and optionally different tags and different The coupling and distribution steps using It includes a step of repeating 1 to 20 times or more. Typically, a substantially equal number of A body support is distributed to each reaction vessel. Different cups on the same chemical building block It can be used in the ring process, and the same chemical building block can be Can be used in two or more coupling reactions (reaction vessels) in the coupling step of Those of ordinary skill in the art will understand that they can.   In order to make the method easier to visualize, first, three different monomers, A, B And all oligos of three residue length assembled from the monomer set of C It is possible to consider probabilistic synthesis of untagged libraries. Wear. The beads were divided into three parts, which were distributed to three reaction vessels, and the monomer was added. A is linked to the beads in the first reaction vessel, B in the second vessel and C Connected in a third container. The beads from all reaction vessels were then collected. Collection The pool contains approximately the same number of three different types of beads, each Each type is characterized by a monomer linked to the beads. Pool Mixed and then contains A, B and C respectively as the next monomers to be linked It was redistributed into a separate monomer reaction vessel.   Following this coupling reaction, each reaction vessel had all three With two different monomers and in position 2 in each specific second reaction vessel With beads having the monomers contained in. Re-install all beads Collect and produce a mixture of beads, each with one of the 9 possible dimers. This The pool of 3 again into 3 reaction vessels and into 3 different monomers Linking all trimers of all three monomers (3^Three= 27) complete set Build. As can be easily seen, the use of a sufficiently large number of synthetic beads Are the monomers used in this random stochastic combinatorial synthetic scheme Helps ensure that the various combinations of are completely represented.   Modifications of this completely random approach are also possible. For example, monomer The set expands from process to process if coupling chemistry is available, and Can be reduced, or the set of monomers can be reduced to the next step (eg, the first step). Amino acids, other process nucleosides, other process hydrocarbons) Can be changed. A monomer unit for peptide synthesis may be, for example, a single amino acid or May include larger peptide units, or both. One change is the synthetic On a solid support to be distributed between different monomer sets in a process To form several pools of different sequences of This approach To produce different length oligomers with related or unrelated sequences. You can, and change other residues while Immobilizing the monomeric residues of a particular residue or region modified to provide diversity. Can be constructed.   The synthesis of tagged chemical libraries often involves such combinations. Including the synthesis step of. However, the identification tag presents an identification of each oligomer. A tagged chemical library can be easily It can be significantly larger or more complex than the unfilled library. In fact, the method of the invention for synthesizing a synthetic library of compounds comprises a multi-step process. Screening a large collection of non-sequencing compounds produced synthetically Enables learning.   In particular, the use of oligonucleotide tags and A vast library of connected compounds, especially peptides, produced through synthetic synthesis Provide a powerful mechanism for recording the structural identity of each member of This way The method can be widely applied to encode combinatorial assemblies of other non-peptidic structures. However, provided that the parallel composition scheme remains orthogonal and compatible. Combination The net result of the synthetic synthesis is that each yields a very high yield to yield a single product, each Only the proceeding reaction sequence is explicitly defined. In this situation, the standard peptide And the structures of the products obtained, which are almost obtained by synthetic DNA chemistry, Explicitly specified by the building block used and / or the order of ligation reactions .   However, most synthetic organic reactions are more specific and have variable yields. And a wide variety of products, such as regio and stereoisomeric structures. On solid support The use of such chemicals for combinatorial library synthesis in A product mixture results on each bead in the tray. In many general cases, Cryptography may not uniquely identify the chemical structure of related objects. Yes. Rather, the cryptographic process is built by the members of the library In order to encode the exact synthetic method (eg reagents, reaction conditions etc.) It can be certainly considered. The library was screened to identify "active formulations". And then remanufactured on a preliminary scale, and (if necessary) It can be fractionated to isolate the bioactive ingredient. Encoded library tricks Surgery covers the range of combinatorial chemistry and its applications, drug discovery and a wide variety of useful It has considerable potential to extend to the development and isolation of compounds. Tag attached With this overview of the synthesis of tailored molecular libraries, important aspects of the invention, such as The use and selection of solid supports in library synthesis is better appreciated. II. Solid support A. type   Typically, the tagged chemical libraries of the present invention are "solid supports". , For example, consisting of a collection of beads or particles. Such a solid support is It may have any shape, but is preferably approximately spherical. Support size, shape Alternatively, the composition does not have to be uniform, but The carrier is usually, and preferably, uniform. However, some embodiments In particular, a support having a very uniform size is particularly preferable. In another embodiment, Two or more distinctly different populations of solid supports are used for certain purposes. obtain. That is, the solid support may consist of a single particle, or two or more linked particles. sell.   A solid support can consist of many substances, but many of the chemically reactive groups are Of derivatization and oligomer or other molecular synthesis chemistry and tags to bind them It is largely limited by its ability for compatibility with binding. Suitable support materials are , Glass, latex, highly cross-linked polystyrene or similar polymers, gold Or other colloidal material particles, and other materials known to those skilled in the art. Especially described In the absence of these, the chemical reactive groups with which such solid supports may be derivatized are , Which are commonly used for the solid state synthesis of each molecule or oligomer. Yes, and therefore known to those skilled in the art. "Solid support" as used herein Refers to oligomer synthesis, and in some embodiments tag attachment and / or Alternatively, it includes a particle having a site suitable for synthesis. Synthetic oligomer of the invention There are various solid supports useful in the preparation of libraries. Solid supports are Usually, for example, in solid phase synthesis of peptides and nucleic acids, and other oligomers listed above. Used for this purpose and are therefore well known to the person skilled in the art. The solid support of the present invention is Live cells, viruses or cloning vectors, eg Does not include phage vectors or plasmids. Monobeads ™ ( Commercially available from Pharmacia Fine Chemicals AB, Uppsala, Sweden Or equivalents thereof for the purposes of the various aspects of the present invention are solid supports. Is especially useful. Monobeads ™ have good size homogeneity and small Provides a small size, ie 10 μm. In addition, these Monobeads (quotes Standard) does not aggregate in any organic or inorganic solvent, and And a suitable support for peptide synthesis. Finally, Monobeads ™ provides a very high loading of primary amines (100 nmole / mg).   One important aspect of the particular solid support chosen for the practice of this invention is the support. It is the size of the body. With sufficient solid support and efficient coupling, If desired, a complete set of certain oligomers can be produced. Generally, solid The size of the holder is from 1 nm to 100 μm, but larger than 1 mm. Some solid supports may also be used from time to time. A suitable size for the solid support is (1) Number of ligomer synthesis sites and desired attachment sites of identification tags, (2) Differences to be synthesized Number of compounds left (and solids carrying each oligomer that needs to be screened) Number of supports), and (3) the particular screening strategy to be used (eg, Depends on the effect of solid support size on fluorescence activated cell sorting (FACS) I do.   As a specific example, a 1 μm diameter solid support is used in the method of the present invention. obtain. If each reaction vessel contains about 0.2 mL of solid support and the oligomer is If synthesized from a set of 50 monomers (50 parallel reactions), then 10 mL of solid support or about 10¹³Solid support is required. If these If it is desired to make a hexamer using 50 monomers of . 5 × 10^TenThere are over 10 possible sequences, and each particular sequence is approximately 10^ThreeSolid It can appear on the body support. The estimated capacity of each bead is determined by the commonly used peptides Based on the capacity of the synthetic resin, there is about 0.1 pg peptide per bead. Next So, by this approximation, each solid support is about 100 amol or 10⁸Oligomer Has a chain.   To improve washing efficiency, rather than non-porous beads or typical peptide synthesis Other solid supports of low porosity can be used, but for certain applications of the invention In, fairly porous beads or resins work well and are often preferred. Non-porous supports have a low density of growing chains but have a reduced order of several orders of magnitude. Manufactured for sufficient screening, even when accompanied by sufficient oligomer density Can be done. On lower porosity supports, a greater proportion of oligomeric moieties will It is accessible for binding to the receptor during the cleaning process. Also Low porosity support reduces carryover from one reaction to the next And thus improve the decoding accuracy of the dominant (correct) tag.   As mentioned above, another embodiment provides two solid supports physically connected to each other. , Including the use of beads, one of which is a synthetic site for molecules or oligomers ( Or linker), and the other is an attachment for one or more identification tags. The attachment site (or linker). This arrangement allows for identification of the molecule or oligomer and the identification tag. Allows for separation into separate "zones", and greatly different for adhesion It enables the use of chemically reactive groups and chemistries. The solid support is separately derivatized , Then all or almost all of the synthetic solid support is attached to the attached tag-attached solid support. It is connected under the condition of having a carrier. Solid supports may be of different sizes, eg Large synthesis with several (or many) smaller tag-attached beads attached It can be beads. In one embodiment, the first solid support is at least A second solid support having one attached amino acid and at least one With attached nucleotides.   The method of linking two beads is done by oligomer synthesis chemistry. most The obvious means of linking the beads to the predominantly chemically reactive groups on each species of solid support are compatible with each other. Heterobifunctional crosslinkers that interact (eg,Pierce ImmunoTechnology Ca talog and Handbook  pp.E10-E18 (1991)) . Such crosslinkers serve a variety of purposes, as will be shown in the following sections. B. Linker   When attached to a solid support, the oligomer and its associated tag are usually It is attached to the support by one or more molecular linkers. The linker molecule is attached prior to attachment. Because it has suitable functional groups at each end, one group is suitable for attachment to the support , And another group is suitable for attachment to the oligomer or tag. Several In embodiments, the cleavable linker facilitates the assay or detection process. Used for.   Given the wide availability of diverse ligation reagents, the identification tag can be Or any other library compound, or solid support, or pre-existing You can connect to the tag. For example, the identification tag may be a model that is incorporated into the oligomer. Can be attached to a building block incorporated into a nomer or non-oligomeric compound You. For peptide oligomers, the side chains of cysteine residues are convenient for tag attachment. Provide an advantageous part. In another example, the tag covers a small number of oligomer chains (key Can be attached to the desired oligomer, provided that the Provided that a reduction in the amount of synthesis can be easily tolerated. Oligomer on solid support The tag can be attached directly to the linker that binds the-(or other compound of interest). this In an embodiment, the linker is suitable for attachment of the identification tag prior to attachment. It has a third functional group.   Of course, depending on the desired application and effect, a wide variety of Can be attached. For example, characteristics such as coupling or coupling efficiency. In order to achieve the desired effect, the hydrophobic, hydrophilic, or configurational You can choose the linker that gives the product. In one aspect of the invention, branched phosphorus Ker, ie a linker with bulky side chains, eg the linker Fmoc- Thr (tBu) to give hardness or on a solid support in the library Control the intermolecular spacing between molecules or between the molecules in the library and the tag. Used for   As mentioned above, cleavable linkers can be used for useful effects. The preferred photocleavable linker of the present invention is 6-nitroveratryloxycarbo. Nil (NVOC) and other NVOC-related linker compounds (PCT Publication No. WO See 90/15070 and WO 92/10092, and U.S. patent application serial number 971181. No. (filed Nov. 2, 1992), which are incorporated herein by reference. Including). In other embodiments, the linker has one or more restriction sites. Nucleic acid, and thus a portion of the library member (tag, oligomer or Are other compounds of interest, or both, or solid supports) It can be selectively disconnected from others by the element. This novel nucleic acid linker is Examples of a wide variety of linkers that can be used with useful effects for a purpose Testify. C. Molecular support   As mentioned above, the present invention also lacks a solid support and the tag is synthesized. Method of attachment directly (typically through a linker) to an oligomer or other molecule Can be done by. Alternatively, the oligomer or other molecule and its associated tag are Can be synthesized on a solid support and then used for screening or other uses. It can be previously cleaved or removed from the solid support. Such a method More fully below. The library may or may not have a solid support present. The size and composition of the reel depends on the number of coupling and mixing steps used during the synthesis. , And the monomer or other building block. III. Chemical building blocks A. Oligomer and monomer   The wide applicability of the present invention is due to the huge library of diverse oligomers and polymers. Probably most easily understood by synthesizing and screening Lee . Oligomers are polymeric compounds consisting of monomers, such as biopolymers. And the arrangement of the monomers in the oligomer often characterizes important biological properties. Set. Preferred oligomers of interest are peptides, oligonucleotides, oligos   Includes N-substituted glycines and polycarbamates. As mentioned above, the object of the present invention In order for the monomers to be linked together to form an oligomer or polymer Can Any member of the set of monomers, such as amino acids, carbamates, sulfos , Sulfoxide, nucleoside, hydrocarbon, urea, phosphonate, lipid, s Tell, a combination of the above, and the like. Therefore, the monomer is not Which can be activated appropriately or acceptable for enzymatic coupling Type may be used.   Methods of assembling oligomers from many monomer types include monomer units or Use the appropriate coupling chemistry for a given set of building blocks Request. Any of the building blocks that can be attached to each other in a sequential fashion The set can also act as a monomer set. Attachment can be chemical, enzymatic or otherwise By any means, or by any combination of these means. The resulting oligomers can be linear, cyclic, branched or various other structures envisioned. It is possible and these will be apparent to the person skilled in the art. B. Other building blocks   The present invention relates herein to the preparation of molecules which primarily comprise a sequence of amino acids. Although described, the present invention provides for the preparation of other oligomers and a sequential mode for each component. Can be readily applied to the set of compounds that can be synthesized by Should be understood by the vendor. For example, benzodiazepines, hydantoins And compounds such as peptidyl phosphonates can be prepared using the methods of the invention. (See US patent application serial number 08/119700 (filed Sep. 9, 1993)) , Which is now abandoned US Patent Application Serial No. 081577 (1993 Filed on June 21, 2014) and a part of US Pat. No. 5339115 Which is a genus application, each of which is hereby incorporated by reference).   In one embodiment, the method of the invention provides a library of branched polymers. Can be used to produce In many instances, linear polymers such as , A library of peptides is quite useful, while with residues larger than 3-4 , The shape of these linear molecules becomes longer and narrower. Most drugs are probably Some do not have such long shapes, due in part to the high degree of flexibility of the molecule. Branched skeleton The polymer may result in a molecular form similar to known drugs. Therefore, one embodiment In accordance with the present invention, the present invention provides at least three utilities to which other monomers can be attached. The present invention relates to incorporation of a monomer having a functional group.   However, if such a monomer is used exclusively, then Fully branched syntheses are always used in the synthesis of the last monomer linked. (Relative to other monomers) results. Of course, you can change this ratio For this purpose, it is possible to incorporate a mixture of different branched monomers, At that time, identification of the structure of the compound of interest will be more difficult. That is, a branched monomer -The more complex the mixture, the more It can provide less information about a particular compound. Improved version of the invention In the method, a mixture of two monomers was introduced and at each monomer linking step , One can branch but another cannot, and this gives a lot of information A library containing a large diversity of shapes with a tag is produced. In this case The tag identifies the monomer present at each coupling step, which is , It does not specify whether the monomer can be branched. However, the first Of those monomers contained in the set of compounds selected from the library of Simple resynthesis using only the compounds readily identifies the structures of those compounds. IV. tag   The identification tag can be, for example, microscopically or otherwise, shaped, size, weight, charged or charged. Has distinguishable characteristics in color. This recognizable property is the tag From the optical, chemical, electrical or magnetic properties of, or some of these properties Can result from combinations. In essence, a tag labels a molecule and Encode readable information at the level of the molecule (or molecules) or solid support Function to do so. Follow the synthetic pathways that each member of the chemical library has undergone By using the identification tag to identify any chemical in the library. The structure (ie, the monomer sequence of any oligomer) is determined by reading the identification tag. Can be deduced.   Small recognizable different sizes, shapes or colors, or marked with a barcode Microscopically identifiable tags, such as simple beads, can be constructed. The tag is "mechanical Can be a "readable" "luminescent or radiolabel. Identification tags can also be encoded It can be an effective molecular structure. Information can be molecular size (polymer length) or composition Can be encoded into. Perhaps the best example of the latter type is the nucleic acid sequence , RNA or DNA assembled from natural or modified bases.   Played by tags in the synthesis and screening of chemical libraries Explaining the role is, for example, attaching to each bead in oligomer synthesis. The use of tags with a combination of microscopically recognizable letters and numbers That is. For example, for the tag “A1”, beads participate in the A monomer reaction in step 1. "C2" means that the beads participated in the C monomer reaction in step 2. And "B3" means that the B monomer was added in step 3. I do. At the end of the three-step synthesis, beads are attached to three tags, eg, For example, it has A1, C2 and B3, which means that the peptide sequence on the bead is AC B is shown. This scheme is based on the number of different monomers and the number of synthetic steps. It requires a number of different identification tags, at most equal to the product (9 in this example). Also If the symbols are attached to each other in the order of steps A, A-C, A-C-B, identification is performed. The number of tags is reduced. in this case Requires as many identification tags as there are monomers, and which identification tags are A method to keep a record of which process was added and in which additional process Can be assembled.   In another example, the tag is a molecule that is addressed by a variety of light, such as a fluorescent or Is a phosphorescent compound whose spectral properties can change (eg, Fading), and therefore used to store information, Used to mark each bead or other solid support in the substrate. One of this In such a method, the beads incorporate various fluorophores, each of which Selectively photobleached, rendering fluorescence or reduced fluorescence unavailable . During each coupling or chemical reaction step, beads are packed with one or more specific types of beads. It is (not) illuminated to photobleach (or not) the lorophore and thus The monomer identification is recorded in the oligomer formed.Science 255: 1213 (1992 March 6, 2013) (which is incorporated herein by reference).   Therefore, the identification tag can be used for each monomer coupling, or for individual library members. Identify the bars or other reaction steps experienced by the solid support, and attach each monomer to Record the steps in the synthetic series in which additional or other chemical reactions took place. The tags are Immediately before, during or after the monomer addition or other reaction, the tag of the identification tag is Attached in a manner compatible with the chemistry of attachment, attachment mode and oligomer or other molecular synthesis. U You. As mentioned above, the identification tag directly passes through the linking molecule through various mechanisms. In situ or through a solid support on which the oligomer is synthesized. Can be chained. In the latter mode, the tag can be attached to another solid support. And the second solid support is a solid on which the oligomer is synthesized. It can be attached to a support. Solids that undergo specific monomer additions or other chemical reaction steps When the supports are physically brought together, an identification tag is added, and so Thus, tags can be added as a group, ie prior to the next pooling step.   Of course, in some cases only a few of the monomer units of the oligomer will change. If so, it is necessary to identify only the changing monomers in the oligomer. This can be important, for example, if you want to change only a few amino acids in a peptide. You. For example, changing only 3-6 amino acids in a 6-12 amino acid long peptide No, or changed as few as 5 amino acids in a peptide up to 50 amino acids long If not. For each solid support, specify only the amino acids that are changed in each sequence Uniquely identify the sequence of each peptide by providing an identification tag Which is easily recognized by those skilled in the art. In such a case, all The body support can remain in the same reaction vessel to add common monomer units, , Divided between different reaction vessels due to the addition of distinct monomer units .   Synthetic oligodeoxyribonucleotides are particularly preferred It is an information transmission identification tag. Oligonucleotides are natural, high-density information storage media. It is the body. Related to the process of monomer type identification and addition, or chemical synthesis Other information is easily encoded in the short oligonucleotide sequence. next, Oligonucleotides include a wide variety of solid supports, oligomers, linkers and Easy to follow for attachment to other molecules. For example, an oligonucleotide can be a peptide It can be easily attached to synthetic beads.   Significant advantages inherent in the use of oligonucleotide-based coding schemes Is a polymerase chain reaction (PCR,PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., Academic  Press, San Diego 1990), and also US Pat. Nos. 4,683,202 and 4,965,188. , Which are each herein incorporated by reference) and other nucleic acids. The ability to achieve target amplification at very high levels through replication and amplification techniques. You. Most commonly usedIn vitroThe DNA amplification method is PCR, but a suitable alternative Alternative amplification methods include, for example, amplification based on nucleic acid sequences (Compton, 1991,Nature 35 0: 91-92, which is incorporated herein by reference) and amplified antisense R NA (Van Gelder et al., 1988,Proc. Nat. Acad. Sci. USA 85: 7652-7656, quote And a self-sustaining sequence. Replication system (3SR, Guatelli et al., 1990,Proc. Natl. Ac ad. Sci. USA  87: 1874-1878, which is incorporated herein by reference). only A small amount (a single copy is sufficient with a high selectivity and efficiency method) DNA template of (a) is required for PCR, solid support of microscopic diameter The body can be used to obtain a huge library.   The use of nucleic acid tags is a versatile technique available through other constrained library technologies. Facilitates the construction and screening of synthetic libraries far beyond sex. Furthermore, these libraries use a manageable amount of bead material, and Thus, experimental binding of biological reagents can be used to assay for receptor binding. An improved method of the present invention is an ESL library using oligonucleotide tags. Steps in the processing of DNA-amplification, strand separation and tags from individual beads Involved in sequencing. Method increases sequencing efficiency by at least one order And is involved in the incorporation of the tag chain process, where typically Many different tags, typically amplified from a selected set of Braly members. Tags to each other prior to cloning or sequencing the oligonucleotide tags. Is tied.   In one embodiment of the method, the amplified tags are linked and Later, in a conventional sequencing vector, 10-20 (or more) tags of linear Cloned as a sequence. Preferably, suitable restriction sites are Adjacent to the "coding region" (sequence with information content) of a gonucleotide tag The restriction sites were cleaved, and the fragments were cut after amplification of the tag, placed and tagged on a panel of beads. The lagments are linked to form a chain. The linkage is then sequenced appropriately. Cloned into the computer. After that, each template is, for example, 500 to 800. Used for bidirectional sequencing of entire bases, less per template Both allow identification of more than 10 tags. This approach is also FACS To provide the option of avoiding the isolation of individual beads. Beads and tags The assigned compounds are sorted into pools and the pool of tags amplified and linked. , And cloned for sequencing. In addition, handling individual beads Requirements for library construction and screening Beads smaller than 1 μm (typically this size is comparable to conventional FACS analysis). Too small for) can be used. The selection is conveniently an affinity purification method (Panning, magnetic beads, etc.) The bead-enriched pool was then amplified and cloned as described above. Is done.   The identification tag of the oligonucleotide is the corresponding mono (for oligomer synthesis). Assembling one base at a time before, during or after mer coupling or other chemical reaction steps Be taken. In the case of synthesizing an oligonucleotide tag for each base, the tag for each step Is a single nucleotide or many With a small number of nucleotides (ie, a block of 2-5 nucleotides). Block In a step-by-step sequential approach, 2-5 to 10 or more bases An encoded set of nucleotides (“codons”) is protected and activated Added as a blocked block. Each block is a monomer type or other information And the order of addition of one tag block to the next can be monomer addition or other Indicates the order of reactions. Alternatively, the block is the number of oligomer synthesis or other reaction steps, And may encode monomer type or other building block information. This trick Is a linear array of oligonucleotide chains grown in parallel with oligomers. Save the order of steps in. Parallel synthetic steps (eg, oligonucleotides and peptides Standard synthetic chemistry can be modified to preserve the chemical compatibility of However, important aspects of the invention are discussed in further detail below.   Amplification primer site, containing monomer-specific information and reaction sequence information, 50- Protected (or unprotected) oligonuclides 150 bases (nucleotides) long Cleotide may be attached at each step. A series of oligomer synthesis (monomer Coupling) or other chemical synthesis step at the end of each oligomer in the library. -A different n of oligonucleotide identification tags associated with sequences or other chemicals There is an encoded set. After identifying the oligomer using ligand activity , Related oligonucleotides are amplified by PCR, and Is another compound Sequenced to decipher the identity of   Used in oligonucleotide identification tags, as discussed more fully below. The choice of base depends on the chemistry of the oligomer synthesis or other chemical reaction conditions that expose the tag. Directed by For example, the use of strong acids depurinates nucleic acids . Therefore, when using chemicals that require the use of strong acids, pyrimidines C and T It is important to use only oligonucleotides and binary codes I can prove it. In a similar manner, the instability of purine nucleotides to strong acids is Purine nucleoside analog analogues, such as 7-deaza-2'-deoxyade Nonosine and 7-deaza-2'-deoxyguanosine (Barr et al., 1986,BioTechniq ues  4: 428-432 and Scheit,Nucleotide Analogs: Synthesis and Biological Fu nction  See pp. 64-65 (John Wiley and Sons, New York), which are cited together. Can be overcome through the use of (as part of this specification). These or The use of other analogues has the opposite effect of four elements or Allows other uses. Therefore, in a preferred embodiment, the identification tag is about It is an oligonucleotide with a nucleotide length of 50 to 150 Pirimi, which does not decompose under the coupling conditions used to assemble the rally Gin or pyrimidine and purine analogues, or any type of nucleoside Consists of The oligonucleotide identification tag includes 5'and 3'amplification sites, and can be any DNA sequencing May contain primer sites, which are specific for each step of oligomer synthesis Can be.   Encoding combinatorial synthetic procedures using oligonucleotides is a huge Encountered in the direct structural analysis of trace amounts of ligands isolated from various libraries It provides a mechanism that addresses the major limitations of ambiguity and sensitivity. For information storage The high capacity of the DNA in the DNA to store the exact details of library construction Can be used for. In Example 1 below, three bases (c⁷dA, dC, T) included The “codon” structure of two adjacent nucleotides is 3²= Amino acid structure up to 9 It was able to encode a composition that takes in building blocks and was used (only 7 building blocks Lock was used in the synthesis of this library). If c⁷dG is also If included in the coding template, then 1000 different Combinatorial synthesis using monomers results in a "codon" sequence of only 5 nucleotides. (4^Five= 1024).   The information can be the sequence of the oligonucleotide or other polymeric or Encoded in length rather than, or in addition to, the sequence of lygomeric tags sell. If only the length is used to represent each specific monomer addition to the oligomer Is used, then the identification of the oligomer is determined by, for example, Amplifies the ligonucleotide tag, and various size separation techniques (polyacrylic Identification of tags through mid gel or capillary gel electrophoresis) Can be deciphered. Given step of oligomer synthesis or each different chemical reaction step Each different monomer added in the is represented by a unique length nucleotide tag. Is done. Oligonucleotide tags are used for amplification sites, such as PCR priming Columns, the sequences featuring a given number of steps in an oligomer or other chemical synthesis Designed to attach. Then the orientation at any given location in the array Determination of gommer composition is dependent on PCR priming sequence characteristics for the steps in synthesis. Amplification of tags using techniques known in the art, such as (tag as standard Gel or capillary electrophoresis (using an oligonucleotide that imparts Including separation by size of amplified products. This embodiment relates to lead sequences It is especially useful if you want to create a library of compounds. Similar parts Only tags are required during the process of synthesis.   In addition to length, oligomer sequence information is also It can be encoded in a nucleotide sequence containing a reotide tag. This type of cipher is different In the embodiment in which the resulting oligonucleotide tag is attached at each coupling step Not only the pre-existing oligonucleotide tags It is also valuable in embodiments that extend in moderation. For example, as described below, Approximately 100 bases (or somewhat longer), each with 7 (or more) regions Up to the oligonucleotides) can be used.   Region 1 is the 3'-PCR primer site (20 to 25 bases). This part Position with other PCR sites (oligonucleotides) to prime amplification by PCR. Used at the 5'end of the reotide) linked. Other amplification methods also used Can be done.   Region 2 is a "step-specific" DNA sequencing primer site (15-20 bases) It is. This site corresponds to a specific numbered process in the synthetic series. Is specific. All oligonuclides attached to all beads in a particular process Leotide usually has this sequence in common. The process given each number is It has a very specific primer site that represents   Region 3 is a spacer (20-30 bases). Of various lengths, preferably Spacers with a length of 20-30 bases span the monomer encoding or identification region. In order to give a good "reading", separate from the sequencing primer site Put the code part.   Region 4 is a monomer identification region (8 bases). In this exemplary embodiment And each base in the 8-bit symbol string represents a 1-bit binary code, Then, for example, T = 0 and C = 1. Each set of process-specific identification tags It consists of 8 bases with 1 (C) or 0 (T) at each of the 8 positions. This They are switches that are set to "on" or "off" at different locations. Can be considered. Each monomer type has a mixture of 1-8 of these "switches" Therefore, it is encoded.   Region 5 is a process number information region (4 bases plus 2 salts on both sides for distinguishing regions) Group). The 4 bits in this short length encode the number of steps. this is, Redundant but not suitable primer for sequencing primer Can be used to confirm that the correct process has been decrypted.   Region 6 is a repeat of the monomer identification region (8 bases). This area is It has the same information as 4 and is used to confirm monomer identification. This first Setting the secondary monomer encoding region also provides good sequencing " Increase the probability of acquiring a "read".   Region 7 is a 5'-PCR primer site (20 or 25 bases). This part The position is a part for annealing the second PCR primer for sequence amplification. Work as a rank. Have all seven of these features, some of which are optional The length of the oligonucleotide It has 125 bases.   The 8-bit format can encode 256 different monomer types. EN The number of steps that can be encoded depends on the step-specific set of oligonucleotides at hand. It is determined by the number (8 per set). 10 sets (80 oligonucleoti 256) assembled into oligonucleotides up to 10 units long Of different monomers can be encoded (hence 256^Ten= 1.2 × 10^{twenty four}Ma Encoding capability is provided for the oligomer sequences in. Coded identification Tags are assigned a unique binary number for each monomer (eg, Ala). = 00000001, Gly = 00000110, etc.). Suitable oligonucleo Chid is combined to give the correct binary code.   There are various options to facilitate oligonucleotide tag identification. example Read tags directly from beads, for example by sequencing or hybridization be able to. Also, in order to facilitate tag identification, oligonucleotide tags are Can be amplified. Oligonucleophiles carried by a single solid support or oligomer Tide identification tags can be inserted in vivo by cloning or by, for example, PCR. Can be amplified in vitro. If the limit of detection is on the order of 100 molecules, Then, at least 100 or each of the oligonucleotide tags on the beads. More copies are required. A copy of the tag, that is, a single-stranded oligonucleotide Either a cleotide, a double-stranded nucleic acid, or a mixture of single-stranded and double-stranded nucleic acids , Manufactured by any of a variety of methods, some of which are described below, The amplified material is then sequenced. Separate and distinct oligonucleoti De-tags add each monomer (rather than extending the existing tags at each step) In an embodiment of the invention that is added in steps, all tags can be amplified at once, And then (use different sequencing primers for each type of tag ) If there is an oligomer synthesis step, the amplified material can be used in many separate sequencing reactions. Can be divided. In this embodiment, the primer sequences are also selected appropriately. By choosing, the tags can be designed so that each tag can be amplified separately from the other tags. You. Sequencing reactions were performed and run on a standard sequencing gel to remove oligomers. The sequence is derived from the code revealed in the sequence information obtained.   Other strategies include conventional PCR primers and conventional sequencing primers (sequencing The constant primer should completely or partially overlap the PCR primer site. Step-specific in the oligonucleotide from the beads Hybridize to an oligonucleotide probe that is complementary to a unique sequence To identify the process. A single set of sequencing reactions Made on all amplified oligonucleotides from a single bead and The reaction product is a single set of Run in lane. The reaction product is then subjected to appropriate hybridization. It is transcribed into a membrane and hybridized to a single process-specific probe (Mani See atis et al., Cold Spring Harbor Laboratory, Cold Spring Habor, NY (1980), This is incorporated herein by reference). After detecting the obtained signal, The lobe is washed from the membrane and hybridized with another process-specific probe . European Patent Application Publication No. 237362 and PCT Publication No. 89/11548 ( Each of which is incorporated herein by reference). You can also.   Parallel hybridization is an alternative to sequential hybridization. provide. The sequencing reaction was divided into equal numbers equal to the number of peptide synthesis steps, and And run on a sequencing gel in separate sets of lanes, one for each Is done. After transferring the reaction product to the appropriate membrane, the membrane is cut and the lane set is removed. To separate. Each lane set is then loaded with multiple process-specific oligonucleotides. Hybridized to one of the probes (Plex Luminescent Kits Product Cata log,  "Uniplex DNA Sequencing" in Bedford, MA, 1990. And "multiplex DNA sequencing").   As mentioned above, a single synthetic solid support (or attached beads carrying tags). , Or in solution in “wells” is a few hundred copies of each oligonucleotide tag. Including pea Only. These tags may be labeled, for example, by PCR or other means known to those of skill in the art. Can provide sufficient DNA to be amplified and correctly sequenced. Oligomer Ability to decipher the number of available oligonucleotide identification tags, available Depending on the level of amplification that can be achieved from the tag and the accuracy of sequencing the DNA was amplified. Exist.   If PCR amplification of the oligonucleotide identification tag is used, "PCR We often encounter "product contamination", which means that the products of a PCR reaction are the same PCR The following PCR reaction mix designed to amplify other tags with immer binding sites: It is caused by polluting the compound. Introducing instability into the product sequence, and The next reaction may be treated to destroy any potential contaminants introduced from the reaction. This can prevent this problem. A specific example of this strategy is Are sold by PECI and Life Technology), dUMP Introduced in the product. Add each new PCR reaction to uracil-N-glycosidase Treatment with lysate degrades any dU-containing DNA that may be present and amplifies contaminants. prevent. Template DNA that does not contain dU (dT only) is unaffected. Rice cake Of course, glycosidases are removed or inactivated before amplification begins.   Some of the tags mentioned above for peptide synthesis are said to contain only pyrimidines. It has unusual characteristics. This is due to the uracil glycosidase strategy (Perkin  Elmer Cetus Instruments (PECI) Catalog, Alameda (1991), which is hereby incorporated by reference. Only part of the chains (which contain T (or U)) manufactured by Act on. However, dUMP is introduced into the complementary, purine-only strand. , The purine chain is acid depurinated and scaffold alkali-mediated. Very sensitive to cutting. The combination of these treatments results in product contamination. The problem of can be greatly reduced. Another approach to prevent the introduction of contamination is the restriction site (EarI can be used for the polypyrimidine tag), oligonucleosides Measures to be taken prior to the introduction into the CidTag and the amplification reaction suspected of being contaminated with the Tag Including digestion with restriction enzymes. If the tag to be amplified is cleaved by the enzyme This method is performed only in the absence of a single-stranded oligonucleotide tag. Is the case.   Single-stranded templates are usually produced for sequencing of amplified DNA. Is desired. This manufacturing is accomplished by any of several means You. One such means is asymmetric PCR, where an excess of one In order for the Limer to amplify one strand to a level 10-100 times higher than the other Used (eg, US Pat. No. 5,065,654, which is incorporated by reference) Part of the specification). Other means for providing a single-stranded template are: One of the primers was biotinylated and absorbed onto immobilized streptavidin. Depending on the clothes Purification or removal of the resulting chains (Pierce Immunotechnology Catalog and Handbook,  1991, which are incorporated herein by reference). Ma Alternatively, RNA transcripts from RNA polymerase promoters (one of the strands (Expressing only) and sequencing transcripts using reverse transcriptase (Somm er et al., Chapter 25,PCR Protocol: A Guide to Methods and Applications, I mentioned above Which is incorporated herein by reference). If the tag is Pyrimidine Nucle If it consists only of octides, then all purine chains are treated with acid / base. Can be removed, leaving the pyrimidine chain for sequencing.   Use separate sequencing primers for each step-specific oligonucleotide This requires a separate, conventional sequencing reaction for each step-specific primer. Request. The use of differently labeled primers is the same from a single solid support. Constant tags sequenced in a single reaction and run on gel in a single lane set (2 lanes if only polypyrimidine was used) , 4 lanes if 4 different bases were used). Currently suitable for this purpose Distinguishable fluorophore-labeled primers are commercially available (ABI catalysts). Logs, which are part of this specification by reference). Currently on the market A set of chemiluminescent labels can also be used (Bronstein et al.,BioTechniqu es  8: 310-314 (1990), This is incorporated herein by reference).   The amplified product can be easily sequenced or otherwise identified and Peptide or other molecule attached to an oligonucleotide tag Decipher the identification of. For this purpose, various sequencing methods (which are sequence-specific (Including sequencing by probe hybridization) obtain. DNA sequencing enzymes that can be used in the present invention includeTaq  DNA poly Melase,E. FIG. coli  DNA polymerase I (or Klenow fragment) , T7 polymerase, Sequenase ™ and Sequenase II ™ ( Modified T7 DNA polymerase),Bst  DNA polymerase and reverse transcriptase (From AMV, MMLV, RSV, etc.,USB Enzymes for DNA Sequencing,  U.S. Biochemical Corp, 1991, Cleveland OH, by reference (Which is part of this specification). The sequence of the oligonucleotide tag is also high It can be identified by real DNA hybridization techniques. For this purpose Therefore, very large scale immobilized polymer synthesis using oligonucleotides May be useful (see PCT Patent Publication Nos. 92/10587 and 92/10588, see Are incorporated herein by reference).   The choice of tag is whether the tag is an oligonucleotide or other molecular construct. However, the nature of the molecules that make up the library and their properties as described in the previous chapter Molecule synthesized Depends on the method used.   Synthetic chemistry is not compatible with reagents and reaction conditions that are incompatible with the oligonucleotide tags described above. If it involves use, it may be desirable to use an alternative tagging method. Yes. Therefore, the method for synthesizing the tagged molecular library of the present invention also , Can be solved separately by various methods, eg chromatographic methods, The idea is to use a hydrocarbon-tagged molecule that is biologically inert.   The use of such inactive hydrocarbon tags in molecular libraries has been reported. I have. Michael H.J. Ohlmeyer et al.Proc. Nat'l. Acad. Sci. 90: 10922-26 (1993 Dec.), and PCT application number WO 94/08051, both for all purposes. Incorporated herein by reference).   The described tags use the binary code mechanism described herein. Especially , The binary code is to be added in the synthesis, each chemical building block, eg For example, it is assigned to an amino acid. The length of the code depends on the build block to be added. It can depend on the total number of locks. For example, only seven full building blocks are added In some cases, a 3-bit binary code may be used. This is seven separate Specific code, 001-111, is assigned to one of the seven building blocks. , For example, lysine = 001. The number of building blocks If larger, use a larger code, such as the one described herein. For example, an 8-bit binary code can be used.   Each is different from the others when using chromatographic methods. Many tags with tags are prepared. If a particular tag exists, it Represents a "1" at each place in the final tag code for a given synthesized molecule. Therefore, the molecule has four building blocks and is coded in a three-bit code system. If so, there are 12 possible code digits, each building block. Has 3 bits. For each step in the synthesis, the solid on which the molecule is to be synthesized The support is tagged so that it can be attached to the building block as well as the attached building block. The steps performed are also shown. For example, the presence of tag No. 1 or "T1" is added in the first step. For the added building block, there is a "1" in the first place of the binary tag code. It indicates that it exists. Similarly, the existence of T7 is the 7th place of all codes. Indicates that there is a "1" in the place, which also , Corresponding to the first location of the third added building block. Therefore, The building block to which code 111 is assigned, if added at location 1, It is encoded by the presence of tags T1, T2 and T3. Or, if step 2 , The same building block is added by the presence of T4, T5 and T6. Encoded.   The specific hydrocarbon tag reported by Ohlmeyer has the structure: (Where n is 1 to 10) Different lengths of hydrocarbon chains and different halogenated groups can be analyzed by chromatographic methods. Allows for physical separation and analysis of tags, especially by electron capture gas chromatography Noh.   However, detection of these hydrocarbon tags is limited by the sensitivity of the detection method. Is done. Therefore, a larger amount of tags should not be used to ensure tag detection. I have to. This requires larger size beads and therefore It reduces the size of the total library that can be made and also maximizes the tag to solid support. Increases the increased time to ensure coupling. Finally, a larger More hydrocarbon tags are added to the solid support when the offspring are screened Is done. For example, too many tags The binding of large amounts of hydrocarbons onto a solid support for large synthetic compounds with In a continuous synthesis and / or screening of compounds on a solid support It seems to have an adverse effect. This is due to steric, hydrophobic or ionic interactions. It is.   Detectability, tagging time and hindrance to synthesis associated with these hydrocarbon tags The problem of can be avoided using the method of the present invention. In particular, the invention is in one embodiment , A method of tagging with a hydrocarbon tag is provided. Tags such as, instead of the detectable electrophores described in Ohlmeyer et al. It has a “molecular hook”. This “molecular hook” is herein amplifiable , Is defined as a functional group on the tag that allows the attachment of a detectable group, Allows detection of smaller amounts of tags on the body support. Hooks generally amplified Possible, forms a covalent bond with a detectable group or has a high affinity for a part of said group With stable functional groups or molecules. An example of such a hook is, for example, A treptoavidin-linked, amplifiable, detectable group to which a detectable and detectable group can be attached. Includes complement with otin or highly associated peptides. Such highly associated peptides are Generally, each comprises a complementary pair of peptides having a high affinity for the other. Therefore, one Complement represents one peptide of such a pair. Highly associated peptides are generally , Serial No. 08/321933, US patent application (filed October 12, 1994) It is listed in the US Permission application Serial No. 08/067387 Specification (filed on May 24, 1993) partially continued Wishes, which are incorporated herein by reference. Or this frame The lock contains a protected activatable group which is an amplifiable and detectable group. Can be activated to covalently attach the tag to the tag. Light protected and activated The groups are particularly useful for this application. Activated groups are generally known in the art. Well known, and carboxyl, hydroxyl, amino, thiol And other groups. These groups are photolabile protecting groups such as PCT application WO 93/2 2680 (which is incorporated by reference in its entirety) Can be protected with. The resulting groups can be activated by light.   In addition to the above, the molecular hook can include a polyfunctional group. For example, the molecular hook May contain two or more different functional groups that can be linked to two separate ones. this For example, in other solid supports, such as in microtiter plates for detection purposes. It may be the case when it is desired to reconnect the tag to the reaction well. The first functional group is , Can be used to selectively attach complementary groups on a solid support. Once Once attached, the second functional group provides for selective attachment of the amplifiable, detectable group. Can be used. Such hooks generally include functional groups described herein, Or, it may include combinations of other groups that can be selectively attached to other such groups. As an example, such a hook is a hydrocarbon tag Can include both biotin and digoquin, orthogonally linked to. Once separated Then, these tags are labeled with one of the functional groups on the tag, such as anti-digokin antibody. Contact with a solid support, such as a microtiter well, coated with a bondable group Made and bound to it. After repeated washing steps, the solid support , A second functional group, eg, a strand linked to an oligonucleotide as described above. Contacted with an oligonucleotide linked to a group capable of binding to leptavidin You. The bound oligonucleotide is then detected as described above. . The synthesis of the hydrocarbon tag of the present invention is performed by methods well known in the art. Can be done. For example, March, Advanced Organic Chemistry (Jhon Wiley & Son s, 3rd edition, 1985), Larock, Comprehensive Organic Transformations (VCH Publ See ishers, 1989).   The inert hydrocarbon tag of the present invention provides more sensitive detection of hydrocarbon tags Therefore, the amount of a particular tag on a solid support can be reduced without affecting its detectability. Can be reduced. In addition, by reducing the amount of tags on the solid support, the tag on the support is reduced. The time required to connect the links can be reduced.   Tags useful in the present invention generally include various hydrocarbon regions and molecular hooks. In particular, such tags include cleavable linkers that attach the tag to a solid support, Described molecular hooks, and various lengths that connect the molecular hooks to the linker Including hydrocarbon chains. Various tags allow their physical separation and detection Thus, they have different lengths of hydrocarbon chains, or different molecular hooks.   Tags having the following general structure are preferred: (Where n is 1 to 10 or more, X is a cleavable linker, And R is a molecular hook). Preferred molecular hooks are, for example, biotin, Highly associated peptides, and activatable groups such as photoactivatable groups, or their Including combinations.   Examples of cleavable linkers useful in the present invention are, for example, US Patent Application Serial No. No. 08/265090 specification (filed on June 23, 1994) that can be cut by light. , Which is hereby incorporated by reference for all purposes. It is a department. Examples of such light-cleavable linkers have the following structures including:   Following synthesis and tagging, the tag is labeled, for example by photolysis of the linker, Removed from solid support. Then the tag is a way to keep the tag's separation pattern, eg For example, HPLC with fraction collection, or other chromatography -Separated from each other using methods such as gel or capillary electrophoresis. Ta Are generally present in amounts that cannot be detected by conventional means, such as absorption, They provide a way to enable subsequent detection and comparison with their separation patterns. Must be separated. As an example, the tag cleaved from the solid support is Separated on an HPLC column and collected in a fraction collector. If the tag is finally detected as described in further detail below, the tag The fraction indicating the presence of all tags used in tagging / synthesis With respect to known elution profiles or separation patterns.   The separated tags are then immobilized according to their separation pattern. This Immobilization, such as, is for separation using gels that spot individual fractions. Blot or immobilized on reaction wells, ie microtiter plates It can take the form of   Once immobilized, the tag is "hooked" into an amplifiable, detectable group. Can be Amplifiable / detectable groups are generally or can be amplified. A compound or structure that produces a signal that Particularly useful amplifiable and detectable The functional group is an oligonucleotide sequence. Hooking of amplifiable and detectable groups Can take various forms. For example, if the hook contains a biotin group, the oligo The nucleotide is linked to streptavidin which binds tightly to biotin sell. Alternatively, streptavidin is added in an intermediate step and then biotin The addition of derivatized oligonucleotides can be performed. In another embodiment, the tag is high. It may include the complement of associated peptides. In this case, the oligonucleotide is the peptide It can be linked to other complements to the tag and bind tightly to the tag. Also other implementations In an embodiment, the tag is a photolabile protecting group, such as PCT application WO 93/2. Protecting groups described in JP 2680, which has already been incorporated by reference. If it contains an activated group that is protected by Once activated by photolysis of certain groups, the oligonucleotide is Can be linked to the tag by methods well known in. In this case, if the light is unstable When a protecting group is used, a photolabile protecting group having various photolytic properties is used. It may be desirable to select from photocleavable linkers. This is the molecular hook Allows the selective cleavage of tags from a solid support without activation You.   Once the oligonucleotide sequence is hooked to the tag, the oligonucleotide The sequence can be amplified using the PCR techniques described herein. Immobilization If it is a tiling format, the amplification will retain and increase local concentrations. Avoid the spread of widened oligonucleotides, resulting in their detection and separation patterns. Must be done so as to allow comparison with This is an amplification reaction Can be achieved, for example, by performing in a gel overlay of the blot.   Detection of the hooked oligonucleotide, and thus the tag, amplifies the label. Have been introduced into the oligonucleotide, or such sequences are known. In some cases it can be done by examining the amplified oligonucleotide sequence . Tag detection is correlated with known separation patterns for tags. Then The tags so identified are incorporated into the entire tag code for the synthesized molecule. It is. For example purposes only, if HPLC separation, hooking, amplification and probe Following Bing, if Fraction # 17 indicates the presence of tags, this means Fraction # 17 Correlated with tags known to separate in 17. If, for example, this If the group is # 5, then for molecules synthesized on a particular support, 1'can be assigned to location # 5 of all binary code. Described in this specification The code schemes provided are for illustrative purposes, and those skilled in the art will appreciate that the various code schemes are It will be appreciated that it is applicable to the method of the invention.   Those of skill in the art will also recognize that the code indicating the sequence of the synthesized molecule is a single It will be appreciated that inclusion in the mar sequence is not required . Instead In addition, the code depends on the presence or absence of individual, distinct tags on the solid support. Can be embodied. These tags could potentially be serially linked to each other. However, one of ordinary skill in the art would have each different tag individually attached to the support. You will recognize the benefits. In particular, only one coupling chemistry is optional or It can be required for all tagging steps. In addition, tags with different reactive groups Complex protocols of protection / deprotection reactions to protect can be avoided. V. Synthesis method   The method of the present invention is a synthetic process performed sequentially to produce a wide variety of compounds. It can be applied to any set of scientific reactions. The present invention is typically a chemical construction block. The present invention, although more typically described using monomer building blocks The general nature of should be understood. The majority of synthetic chemical reactions are typical It proceeds quite differently from the mer coupling reaction, and the typical organic chemical reaction is To give different yields and multiple products such as regio and stereoisomeric structures. Bring a creation. The present invention identifies useful products of such a series of chemical reactions. Can be used to determine Because the tag clearly identifies the structure of the reaction product Instead of encoding, the present invention is designed to encode the protocol of compound synthesis. Because you can do the law.   However, in order to simplify the discussion, the present invention is described as a monomer coupling process. This is the most easily understood series. Various couplings of the method of the invention Reactions can be carried out at different times and in different reaction vessels, thus allowing very different capacities. Even building blocks with pulling properties, such as monomers, in the library Can be used to assemble compounds of interest. The present invention comprises a solid support Building blocks and identification tags, either simultaneously or sequentially (building blocks then tags, or Tag and then in any of the building blocks) exposure The arch can provide one additional flexibility with respect to coupling chemistry. Anyways However, the preferred configuration for carrying out the coupling reaction is such that various coupling reactions are It is an arrangement done in rows.   After each parallel series of coupling steps, the library oligomers -Or other compounds are pooled on a solid support on which the Due to the pulling process, they are mixed prior to their redistribution into individual containers. This mixture The combining step comprises the separate members of each library on a separate solid support. Is to produce a huge library of compounds. If each synthesis step However, if it has a high coupling efficiency, then it can be loaded onto a single solid support. Qualitatively all compounds have the same structure, or if the compounds are oligomeric If present, have the same monomer sequence. The structure and sequence are synthetic Finally, the synthetic route (monomer type and sequence, for any given solid support, Or other building block coupling reaction). Of oligomers Larger lengths are typically less than about 20, usually 3 to 15 monomers long. However, in some cases a length of 8-12 monomers (residues) is preferred.   Given a number of tags and building blocks suitable for use with the present invention, Many chemical methods by which the chemical libraries of the invention can be prepared Exists. However, each coupling step is either a tag or an oligomer. But does not produce unacceptable levels of unwanted reactions and is already present on the support It must be ensured not to destroy the tag or oligomer that does. One In embodiments, only the desired reactions are of two different or "orthogonal" types. Chemically reactive groups for attachment of tags and oligomers protected with other protecting groups Is guaranteed to occur by using a solid support having Solid support Is exposed to a first deprotecting reagent or activator and the first type of protecting group is, for example, , Are excluded from chemically reactive groups that function as oligomer synthesis sites. First monomer After reaction with the solid phase and after the optional blocking step, the solid support is Exposure to a second activator that removes protecting groups of Expose chemically reactive groups that function as sites. Then the tags are concatenated, and this These steps are typically repeated 1 to about 20 times. You. A. Peptide library with oligonucleotide tag   In one important embodiment, the present invention provides a giant library of diverse peptides. Regarding the composition of Lee. Many other compounds and oligomers have been developed by the method (Gait,Oligon Nucleotide Synthesis: A Practical Approach,  IRL Press, Oxford (1984); Frie sen and Danishefsky, 11989,J. Amer. Chem. Soc. 111: 6656; and Paulsen, 1986. ,Angew. Chem. Int. Ed. Engl. twenty five: 212, these are quoted (Which is part of the textbook), while solid-state synthesis techniques for peptides are Particularly important and well known (Merrifield, 1963,J. Am. Chem. Soc. 85: 2 149-2154, which is incorporated herein by reference), and Pep Tide libraries are very useful for various purposes. Merrifield method smell For example, the amino acid is covalently bound to a support made of an insoluble polymer . Other amino acids having an alpha-amino protecting group have covalently attached amino acids. Reacts with mino acids to form dipeptides. Protecting groups removed, and alpha protection A third amino acid having a group is added to the dipeptide. This procedure has the desired length And so on until a peptide of sequence is obtained. Protecting groups known to those skilled in the art are pseudo To prevent coupling (The Peptides, Vols. 1 & 3 (eds. Gross, E., and J . Meinhofer, Academic Press, Orlando (1979 & 1981), which are hereby incorporated by reference), or cups. It can be used to control the ring. Light-labile, base-labile and acid Are labile protecting groups, and combinations thereof, all serve various purposes in the present invention. Can be used for   In addition, the L and D forms of the amino acid are used in the peptide synthesis described herein. Can be put. Using D amino acids is described in US Patent Application Serial No. 08/3094. No. 51 (filed Sep. 21, 1994, which is hereby incorporated by reference) "Retro-inverso peptides". Is useful in the synthesis of Such retro-inverso peptides have the same identity. The opposite stereochemistry from a peptide containing a mino acid sequence but synthesized with L-amino acids. Have chemistry.   The present invention was used to produce and screen peptide libraries. If selected, the selected tag is a nucleic acid. Various compatibility for peptide synthesis Chemistry and sequential attachment of oligonucleotide identification tags Exists. However, during peptide synthesis, the integrity of the oligonucleotide tag In order to avoid degradation of the synthesized tags or oligomers, It may be necessary to use a combination of protecting groups and / or synthetic nucleotides Yes. Generally, the polypyrimidine oligonucleotide tag is a natural purine nucleoside. Unlike peptide tags containing tide, typical peptide synthesis Although relatively stable under conditions, polypyrimidine nucleotide tags It may be difficult to process by amplification by PCR. To achieve the desired amplification efficiency , Purine bases, or analogs such as 7-deaza-deoxyadenosine and 7 -Deaza-deoxyguanosine (these are peptide coupling (and deprotected ) Tested for ability to withstand conditions) may need to be incorporated into the tag Not. For purposes of the present invention, the tag is optionally 10-90%, more preferably 35-50%, most preferably 33-35% of purines or purine analogues It may include leotide. Oligonucleotide tags can be used for purification and hybridization. Biotin or other recording group is optionally used to facilitate amplification, amplification or detection. Can be incorporated (Pierce Immuno Technology Catalog and Handbook, See 1991, Which is incorporated herein by reference).   Therefore, a peptide library provided with the oligonucleotide tag of the present invention (1) having suitable functional groups in the choice of chemistry used to produce Selection of solid support, (2) selection of amino acid coupling chemistry, (3) oligonuc Selection of Rheotide Tag Coupling Chemistry, (4) Various Tags, Monomers and Oligos Of protecting groups for mers, and (5) selection of deprotection, and some embodiments In some cases, the choice of cleavage chemistry (either for the tag or the peptide) There must be. Those skilled in the art will appreciate that some applications do not give the same results as others. As well as on We recognize that not all of the choices need be made in all cases. Was For example, one or more protecting groups may not be required for all applications. However However, in the general case, each of these choices is important.   Peptide library with coupling chemistry and oligonucleotide tag To consider the factors involved in the selection of protecting groups for the synthesis of Li, commercially available Fmoc The protected amino acids were ligated using standard Merrifield chemistry and the oligo Nucleotide tags can be linked using standard phosphoramidite chemistry Think. The procedure can be considered as having the following steps: (1) Attaching to beads Removing the amino-terminal Fmoc protecting group from the modified linker or peptide, (2 ) An Fmoc-protected (side chain may be similarly protected) amino acid was prepared in step (1). Coupling to the produced free amino acid, (3) unreacted free amino Optional acid capping step, (4) bead to which the nucleotide tag is attached Removal of the DMT protecting group from the hydroxyl group on the tag or tag, (5) 5'-D MT protecting groups, and protecting groups and nucleosides on phosphates and exocyclic amines of beads Coupling tide phosphoramidite, (6) optionally, any unreacted Capping free hydroxyl groups of (7) oligonucleotide tag Oxidation process of phospholipids and (8) Deprotection of peptide and oligonucleotide tags Process. Each of these steps is discussed below.   (1) Amino-terminal Fmoc protection from linker or peptide attached to beads Removal of protecting groups is required prior to subsequent attachment of amino acid monomers. Typically , Treatment with 30% piperidine in DMF for about 1 hour allowed this deprotection (step 8 is also used to achieve the above), but one aspect of the present invention is Piperidine for the synthesis of nucleotide-tagged peptide libraries Use of reduced concentrations or reduced deprotection times. Piperidine is methyl Can cause deprotection of triester-protected oligonucleotide tags , And O-methyl phosphate protecting groups are susceptible to piperidine cleavage. It has a greater base stability than the known standard beta-cyanoethyl groups. Preferred The Fmoc deprotection conditions of the present invention are 5-15%, preferably 10% piperidine. 5 to 60 minutes, preferably 10 to 20 minutes, and 1 to 15 to 30% piperidine 5 to 30 minutes. Another known process that results in Fmoc removal is DBU (1,8- Treatment with diazabicyclo [5.4.0] undec-7-ene (eg 5% DB U for 5 minutes). However, Palom et al.Tetr. Lett. 34: 2195-2198 (This Which are part of this specification by reference) Suggest that it may result in methylation of thymidine at N-3. Mediated by DBU Fmoc removal, which may be effective in some applications, is The possibility is also recognized.   (2) The Fmoc-protected amino acid is used as a free amino acid on beads or peptides. Coupling to groups can be accomplished using standard BOP coupling chemistry (The Peptides, , See). Typically consists of 1: 1 DMF / DCM A mixture of Fmoc-protected amino acids (110 mM), HBTU (1 00 mM), HOBt (100 mM) and DIEA (300 mM) Used to perform amino acid coupling. Other activation chemistries in this example Can be applied, for example in the replacement of HBTU / HOBt with HATU is there. However, in one embodiment of the invention, the reaction mixture is 3: 1. 55 mM Fmoc-protected amino acid in a solution consisting of DMF / DCM, 5 Composed of 0 mM HBTU and 150 mM DIEA, this embodiment It is preferred for use with devices where the recovery bottle may be limited. The side chains are the same Can be protected like Fmoc /^tBu protection is a commercially available building block Preferred for most purposes because of its sex. Other useful amino acids with side chain protection Building blocks are Arg (Pmc), Gln (Trt), His (Trt), A sn (Trt), Asp (O^tBu), Glu (O^tBu) and Lys (^tBoc ), And amino acids with side chain protection provided by a photolabile protecting group. No.   (3) Optional capping of unreacted free amino groups is performed with acetic anhydride and 1- Treatment with methylimidazole, Alternatively, it can be achieved by other methods known in the art.   (4) beads on which the nucleotide tag is to be attached or a hydroxy on the tag. Removal of the DMT protecting group from the sil group is accomplished by trichloroacetic acid (TCA), ie CH₂C l₂Can be achieved by treatment with 1% TCA in. If phosphate and And nucleotides (ie, deoxycytidine, 7-deaza-deoxyadenosine and And 7-deaza-deoxyguanosine) using an acid labile protecting group on the exocyclic amine , Then those groups are used in the 5'-O-detritylation. It should be sufficiently resistant to TCA (typically 1-3%).   (5) Exocyclic amide on the phosphate oxygen and on the base of the oligonucleotide tag. The need for protecting groups on the 5'-DMT Coupling of a nucleotide phosphoramidite with a protecting group is accomplished using standard phosphor It can be achieved using foramidite chemistry. For light-labile protecting groups for nucleic acids Regarding PCT Patent Publication No. WO 92/100092, and Baldwin et al., 1990,Tetr .  Lett. 46: 6879-6884 (these are incorporated herein by reference) ). As noted above, suitable phosphate protecting groups are O-methyl and beta-sia. Containing an ethyl group, but containing O-allyl and / or N-allyloxycarbonyl groups (immediately Then, for incorporation of 3- (allyl N, N'-diisopropyl) phosphoramidite According to Can be used to protect sulphate oxygen and exocyclic amines of nucleotide bases (Hayakawa et al., 1990,J. Amer. Chem. Soc.112: 1691-1696, these are quotes Is part of this description). The allyl protecting group is tris (dibenz (Rydenacetone) dipalladium-chloroform complex, triphenylphosphine and And THF containing n-butylamine / formic acid, followed by THF washing, aqueous N 2 , N-diethyldithiocarbamate sodium washing and water washing Can be excluded. Phosphoramidite coupling is performed with reagents such as 1H-teto. Razole, 4-nitrophenyltetrazole, pyridinium hydrochloride / a Mediated with midazole. The latter phosphoramidite activator is a peptide The pseudoreaction with nitrogen on the cathode or oligonucleotide occurs at low levels. At a price, it results in selective 5'-O-phosphorylation (Gryaznov and Letsinger, 19 92,Nucleic Acids Research 201879-1882, which is quoted in the book Part of the specification).   (6) The optional capping of unreacted free hydroxyl groups is carried out with acetic anhydride and And treatment with 1-methyltetrazole or acetic anhydride / lutidine / DMAP Can be achieved by treatment with   (7) Oxidation of the oligonucleotide tag phosphorous is treated with iodine and pyridine. Or by I₂, Collidine, H₂This can be achieved by treatment with MeCN in O. Or phosphorus Oxidizer, which is mild for oxidation in^tBy using BuOOH, the amino acid , That is, the oxidation of methionine, tryptophan and histidine can be minimized (Ha yakawa et al., 1990,Tert. Lett. 27: 4191-4194, this is a book by quoting Part of the specification).   Deprotection of peptide and oligonucleotide tags can be achieved with 1% T in dichloromethane. CA, then thiophenol / NEt_Three/ Use dioxane (1: 2: 2) Sequentially, using ethylenediamine / EtOH (1: 1) at 55 ° C Upon treatment, the tag is stripped of protecting groups and then trifluoroacetic acid. (95: 5 TFA / water, combined with cation scavenger) is an acid-labile amino Used to remove acid protecting groups. Purinuk against strong acids (eg TFA) The instability of leotide is due to the purine nucleotide analog phosphoramidite, 7 -Deaza-2'-deoxyadenosine and 7-deaza-2'-deoxyguanosine Can be avoided by usingBioTechniques Four: 28-432, And Scheit,Nucleotide Analogs: Synthetic and Biological Function pp.64- 65 (John Wiley and Sons, New York), both of which are incorporated by reference in the book. Part of the specification).   The next chapter is about the synthesis of peptide libraries tagged with oligonucleotides. One preferred embodiment for will be described. B. Improved combination of oligonucleotide-tagged peptide libraries Method   Practical bead-based oligonucleotide-encoded peptide library The establishment of Brary requires meeting several key technical criteria. these Are (i) mutually compatible for the parallel assembly of peptides and oligonucleotides. Of chemistry that is sexual, (ii) selection of bead materials with suitable physical characteristics, (ii i) simple isolation of small beads bearing the ligand that binds to the target receptor, and (Iv) Sequence of template tag DNA from, for example, PCR amplification and single beads Includes successful decoding of tags from single beads by determination. The present invention provides such To provide improved synthetic methods for libraries, and to As explained in Example 1, this means that polystyrene beads with a diameter of 10 μm Single-stranded oligonucleosides to encode combinatorial peptide synthesis. It showed how to use Cidtag.   In this improved method, peptides and nucleotides are assembled in parallel. Each bead contains a unique peptide sequence and unique oligonucleotide identification Alter the composition to carry multiple copies of both of the groups. Oligonucleotides The common 5'and 3'-PCR priming sites are shared, therefore the beads are Can serve as a template for R. To explain the method, about 8.2 × 10^FiveA synthetic library of encoded hepta-peptides of Inorphin B monoclonal antibody D32.39 (Cull et al., 1992,Proc. Natl. Acad.  Sci. USA 89: 1865-1869, which is hereby incorporated by reference. Fluorescent activity to select individual beads that bind strongly to the antibody for binding Screening is performed using an activated cell sorting (FACS) device. Single out Of the peptide ligand after PCR amplification of the oligonucleotide tag on the To determine the identity, the DNA was sequenced as described in more detail below. Is determined.   One important aspect of the method of the present invention, which is described in further detail in Example 1 below. (Listed) is the solid support chosen for the synthesis of peptides and tags. You. The solid support, i.e., the beads with a diameter of 10 [mu] m, is a macroporous styrene-divinyl chloride. Formed from rubenzene copolymer and induced with a dodecylamine linker. It is preferably a conductor. The amino groups loaded on these beads are , Exhaustive acylation with Fmoc-glycine, followed by pipetting of the Fmoc group. Spectrophotometric determination of lysine cleavage and liberated piperidine-dibenzofulvene adducts Quantity (e₃₀₂= 7800 l mol^-1cm^-1) Is estimated to be about 100 μmol / g Was. 5 × 10⁹With beads / g, this is a maximum of about 20 fmol / bead. Corresponds to a large peptide load. Of appropriately protected amino acids and omega hydroxy acids Mixed Acylation of beads with a compound from which peptide and nucleotide chains It provides orthogonally different amino and hydroxyl groups that can be extended in each case. Bi The average stoichiometry of peptide to oligonucleotide per dose is Controlled by changing the ratio of amino and hydroxyl acids linked to the beads Will be done (see below). Cleavable with trifluoroacetic acid using standard Fmoc chemistry Knorr Linker (Bernatowicz et al., 1989,Tetr. Let. 30: 4645-4648, this is a pull Test beads on these beads, which are incorporated herein by reference). It was found that the peptide synthesis (pentamer-12 mer) proceeds with high accuracy, and this high accuracy As determined by HPLC analysis of the crude truncated peptide carboxamide, No distinction is made from the synthesis carried out on customary peptide synthetic resins.   The parallel synthetic strategies are orthogonal to each other, and each of the polymer chains has a second Amino acids and nucleosides that are stable to the reagents used in synthesis and deprotection Requires the use of a set of protecting groups on the Leotide building block. As a rule, seeds Although different protection / deprotection mechanisms can be used (as described above), the peptide structure Fmoc / on the building block^tBu protection is preferred. Because of the wide range of commercially available heavens Natural and unnatural amino acids are protected in this way. However ,^tThe Bu-based peptide side chain protecting group is a strong acid (typically Treatment with 2'-deoxyadenosine (dA) or Of oligonucleotides containing either 2'-deoxyguanosine (dG) Conditions that can lead to rapid depurination (Capon, 1969,Chem. Rev .  69: 407-409, which are incorporated herein by reference. This problem is due to the 7-description in the template oligonucleotide tag instead of dA. Aza-2'-deoxyadenosine (c⁷by using dA) You. The glycoside linkage of deazapurine nucleosides resists acid-catalyzed hydrolysis. Sex (Scheit, 1980,Nucleotide Analogs:Synthesis and Biological Fun ction (John Wiley and Sons, New York) pp. 64-65, by reference And part of this specification) and oligonucleotides containing these monomers. Is accurately replicated by the temperature-stable polymer used in PCR. (McConlogue et al., 1988,Nucl. Acid.,Res. 16: 9869, and Barr et al., 1986,BioT echniques  Four: 28-432, which are hereby incorporated by reference) . Acid-resistant guanosine analogs can also be incorporated into the template DNA.   5'-O-dimethoxytrityl 2'-deoxynucleoside 3 '-(O-methyl Ru-N. N'-diisopropyl) phosphoramidite is used in all parallel syntheses I was. In a DNA synthesis protocol, a nucleotide phosphite intermediate Reagents used to convert phosphotriesters (I₂/ Collidine / Acetonite Ril) is easily oxidized Residue, any of Trp and Met, or any other protected amino acid Was not found to have a negative effect on. From a growing oligonucleotide chain Complete removal of the 5'-O-DMT group of 1% trichloroacetic acid in dichloromethane Was achieved in about 40 seconds using (TCA), while^tBu ether Fmoc / excluding derivatives^tAll acid labile sides conventionally used in Bu chemistry The chain protecting group is inert to treatment with 1% TCA for 1 hour. Fmoc-Ty r (O-Bz) demonstrates a suitable alternative in the synthesis of tyrosine-containing peptides, O-benzoyl ester is alpha-N-Fmoc protected in peptide synthesis The group resists both the TCA and piperidine used for removal. A Quantitative deprotection of rufa amino acid residues was determined by piperidine / DMF (10% v / v ) For 5 to 10 minutes and also protected polynucleosides Partial demethylation of the tide phosphotriester resulted (t_1/2  About 45 minutes). versus Experiments show that the phosphodiester species obtained during the subsequent nucleotide chain extension , Aberrant phosphite reversal by the final oligonucleotide deprotection step (Lehmann et al., 1989,Nucl. Acids Res. 17: 2379-2390 visit , Which is incorporated herein by reference). Smell of completion of parallel synthesis For example, DNA is treated with thiophenolate (phosphate O-demethylation). ) Then treatment with ethanolic ethylenediamine '(protected cyt Deprotection by debenzoylation of gin and 7-deaza-adenine residues) Is done. These mild, anhydrous aminolysis conditions use TFA under standard conditions. And deblocked protected peptide sequences are not adversely affected (Juby et al., 1991,Tetr.  Lett. Three: 79-882, which is incorporated herein by reference) .   The carboxy-terminal region of the opioid peptide dynorphin B (YGGFLRR QFKVVT) (SEQ ID NO: 1) is an anti-dynorphin B monoclonal antibody D3 It has already been shown to represent the epitope of 2.39 (see Cull et al., Supra). Yes The soluble heptapeptide, RQFKVVT (SEQ ID NO: 2), has a high affinity (Kd is approximately Binds to D32.39 at 1 nM). 69 base oligodeo of this peptide A parallel synthesis of xynucleotides is an acid-cleavable Fmoc-protected carboxa. Made on orthogonally distinct beads bearing a Knorr linker. After the addition of the first 20 nucleotides, the beads were treated with piperidine / DMF , And the first peptide residue (Fmoc-Thr (^tBu) -OH) is a free Connected to Min. The beads were then subjected to two cycles of phosphoramidite chemistry and then Was subjected to the coupling of the amino acid (Fmoc-Val-OH). This procedure Repeat until the heptapeptide sequence and nucleotide coding region are completely created , Then the DNA was extended by an additional 35 nucleotides to allow space for PCR. It provides a sir region and a 5'-priming site. The beads are finally subjected to complete oligonucleotide and then peptide deprotection conditions. The TFA supernatant containing the exposed and cleaved peptide was subjected to reverse phase HPLC. Therefore analyzed. The HPLC results show that the crude peptides from parallel inclusions are the single major Component (eluted with authentic RQFKVVT (SEQ ID NO: 2)), This crude product is then used as a control only peptide where oligonucleotide chemistry does not occur. It was shown to be not significantly different from that produced in the synthesis.   T, dC and c⁷For parallel synthetic chemistry of template DNA containing dA Stability was compared to an analog target containing the standard purine nucleotide dA. Uses the single bead cloning capability of the FACStar Plus cytometer The individual deprotected beads from the two syntheses were then loaded onto a microfuge tube. Oligonucleosides classified into a group and amplified through 45 cycles of PCR Connected to the chid template. A "clean" amplification of the expected size and sequence The product was obtained only from the template containing deazapurine. Therefore, this orientation Gonucleotide integrity is maintained throughout the process of parallel peptide synthesis and is consistent with the single That the template from the beads can be easily amplified and sequenced It has been shown.   823543 (7 attached to 10 μm beads⁷) Different heptape petit The encoded library, designed to contain the Building block, A set using Arg, Gln, Phe, Lys, Val, D-Val and Thr. Constructed by combined synthesis. Alpha-N-Fmoc-Thr (tertiary leave Cyl) -oxybenzotriazole (protected threonine) and succinimidi The 4-O-DMT-oxybutyrate residue was first linked to all beads. To provide orthogonally distinct amino acids and hydroxyl groups for this synthesis. Was. On average, each bead contains 20 molecules of a single peptide per molecule of DNA tag. Responsible for the array. Each amino acid addition constitutes a characteristic dinucleotide unit Encoded by and after 7 cycles of peptide coupling The seeds were collected and DNA synthesis was completed. 35 mg total bead weight (1.7 5 × 10⁸Starting with about 2 of each peptide sequence in the library. It was guaranteed to appear on 00 different beads. Divide the library evenly Microsequencing analysis of one of the It was confirmed that each position in the peptide mixture was stochastically distributed ( Note that L-valine and D-valine are not distinguished by the Edman decomposition method. It should be understood).   Binding of monoclonal antibody D32.39 to control beads and bead library In that case, analysis was performed by flow cytometry. Positive control sequence RQFKV Beads carrying VT (SEQ ID NO: 2) and 69-mer oligonucleotide tags were It was strongly stained by the body, while the blank The beads were not stained. In contrast, less encoded libraries Only the fraction bound to D32.39. 10^FiveEvent analysis is back Represented approximately 2% of the library stained above ground. Importantly, This binding to D32.39 results in a soluble RQFKVVT peptide (SEQ ID NO: 2). Pre-incubation of the monoclonal antibody with It is specific to the mating site. Has fluorescence intensity compared to positive control beads Individual beads from the library were labeled with microbe for tag amplification by PCR. Beads in fluorescent tubes (fluorescent in the top 0.17% of the population) Were collected). The amplification reaction prevents the introduction of soluble products from previous amplifications. In order to include dUTP and uracil DNA glycosidase (Longo et al., 199). 0, Gene 93: 125-128, which is incorporated herein by reference). Nucleotide sequences were obtained from 12 sorted beads. Deduced peptide The sequences are shown in Table 1. Single bead with fluorescence not significantly above background Representative peptide sequences obtained from E. coli are also tabulated for comparison.   The data in Table 1 shows that the preferred recognition sequence of D32.39 is 6 of dynorphin B. Previous work showing that it is localized to the amino acid fragment RQFKVV (See Cull et al., Supra). The positively charged residues arginine and lysine , Strongly favored at the first and fourth positions of the epitope, and phenylalanine It appears exclusively as the third residue of this subject. In the second position, glutamine Is a preferred residue of the library, while the aliphatic b-branched amino acid variant (L-enantiomer only) and threonine clearly have residues 5 and 6, Is preferable. D-Valine seems to be most tolerated in positions outside the matching subject It is. Range of affinity of selected peptides (Kd about 0.3-1400 nM) Binding assay: Design a bivalent primary antibody detection design using a labeled secondary antibody As expected, it was unexpected. Manipulation of valency (eg, directly labeled monovalent (By using a scepter) and the stringency of washing conditions is Improves the ability to isolate only the band. 3. Small molecule synthesis   As mentioned above, it is mainly composed of peptides and other large polymer libraries. Although described above, various aspects of the present invention apply to other chemical syntheses as well. Can be used. In particular, the apparatus and method of the present invention can be used in many synthesis protocols, such as small Various chemical synthesis steps are performed in the synthesis of small molecules. Can be used to Device adapted to synthetic protocol for specific molecule It can be used in chemical synthesis schemes to selectively add reagents.   Furthermore, different synthetic protocols can be performed in parallel, similar to the peptide synthesis described above. Where different reagents are added in different steps to give a solid Generate a diverse library of small molecules on a support. These diverse live Larry is screened for desired characteristics by the methods described herein. Can be done. Furthermore, if a diverse library of such small molecules is desired, , The library is involved in the synthesis of each separate member of the library of molecules. Tags may be added to encode the synthesis steps involved.   Examples of the synthesis of such small molecules on a solid support are described, for example, in thiazolidinediones. None, metathiazolidinone, and their derivatives, which are referred to herein No. 2 of US Pat. Japanese application), which is incorporated herein by reference. D. Method for producing soluble library   For some applications, "bead-free" or "soluble" molecular live A rally is desired. Soluble molecules are tagged and untagged. Both of them have various purposes (measuring the activity of compounds (see Chapter VI.B. (See below) and amplification of tags). Tagged and Producing a non-tagged soluble molecular library and on a solid support Solubilizes tagged and untagged compounds synthesized in There are various ways to do this. Cleavable linkers typically Used.   For example, and as noted in Section II.B, a cleavable linker tagged Cleaves tagged or untagged molecules from beads or other solid supports Can be used to solubilize the molecule of interest. Soluble To produce the tagged molecule, a cleavable linker is attached to the bead or other Attached to a solid support of at least two functional groups, one of which is the molecule of interest. The other one is for synthesizing tags) Will In this way, the molecule and tag are attached to a common linker and synthesized. Which is then attached to a solid support. Once the molecules and tags are synthesized The linker is then cleaved to provide the soluble tagged molecule.   A single, planar solid support is used to synthesize the library And the members are on a very large scale prior to screening. Cleaved from the solid support using immobilized polymer synthesis (VLSIPS ™) technology. Can be refused. U.S. Pat. No. 5,143,854 and PCT Patent Publication No. 92/19982 Reference (these are quoted Is hereby incorporated by reference). In one embodiment, oligonucleosides The sequence of the otide was synthesized on VLSIPS ™ and each oligonucleoside The tide is linked to the piece by a cleavable linker, for example a disulfide bond. Concluded (US Patent Application Serial No. 874849 (filed April 24, 1992)) Reference, which is incorporated herein by reference). Oligonucleotide A tag is a molecule to be tagged, which is typically an oligomer and a A free functional group, such as an amine, for attachment of You. The tag is optionally a pyrimidine base, or pyrimidine and purine analog salt. Only groups can be included. The tag also binds the binding site for amplification, ie the PCR primer. Site, optionally a sequencing primer site, and the oligomer to be tagged. A short sequence that uniquely encodes the monomer sequence of After that, the oligomers A free terminal amino group on the tag or a linker linked to the tag? Then, each oligomer is synthesized so that each oligomer is linked to the tag. tag A collection of oligomers attached with is made into small pieces by cleaving the linker. To produce soluble tagged oligomers that can be released from Can be.   Other advantages can be appreciated by producing soluble molecular libraries. In any bead-based library, the bead size is Assembled Puts a practical limit on the size of the library. For example, a few grams of bee 10⁹Of libraries containing molecules with different tags Can be required to stand. The present invention makes a much larger library Synthesize a tagged molecular library that makes it practically obtainable Provide an improved method for. This improved method provides a means, Thereby, the compound is released from the solid support prior to the mixing step, but each It is attached to a solid support prior to the coupling step.   In this method, tagged molecules are solid-supported by reversible means. Molecules immobilized on the body and tagged from the support during each mixing step of the method Can be released. In one embodiment, the reversible binding is achieved by ultrafiltration membrane (suitable For the membrane, see “Commercial Compatibility Chart” in the Millipore Catalogue. It is inert to various solvents and chemicals used in synthetic methods. Showing a compliant membrane). About 2000-10,000 Daltons Membranes with molecular weight cutoffs (eg Amicon YM5 membranes) are most libraries Suitable for During the coupling process, the molecules of the library are bound by the membrane. Retention, while coupling reagents and other reagents are pulled through the membrane by vacuum suction. Be withdrawn. The vacuum is released so that the molecules are mixed during the mixing process.   In another embodiment of the method, the reversible covalent linkage comprises a coupling step. Meanwhile, it is used to attach the tagged molecule to the support. Suitable reversible Chemical ligation is described in (1) for example, thiolated tagged molecules and N A sulfoester linkage (provided by a hydroxysuccinimidyl support ( This connection is NH₂OH concentration), and (2) Molecules tagged with allylated and 2-pyridyl disulfide supports (eg Disulph provided by thiol sepharose from Sigma) Ligation (this ligation is controlled by DTT (dithiothreitol) concentration Included). VI. Assay method   The use of large combinatorial libraries for ligand development is a powerful and Significantly depends on the availability of compatibility-sensitive assay methodologies. The present invention encodes Provides a number of novel assays for use with selected synthetic molecular libraries , It has a wide variety of uses. As an example, a library like this , Ligands that bind to receptors, such as peptides and nuclei that bind to proteins Recognized by acids, drugs that bind therapeutic target receptors, and antibodies. To identify (natural and synthetic) epitopes, as well as medical, agricultural and medical diagnostics Can be used in assays to identify different compounds in the application You. Given these diverse uses, a wide variety of different assay methods have become Related to. Two important types of assays (some overlap However, it includes bead-based assays and soluble molecule assays.   However, in general, such assays typically include the following steps: . The library is designed so that each different molecule in the library Screened by assaying for binding capacity to. Les The sceptor is contacted with a library of synthetic molecules to allow it to interact with the receptor and assay assay. Binding to any molecule in the library that can bind the receptor under Formed members. The bound molecule then has a tag associated with it. It is identified by investigating. In one embodiment, live under binding conditions The receptor to which the rally is exposed is a mixture of receptors, Each of which is associated with an identification tag that identifies the receptor type, and therefore 2 Tags are examined after the binding assay. A. Bead-based library screening assay   Beads if a particular bead is isolated in a receptor screen Is infinite dilution, micromanipulation, or preferably flow cytometry. Including Lee It can be separated individually by many means. A library of bound ligands Is most efficient in binding assays using soluble labeled receptors To be evaluated. High sensitivity by adopting cell-sized solid support or beads Flow cytometry for accurate receptor binding analysis and easy bead handling Can be used.   Flow cytometry (this is usually fluorescence activated cell sorting or F Referred to as ACS), for the purposes of the present invention, "fluorescence activated molecule sorting" or Should be considered the same as "fluorescence activated bead sorting". cell FA for cloning mammalian cells expressing surface antigens or receptors Those skilled in the CS method can easily perform the assay method of the present invention. In general, These assays are performed on a mixture of beads representing diverse molecules in a molecular library. Binding of the receptor labeled with a fluorescent tag. Unbound or nonspecifically bound After washing away the combined receptors, the beads were sorted using a FACS machine. , And identify and physically isolate into individual beads that exhibit high fluorescence. Methods in Cell Biology, Vol.33 (Darzynkiewicz, Z, and Crissman, H.A. Ed., Academic P ress) and Dangl and Herzenberg, 1982, J. Immunol. Meth. 52: 1-14 (these are Incorporated herein by reference). Once the desired beads are isolated Then the identification of the molecule of interest on the bead (or molecular structure, composition, Or identify the tag to confirm its synthesis status).   Standard FACS equipment is about 10^FourFluorescence analysis of beads (cells) at a rate of events / second And, when operated in single bead cloning mode, The speed is 5 to 10 times slower. Very large libraries (eg 10⁷Bee Affinity-selective prescreening Several modes of ligation preceded isolation of individual beads using a cell-sorter. Can be used. For example, receptor-coated submicron-sized supranormal Magnetic particles were identified from a huge mixed population using magnetic activation sorting Are often used to affinity purify cells of (Miltenyi et al., 1990, Cytometry 11: 231-238, which is incorporated herein by reference). Non In order to always have a high probability of detecting rare binding events, each library Of different compounds must be present on many beads in the library Absent. Of the size of an encoded library constructed from 10 μm particles A practical upper limit is probably 10 given the hundred times redundancy.^Ten-10¹¹On beads 10 compounds synthesized⁸-10⁹Would be a compound of. A larger library , Can be prepared using smaller beads, but conventional cytometry is approximately 1 μm. Detecting or manipulating particles much smaller than the diameter of m is unlikely to succeed. Also Of course, as noted elsewhere in this specification, the present invention provides for the integration of molecular libraries. Success It provides various uses of such small beads in cleaning. for example For example, by using the oligonucleotide tag chain method of the present invention, a library There is no need to use FACS methodology to sort the molecules in.   Nevertheless, the power of FACS devices for the purposes of the present invention should be underestimated. Not. In one assay method of the present invention, a tagged molecular library Are synthesized on fluorescent beads. Beads are smaller than cells and are fluorescent Consists of The library contains cell surface receptors of interest, such as G protein-linked receptors. The sceptor is incubated with a suspension of cells expressing high levels. Of course, the seed For each control, eg, the entire process, use cells that do not express high levels of cell surface proteins. You can also use these controls to identify false positives .   Anyway, cells expressing the receptor represent the ligand for the receptor Can bind to any library member. Fluorescently labeled cells will not fluoresce. From unmatched library beads and unlabeled cells, light scatter, or FACS instruments based on other fluorescent signals from cell nuclei are used to easily distinguish and Can be separated. After sorting, the tags from the beads attached to the cells will Tested to identify specific ligands. Desired depending on the application The cells that express the highest level of receptor are, for example, those of the brightest cells. To maximize the specific binding events. Adjust the binding conditions for Ligands specific for the receptor of interest and other cell surfaces In order to distinguish the ligands specific to the receptor, the target receptor is highly labeled. Associated with beads that bind cells expressing and non-expressing Can be tested.   The method of the present invention is also the smallest bead that current FACS equipment can sort. Sort a library of tagged molecules synthesized on even smaller beads For this purpose, a FACS device can be used. In this way, Encoded synthetic libraries have effector activity on a single conversion pathway Are screened for The synthetic library has several modifications: (1) beads are 1 μm or smaller, and FAC You don't need to be able to sort by S, and in some cases use fairly small beads (B) tag is resistant to the intracellular environment (particularly Is an oligonucleotide that is And (c) the peptide (or other diverse chemical entity) is It is attached to the bead support via a linker that cleaves within. Such a linker External factors that are not harmful to cells, such as linkers that can be cleaved by the application of light, and And a linker that is unstable to the intracellular environment, such as a phosphodiester bond or a disulfide. In some cases, the cleavable linker may include a side-by-side synthesis. Must be stable to the process No.   Library beads are preferably subjected to mechanical processes, such as biolistics. By receptor projection (biolistic projection) be introduced. In some cases, biochemically mediated processes that lead to internalization. Although this process can be used, this route usually leads to undesired intracellular compartments. Result in a cluster (ie, lysosomal localization). Once the beads are inside the cell Then, the peptide or other component of interest is released. 10 μm beads^TenPe If it has the indicated capacity of the peptide (or other) synthetic site, then 1 μm beads of the material were synthesized when the capacity of Do 10⁷Including molecules. If all of the synthesized peptides have a diameter of about 10 μm, When released into one (spherical) cell (volume of about 0.5 pL), then, A free peptide concentration of approximately 30 μM results. This concentration depends on the synthetic density on the beads. Controlled by degree, lower load density means more stringent screening modalities (Ie, screening for more active compounds). Receptor cells Designed to produce a fluorescent signal by activating or inactivating a pathway of interest ing. Individual cells that produce the desired result are selected by a FACS machine and The tags that are still attached to the beads and contained inside the cells Amplified and sequenced to identify compounds.   In another embodiment, large beads are employed and the detectable compound Cleavage of the substrate to produce a fluorescent or other detectable signal Express a receptor that can occur (ie the enzyme beta-galactosidase) It is used to screen a population of cells. Then the beads are cells It is allowed to adhere to the beads, mixed with a population of. If the cell surface If the upper receptors were stimulated by the compounds on the beads, then the Is produced to activate activated cells attached to beads. A basis is provided for classifying non-existent cells. Suitable reagent (ie free label) Maximized selection of high-affinity ligands (with or without receptors) Can be adopted for.   Of course, the flow-through for screening and selecting the library molecule of interest. -There are various alternatives for cytometry. In one embodiment, Encoded synthetic libraries were screened for antimicrobial activity. To bacteria or other microorganisms, such as viruses, that can be plated in two dimensions. Inhibits the growth of infected cells, many eukaryotic cells including cancer and some protozoa You can discover compounds that do or kill them. Related or unrelated chemical structure giants Large libraries consist of beads or compounds synthesized on beads from beads. Controlled release can be screened against cells in agar .   The steps of the method are as follows. (1) Spread the target cells on the agar plate . (2) Cover cells with another layer of agar. Here in the other layers, the synthesized peptides / Beads that carry drugs, individual beads, for example, using a capillary tube Suspended at a dilution that provides a dispersion that can be pulled from solid agar . (3) Initiate peptide / drug release from beads. (4) Incubate the plate Then, from the beads immobilized in agar, containing the surrounding agar and indicator cells Perform diffusion of peptide / drug into lower agar. (5) diffused from individual beads Read the extent to which the test compound affected the growth / morphology / phenotype of the indicator cells. (6 ) Select the zone where the indicator cells show the desired response (eg depth of bacterial growth). And then using a capillary tube or the like, the test agent is then Pick up the zone of agar that contains the original beads that were diffused. (7) Tag , Read by PCR amplification of the encoded material on individual beads, Determine the structure of the peptide / drug that elicited the desired response. And (8) optionally Chemically synthesize an appropriate drug / peptide and confirm the desired effect.   There are various methods of releasing the test compound from the beads. For example, using TFA Partially cleave the peptide / drug from the beads, and the cleaved peptide is Subsequent resuspension in water (agar) on the surface of the substrate results in release of the peptide / drug and To a zone of agar around certain beads, The dried compound is dried in a form that allows localization. For beads environment Conjugating peptides / drugs to beads using chemistry that is sensitive to specific changes in The bead environment may be coated on agar and indicator cells, or coated and Begun after solidification of the agar, the chemistry is, for example, light-sensitive ligation, thiol-sensitive These include sex-linked and period-linked. These chemical reagents should be It can spread by itself through other thin agar overlays. Such emission The science is the integrity of the test substance, the integrity of the code on the beads and the underlying indicator cells. Must be compatible with your health. The particular release chemistry used is, of course, Depends on the type of chemistry used for the synthesis of ibary and the nature of the indicator cell You. Method is a newly evolved bacterial strain that is resistant to existing beta-lactams Of a library of beta-lactam antibiotics for the identification of new antibiotics that kill For screening and for known anti-bacterial peptides such as Specific for screening of analog peptide libraries of magainin. Preferred.   Other methods have also been used to screen bead-based molecular libraries. Can be used for Affinity adsorption technology has been used with the libraries of the invention. obtain. For example, a mixture of beads is exposed to the surface where the receptor is immobilized. Uru (PCT Patent Publication No. 91/07087, which is herein incorporated by reference) Is part of). Substrate After washing to remove unbound beads, the beads bound to the surface were Conditions that reduce the strength of the mer / receptor interaction (eg, low pH) acid treatment Or base treatment) to elute. The process of affinity adsorption is optionally Can be repeated with the beads. These methods and related variants, even if For example, the use of magnetic selection described above can be done in a variety of ways, for example: The solid support can be a resin packed in a chromatography column.   In another method of the invention, a library of "tethered" compounds is related. Affiliation of a family of molecules, such as a family of receptors of pharmacological interest. Used as a source of structural diversity in a form suitable for nity refining . In general, this method involves the addition of tagged or stretched molecular libraries to To screen a second library of untagged molecules Regarding using. Tagged and tethered library molecules are Acts as a nity purification reagent, including soluble proteins, oligonucleotides, carbohydrates, Screen complex mixtures such as the body. Subsequent to affinity purification, Molecules that bind to matched library members can be eluted and appropriately separated and identified. Identified using the method. The combinatorial library is then combinatorially synthesized. Compound into smaller fractions, if necessary through repeated cycles , Which compounds mediate the binding process Is reductively and accurately determined.   In a similar fashion, combinatorial chemical libraries identify new receptors And can be used for cloning. Many receptors have sequence homology The facies of proteins that share sex, usually reflecting divergent evolution from ancestors. A member of Millie, but a structurally related ligand / cognate receptor , Show differences in their specificity / affinity. Receptor Each member of the Amilly (Rn) has different properties, namely localization in the body. , Its specificity, its affinity for a ligand, etc. May represent separate targets for the discovery and development of If another member of the same family Receptors with binding properties that are important enough that identification of sceptors is beneficial To identify the binding site, the following methods should be adopted to identify their binding site characteristics. R in₁Can identify receptors associated with. First, R₁A ligand that binds to Identified and then tagged with a molecule that is structurally closely related to the ligand Create a compound compound library.   Then believe that it will express additional members of the receptor family. Prepare a polysome preparation from the cells that are being treated. Such polysomes are Species for protein synthesis, from growing peptides to almost completely synthesized proteins Ribosole attached to mRNA using pendant receptors at various stages Including Near completion of synthesis Missing receptor proteins bind to one or more members of a combinatorial library Shows specific receptor properties of. Combinatorial library tethered to a solid support , The receptor with affinity for any member of the combinatorial library. Affinity purification of the polysomes responsible for Such affinity Purification entails attachment of receptors and receptors from non-adhesive polysomes. In order to achieve the separation of the solid phase bearing the relevant mRNA encoding, column chromatography Topography, batchwise separation of immobilized components from the liquid phase, or aqueous Includes two-phase separation method.   Then, using standard techniques (reverse transcriptase, etc.) CDNA synthesis from the loaded mRNA, and the cDNA population It is cloned into a suitable vector for analysis. Probable receptor Depending on the conventional knowledge of the sequence and the degree of sequence conservation that can be expected, this method Attempts have been made to use PCR or other amplifications to amplify the enriched DNA. obtain. By sequence analysis of a suitable number of cDNA clones, previously known receptors R₁CDNA showing sufficient sequence homology with the sequence of Have been identified and represent putative additional members of the same receptor family (Rn). Optionally, these new cDNAs (or parts of the problem, such as ligand binding Full-length cDNA clone (of the portion that encodes the extracellular domain involved in The Prepared by standard cloning methods and prepared these cDNAs using standard methods (ie, (As appropriate in eukaryotic expression systems as soluble or membrane bound proteins) Therefore, it is expressed. Use standard formats for testing receptor-ligand interactions Of mixed compounds or individual compounds from a combinatorial library. Test for join. In this way, the newly identified receptor It is possible to identify exactly which compound from ibary binds.   Individual beads can be small, superparamagnetic, for example by limiting dilution or by cells. Incubated with the receptor linked to the beads, and then for the receptor A method in which cells expressing the ligand are extracted using a high magnetic force magnet (Milt enyi et al., 1990, Cytometry 11: 231-238, which is hereby incorporated by reference. Part) can be physically separated by the same method. As mentioned above, magnetically Selected cells can be further analyzed and sorted using FACS . Radionucleotides also serve to label receptors and Beads can be identified and isolated by selecting recognized beads. B. Screening for soluble molecules   Also, the ligand was tagged prior to binding to the receptor of interest. Or, the novel assembly of the present invention, which is solubilized in the untagged form. In Sei To be useful, tagged molecular libraries can be employed. Soluble Browse a very large library of tagged molecules (without beads) Affinity chromatography under weak affinity conditions for leaning It is preferably used. For example, 10¹⁸Offspring library-30 mg contains several hundred μg Single 10 mL affinity chromatographic squid containing receptor of interest It can be screened using rum. Oligonucleotides are A preferred tag for braille, easily PCR amplified and DNA Market is a convenient form for sorting tag information prior to sequencing analysis. It is cloned into a commercially available TA cloning vector (Invitrigen, Inc.). In addition, the oligonucleotide tags are linked as described above and tagged with a soluble tag. Collect pools of given molecules and clone linked tags from selected pools. The tag can then be sequenced to identify the desired compound.   Soluble tagged molecules are also screened using immobilized receptors. Can be leaned. Contact the tagged molecule with the immobilized receptor. After binding and washing to remove non-specifically bound molecules, bound tags are added. The released molecule is released from the receptor in any of a wide variety of ways. tag Are optionally amplified, then tested and bind specifically to the receptor. Decoded to identify the structure of the molecule. With tag in solution The given oligomer is then labeled with the receptor immobilized by attachment to beads, Assayed, for example, by a competitive assay using a fluorescently labeled ligand. Can be The beads bearing the immobilized receptor are collected and tested positive (labeled). The decrease in fluorescence caused by library molecules that compete with the The beads can then be sorted using FACS. Then the associated identification tag is , Amplified and decoded.   The soluble molecules of the library are synthesized on the beads and then prior to the assay. Because it can be cut. In one embodiment, the fine beads of the molecular library are Are "small", very small, in silicon or other suitable surface Placed in individual compartments or wells. On average, 1 bead per well Wells by dispersing them in a volume of filling buffer sufficient to yield Filled inside. In one embodiment, the solution of beads is placed in a well above the well. The beads and the beads are transferred into the wells. Oligos from beads Cleavage of the mer can be accomplished using chemical or thermal systems, but photocleavable systems are preferred. Good. The molecule of interest is the molecule in solution that is not tagged (the tag is Either remain attached) or produce the tagged molecule in solution. In order to be able to make decisions from beads. In either event, the molecule of interest is Cleaved from the beads but remained contained within the compartment along with the beads and identification tag. You.   In one embodiment, the surface of the well or a portion of the surface is coated with a receptor. Have been Binding buffers, and known fluorescently labeled receptors for receptors Ligands are added to the wells for solution phase competition of receptor-specific ligands. Provide a combined assay. The binding of the fluorescently labeled ligand to the receptor is In one embodiment, evaluation by confocal imaging of a monolayer of immobilized receptor. Can be valued. Wells with reduced fluorescence on the surface of the receptor were It shows that Gand competes with the labeled ligand. Well showing competition Beads on the inside tag were examined to reveal the identity of the competing ligands. You.   Recovery of identification-tagged beads from positive wells is optionally performed on individual Performed by a micromanipulator to pull the beads out of the wells. Can be Other formats are either during bead production or through labeling. , Including the use of beads that incorporate fluorescent molecules. Suitable wavelength laser is positive Used to bleach the beads in the wells. Then all the beads Were collected together and bleached by FACS to identify positives. Is sorted. The associated tag is then amplified and specific for the receptor Decoded to identify molecules that specifically bind.   In another embodiment of the invention, beads with relatively large tags are used. Then, the molecule of interest is Is cleaved in the reaction of. In this method, the beads are 50-500 μm. Diameter and has a volume equivalent to 100-500 pmol peptide per bead Preferably about 200 pmo if a peptide library is constructed. 100 μm beads with a volume of 1 are used. Typical of such a library Size is about 10⁶-10⁸, Preferably 10⁷From different molecules of is there. The library is divided into about 100 pools, each containing about 100,000 Including. A certain percentage of the molecule of interest, approximately 25%, was separated from the pool. In the case of peptide libraries, for example, at 50 nM in a volume of 1 mL. Each peptide is manufactured.   The divided pools are then tested in competitive or functional assays. Most Identify the pool with the highest activity, then collect the rest of the original pool and The rest is evenly divided into 100 pools of 1000 beads per pool. Repeat the procedure until one has only one bead, from which the tag And identify the compound of interest. This method is a synthesis and construction of the Houghten method. Avoid the limit of growth and say that the pool is a random, uncorrelated compound Have the advantage. It is active due to the cumulative potential of many low affinity correlated molecules. The chances of the mixture being reduced are reduced. C. Screening a natural product library   The natural product screener using currently available automated high-flow assays The existing limitations of the ring are primarily due to the ability to obtain and handle the sample (partition, lysis, standard Second, and the second is required to characterize the active ingredients of positive samples. This is a considerable effort. The present invention is very useful in a form that is easily screened. The natural product library that provides a large number of samples and identifies the active ingredients in the sample. Provided are methods for making and screening burrries. The basis of the method is “Natural products”, that is, biochemical and chemical polymorphs with metabolic diversity from nature. It is a combination of characteristics. The simplest example is a collection of peptides for microbial Including feeding to the culture. Each microbial strain contains a number of modified peptides (metabolic labels). Ibrary) can be manufactured. Each culture is (potentially) very complex of metabolites. There is a need for efficient methods of screening, including mixtures.   Several approaches are possible and grouped by element or tag Can be orthogonally classified as endowed and soluble or tethered. For illustration, consider a library of soluble peptides as "feedback". Can be One of the even distribution of the library is the microbial fermentation screening program. Cultures with each of the many strains typical for I learned. Positive cultures were then incubated with a subset of the library. And then rescreened You. This process divides most of the active metabolites into factors (factoring). Input that causes Continue until the peptide is identified. Then, the characteristics of the active metabolite Attribution proceeds with the aid of knowledge of similar precursor molecules. Therefore, the first Cleaning identifies active organisms, the next step identifies active precursors, and Finally, active metabolites are identified by standard analytical means.   However, in all that fashion, factoring is a cumbersome process. is there. A resin-free library produced by separate synthesis, cleaved, It produces soluble compounds that are sensitive to intracellular uptake and metabolism by intact organisms. I will. However, the concentration of individual compounds is quite low (various collections Inversely related to sex), inadequate enzymatic conversion and very low concentrations of the resulting metabolites. Bring The concentration of compounds varies depending on whether a subset of the library is produced and Subsets can be increased by separating each microorganism and fermenting separately . Sub-libraries fix one or more positions and randomize remaining positions It is built by doing. For example, using 50 building blocks of 2 fixed positions There are 500 pentapeptide sub-libraries containing all permutations. These services Each of the libraries contains 125,000 compounds. Rye with a tag Although the use of burrries offers major advantages in ease and sensitivity, it Requires modification in the method of exposing the tract to metabolic activity. Combined feed The bag does not need to be a peptide only, it can It can be of any type.   Oligomers and other molecular libraries use combinatorial processes and identification tags And encoded in each step. This means that It can be done through direct linking to tags and parallel synthesis. If oligonucleotide However, if used as a tag, then the complex, although relatively large, Positively enter cells through cell fusion, electroporation, solvent permeation, etc. Small enough to fit. Once inside, the complex is exposed to the cellular metabolic machinery. It is. Using modified nucleotides and nucleotide linkages to prevent degradation Avoid the susceptibility of Rigonucleotide tags to attack. From culture from lysed cells Of active metabolites of E. coli, screening of samples, and Decrypt the tag to reveal. Scaled up with active precursor Fermentation of the active organism produces a quantity of active metabolite sufficient for characterization. Bi Library of compounds made by the above-described encoded combinatorial process Lee may be exposed to lysates of bacteria, yeast, plant cells and the like. In this style, The need to insert the tagged complex into intact cells is avoided, and Relatively few of the many molecules on the bead are detected (eg fluorescent activity). In the sexual binding assay).   Another useful method of the invention is the production of a microbial culture as a source for other cultures. Including the use of things. For illustration, 10 from a large scale culture (about 1 L) Consider a collection of 0 different microbial isolates. The supernatant of each culture was filtered. Collected and divided into 100 mL aliquots. Divide each aliquot into One of 100 strains was inoculated and incubated. 10000 samples (metabolites of metabolism) , Thereby producing from 100 microbial isolates. This method of combinatorial metabolism , Can be extended to sequential metabolism by many different species, even if For example, the products of microbial fermentation may be incubated with exogenous plant lysate or plant tissue extraction. The fractions are incubated with the yeast culture. These methods should be used in combination. Any product of a chemical diversity manufacturing process can be exposed to these sequential metabolites. It can be attached to a process.   In another aspect of the invention, the natural product diversity is combinatorially tagged. Each liposome was screened by creating a mixture of liposomes The library preferably contains only one member of the natural product compound library or Encapsulate the single mixture. The present invention is a chemical compound or natural product extraction It enables the simultaneous assay of 1000-10,000 of the 100 assays of upcoming chromatographically separated fractions were Le positive. In this context, "simultaneous" means that the cells in the readout system are the same. ー Means assayed for each other.   A mixture of combinatorially tagged liposomes was prepared as follows. You. For each individual natural product extract or chemical from inventory, the Separate liposomes encapsulating the stroma substance are prepared. Unique liposome The tag is incorporated into the liposome preparation simultaneously with encapsulation. Liposomes , Because it is stored at low temperature for a long time, it is extremely advantageous for collection, and For storage of natural product samples near the location of the collection, and subsequently Freeze the natural product extract for long-term storage in a form suitable for combination experiments. It can be dried. Lipids in the liposomal preparation are preferably present in all samples. To produce more and more identical liposomes of desired size and integrity Because of its type and composition. Reagents such as trehalose are Can be included at the time of soma formation, freeze-drying and subsequent water Addition may allow the reconstitution of intact liposomes. Caps extract / chemicals Also during the production / re-manufacture of liposomes tagged with cells, high-pressure technology Is used to cover a larger amount of aqueous phase surrounded by liposomes than the calculated value. Can be made into a psel. 3-5 times increase in volume equivalent is encapsulated by this pressure method Larger capacity of test substance, resulting in cell-based reading Allows for larger signals in Mitori.   Current liposome technology has a high percentage (with respect to the efficiency of use of each test substance). Enables the creation of liposomes that incorporate an aqueous phase (> 80%). take The unincorporated aqueous phase can be removed by a variety of "washing" methods. In addition, (with tag Encapsulated aqueous phase (with regard to specificity and lack of mixing of the encapsulated aqueous phase) Liposomes that do not leak or exchange, as well as intercalated into one of their lipids Not be exchanged (the glycolipid / protein antigen inserted as a tag is not exchanged) It is possible to create liposomes.   A wide variety of tags can be used in the methods of the invention. For example, the tag May be: (a) each fluor in the presence of each other or a combination thereof. Different fluorophores with excitation and emission properties that enable the measurement of lophores : Fluorophores are membranes of liposomes in reconstituted or encapsulated aqueous phases In the phase, it may be chosen to partition outwards. (B) Individual by atomic absorption spectrometry Various metal cations of rare earth elements that can be individually distinguished: rare metal atoms are reconstructed Designed to partition as a salt in the encapsulated aqueous phase of liposomes. (C) different antigens, the antigens being appropriate monoclonal antibodies, and Their specific reaction using primary / secondary fluorescence to detect antibodies / fluorophores Can be distinguished by: Antigens carried on proteins, glycoproteins and / or glycolipids Partition in the membrane phase of reconstituted liposomes towards the outside Can be selected for. And (d) a set of antigens, fluorophores and / or metal ions Matching greatly increases the number of possible signatures for simultaneous screening. Can be identified, and different “amounts” of each component in the signature mixture can be identified , Using different levels of fluorophore / metal ion / antigen, different An additional level (increased number) of liposome tagging can be provided.   A common fluorescent tag shared by all liposomes can be used, The liposome is fused with a liposome from a cell that is not fused with the liposome. It enables quick selection of cells. This tag is a combination of individual liposome preparations. Unlike any of the tags used in labeling, liposomally produced lipids, signatures Mixed with an aqueous sample of the drug / natural product at the time of tag and liposome production. High Self-dissipates in density (ie, in the liposome membrane), but does After lateral diffusion of the fluorophore in the cell membrane, fluorescence is emitted in the cell membrane outside the cell. Representing fluorescent tags can also be used. Depends on the mode of liposome fusion with cells Therefore, fusogenic proteins of viral origin, or glycolipids, such as For example, (in the cell line used for reading, To cells) depending on the lectin-like adhesion process mediated by the relevant receptor Those which mediate strong adhesion of the liposome can also be incorporated. Such a point In addition to the liposome-liposome interaction (which should be avoided However, it may increase the efficiency of liposome-cell fusion.   The method involves a reporter gene (eg, luciferase) downstream of the promoter. , A beta-galactosidase) containing cell line And the promoter is an exogenous hormone or ligand such as a sterol. For addition of cells such as ids, cytokines, prostaglandins, antibodies and antigens It is activated in response. Cell surface receptors or intracellular receptors for activating ligands Binding to either of the proteins activates the signal cascade, Kade ultimately leads to the activation of responsive promoters and the transfer of signal genes. Bring a picture. Expression of signal proteins is individually activated, which is detected quantitatively. Results in the generation of signals from the cells. Intracellular signal as an antagonist In the study of compounds that act on any part of the conversion cascade, cells Exogenous signal agonists (cytokines, hormones, etc.) Can be pretreated by adding and, after liposome fusion, individual The decrease in signal output on the cells is measured. Intracellular system as an agonist In the study of compounds that act on any part of the gnaru transformation cascade , No exogenous signal need be added to the cells, and The appearance of the signal on each cell can be measured.   The mixture of tagged liposomes reads cells Mixed with very large numbers over. Excess cell number is due to liposome cell fusion And is fused to the liposome so that only the following products are produced: Cells that were not fused, and (ii) cells fused with one liposome (acceptor -Cell). Efficient mixing is essential for this process and continuously stirred Or a linear flow of cell suspension, to which the liposome mixture can be added at a slow rate Is made using. Fusion may be achieved by standard methods, such as adding PEG or applying high voltage. Is awakened. Fusions are fusogens or ligands, if necessary -Can be enhanced by including a receptor recognition pair in the cell-liposome membrane . This fusion step effectively allows the aqueous phase compartment of a single liposome to be an acceptor cell. Add to the cell. Therefore, aqueous natural product extracts, chemical inventory (inve Chromatographic separation of test compounds or natural product extracts from These fractions now act at any point in the intracellular signaling pathway. it can. The fusion step also provides a specific tag that gives the signature of the particular test compound sample. Cells are added to individual receptor cells. If those tags are the lipid membrane of the liposome If so, then the tag is in the outer cell membrane of the acceptor cell. To be distributed. The antigen at this position is accessible to a panel of specific monoclonal antibodies. Access is possible. The rare earth metal ions that were in the aqueous phase of the particular liposome are Located in the cytoplasm of cusceptor cells. The fusion step also added a common liposome tag. In addition, the tag was used as an acceptor from excess cells that did not undergo liposome fusion. Identify the cells that acted as. Tags are from liposome membrane to acceptor cell membrane Can be a fluorophore that migrates to.   A mixture of cells, cells fused to individual liposomes, and unfused liposomes. The posome then interacts with the exogenous ligand (eg, with respect to the antagonist). Incubated (when tested) or tested (eg, for agonists) Incubation) without any addition). This incubation time is , Determined with control compounds at defined concentrations and incubation times.   Preferably, FACS is used to select the compound (cell) of interest. example If the cells (whether acceptor or not) are not fused any In order to sort out the liposomes, light scattering to the front or to the side must first be used. Can be Large cells can be easily separated from small liposomes. Then, Cells that were posome acceptors were replaced with excess cells that were not liposome acceptors. Can be sorted from cells. Cells that were acceptors were derived from a common liposome Of the fluorescent label, whereas non-acceptor cells are non-fluorescent with respect to this label. Light. Of course, this step is optional, but if you If desired, acceptor the (typically) major cells that are not relevant for later analysis. It is possible to separate from the few that were there. For identification of antagonists , Receipt From a putter protein (eg, beta-galactosidase or luciferase) Light-emitting cells can be sorted based on the major fluorescence-positive cells (earlier exogenous Riga This can be done by adding a small number of fluorescent negative or small fluorescent cells. Can be separated from. The latter two cell categories fuse with these individual cells Hypothetical antago of the compound encapsulated in specific liposomes This is the result from the Nist effect. Is it a receptor protein to identify the agonist? Can be sorted based on the light emission of the Can be separated from cells. The latter cell is a hypothetical agonist of liposome-derived compounds. This is the result from the Nist effect.   In some embodiments, all cells of interest are populationed according to the above criteria. And then sorted and cloned individually using standard FACS methods. You can collect cells from time to time. These individual cells depend on the characteristics they carry. Specific as analyzed as a single cell for a given tag and mediates the desired effect Enables accurate identification of the liposomes. For other uses, sorted events The distribution of tags in the positive-positive population can be analyzed and the experimental design and different Depends on the specific tags incorporated into the sample from time / location / inventory First, the ability to determine the diversity of tag types in all events-positive populations You.   The single cell or population of cells that has been collected can be characterized by the features used. It can be analyzed by a method suitable for a given tag combination. Fluorescent tag is FACS And / or can be analyzed by traditional spectroscopy. The antigen tag should be labeled appropriately. Addition of monoclonal antibodies, and ELISA, FACS, radioisotope or It can be analyzed by fluorescence analysis. Finally, the metal ion tag can be analyzed by atomic absorption spectrometry. Can be analyzed. After deciphering the tags, the test Purpose added using a mixture of only somes or to separate cell samples Can be repeated with any of the pure liposomes of each member of.   These and other methods of the invention are practiced in the invention, as discussed in the sections below. Can be automated to facilitate VII. apparatus   The coupling steps for some of the monomer sets (eg amino acids) are , Requires a relatively long incubation time in some embodiments, and this And for other reasons, a system for making many monomer additions in parallel is desired. The present invention is directed to the production and screening of encoded synthetic molecular libraries. It relates to an automated device for use. One preferred device, which is 50-100 Or more parallel reactions can be performed simultaneously) in US Patent Serial No. 08/14967 No. 5 (filed Nov. 2, 1993, which is hereby incorporated by reference) Is). Such an apparatus may be used for reaction mixtures or synthetic solid supports. The slurry is under program control under various controls for pooling, mixing and redistribution. Can be distributed to channels.   However, it generally produces a synthetic library of tagged molecules. The equipment for the use of a typical peptide synthesizer, monomer diversity and use Multiple reservoirs for the number of tags and the number of simultaneous coupling reactions desired Requires to be plumbed with. Tag distributive ability, simple command, suitable tag Of the mixture and partition the mixture. Monomer building block desired To dispense as a specialized mixture. Control of reaction agitation, temperature and time You are provided. Appropriately designed devices also have 1-50 for assay purposes. Multi-channel producing up to 100 (crude) specific peptides Work as a peptide synthesizer. PCT Patent Publication No. 91/17823 (This is a reference Incorporated herein by reference).   Typical devices include (1) synthetic reagents such as peptides and oligonucleotides. Accumulation of synthetic reagents, mixing and delivery means, (2) Various reagents are delivered , And in which sealed reactions where various reactions may proceed under an inert atmosphere. Appropriate number, (3) sealed reaction vessel matrix, (4) reagent flow (5) Small beads (0. 1-100 μm) And, means for dividing, and (6) the beads in each reaction vessel to the end of each chemical reaction. It also includes a means for cleaning. The reaction vessel matrix is one of several designs. Including any one of. For example, the containers may be arranged in a ring so that the containers are It can be spun (ie, a centrifuge). Alternatively, the container is a 12x8 matrix ( 96-well microtiter plate format). Automatic device deli Barry, suction, and locomotion features allow for any accessibility sensitive Devices are also useful for some applications.   The system used for particle combination and redistribution is one of several designs. Have one. For example, beads can be combined with an automatic pipetting machine. Suspended in a solvent of suitable surface tension and density so that it can be used to transfer Can be done. After mixing, the beads will have the same automatic pipetting Can be redistributed to the reaction vessel by means of a ring device. Alternatively, the beads are specially valved Can be combined by using a reaction chamber that is eclipsed. Valve open The solvent stream is then allowed to transfer the beads to the combination vessel. Bee after mixing The chips are subdivided by returning the stream to each reaction vessel.   In another embodiment, the beads are closely spaced, top and bottom. Combined using open reaction vessels. Pour a large amount of flow into the container, Allows mixing of beads. If the beads are magnetic, then the beads The beads are subdivided by applying a magnetic field and pulling beads on the bottom of the container. Non-magnetism The sex beads are subdivided by vacuum suction through the bottom of the reaction vessel. Again In an embodiment of, the beads distribute them on a flat surface, which is then Cover them with a device in the shape of a "cookie cutter" to cover them It is divided by limiting the area. This is more fully described below.   The system for bead washing can also have one of several designs. beads Can be washed by a combination of liquid delivery and suction tube. Each reaction vessel Has its own set of tubes, or a single set has all reaction volumes. Can be used for vessels. In the latter case, the liquid delivery and suction lines , Mounted on a robot arm and can be directed to each container separately. each The beads in the container are either centrifuged or the use of magnetic beads and the application of a magnetic field. Can form a single pellet. In addition, the reagent is Use a reaction vessel whose bottom wall is a chemically inert membrane so that it can be removed from the vessel You can also. The reagents are also containers that can provide a continuous flow of reagents and wash solutions, i.e. Remove from each container, using a container with a luer fitting and a membrane on each end. sell.   Any automated tethering device that relies on individual reaction chambers We are facing very important practical limits. Where each chamber is a reagent Connected to the delivery system and "mother pot", beads are for pooling Pumped to the "mother pot", from which the beads of monomer addition Redistributed between reaction chambers for continuous repetition. Large amount of monomer or Has other building block units, and in potentially large reaction numbers, beads And the difficulty of partitioning the reagents makes such devices less than 100 separate arrays. It can be limited to column reactions.   The present invention provides a method for pumping beads to mix and redistribute between chambers. Avoids the need, simplifies reagent delivery, and very few small beads An apparatus that enables simple and accurate division of Basic design placed on the surface A plate with an array of reaction "sites", the surface of which may be planar. Or form a reaction site It can consist of an array of shallow wells. In one embodiment, a 16x16 array There are 256 sites. Each reaction site is a spot or well, with a single spot on its surface. A group of synthetic beads are attached. The attractive force is magnetic force, vacuum filtration, positive mechanical sorting Gravity with swing, or various other simple means. Beads are initially diluted It is evenly dispensed as a slurry into a shallow reservoir to cover the array of reaction sites. When an attractive force is applied, the beads are concentrated at each site.   After placing all the beads on the reaction site, the sites were separated by mechanical division. , Create (temporary) individual reaction chambers as shown in FIG. The change is It provides a compartment that is permanently attached to the surface to form a shallow well. The reaction components are Divided into each chamber, the beads are released into suspension and the reaction begins . If desired, the beads can be reattached to the surface and the reagents removed. cup After all steps of the ring cycle are completed, the chamber compartment is removed and the Are released into a common reservoir above the array of sites.   Mixing of the beads is caused by inducing convection of the reservoir fluid, and , Then the beads are pulled back to the surface site for the next coupling. You. Subsequent steps, including washing steps, are done in a similar fashion. Addition and removal of reagents Is done using a combination of plumbing and automated pipetting. Addition of reaction-specific reagents (eg monomers) is automated with a multi-pipettor ー Can be made using. Addition of common reagents and removal of all reagents It can be done using an immobilized plumbing system that does not require valve actuation on the bar. Some common steps, such as cleaning, can be done before installation of the chamber compartment or after removal. , All together on beads.   The use of multiple monomers or other building blocks adds to the encoding process. Give a burden. In one overview of the encoding of oligonucleotide tags, The basic set of 1000 monomers is a 5 base sequence to tag each reaction step. And a larger set of 1024 monomers requires 6 bases for encoding. You can request. In order to reduce the complexity of plumber synthesis (ie A particular encoding strategy (in order to reduce the number of reactive additions) is proposed by the present invention. Provided. To explain this method, perform 256 different reactions at the time Consider a sequence of 16x16 reactive sites that can With multiple base couplings Encoding each reaction individually is a difficult task.   The array consists of 16 columns and 16 rows, where each part of the array is a unique geographical address With. Each row of sites can be tagged as a base and all 1's Six columns can be uniquely encoded with a two base codon ("subcodon"). The strip-shaped template or channel block contains these two base additions. Can serve to form 16 reaction chambers for I But not as a monomeric phosphoramidite. And)). See PCT Patent Publication No. WO 93/09668. Add reaction site This mode of resizing is another analog, for example, optical methods are used to label beads. Strip-shaped mass described in U.S. Pat. As in the King process (which is hereby incorporated by reference) Is). If a template or channel block is used, When, the template directs individual reactions during the synthesis of library molecules. It is lowered onto the synthetic surface when it is an isolated grid template.   However, beads do not allow their spatial separation during the tagging reaction. , Will not be released because it must be maintained throughout the next step. Column is sub When tagged with a codon, the template is lifted, 90 ° It is rotated and lowered to form a strip covering the rows. Then 1 6 rows labeled with a 2-base subcodon and uniquely tagged 4 salts Results in each of the 256 reactive addresses with the group "supercodon". Reaction By identifying the loess, the supercodon can also be added to the monocoupling during each reaction. Identify the mar. VIII. Equipment for parallel coupling synthesis reaction   In general, the device of the present invention allows the deposition of a wide variety of materials on a solid support. Provides synthesis. By way of example, the present invention is directed to beads, such as those described herein. To use. A variety of peptide synthesis examples are discussed below, with a discussion of synthesizers. Provide the skeleton for.   The present invention utilizes many substrates (denoted as "S") in which synthetic reactions occur. To use. The substrate is optionally a linker molecule "L" in which the coupling reaction takes place. "I will provide a. The substrate is partitioned and contains a variety of monomers, such as "A" and React with "B" to form, for example, the following collection of substrates:     S-A and S-B The substrates are then recombined, mixed and redistributed. like this After the mixing and dispensing steps, a collection of two or more substrates is formed, each of which is , S-A and S-B.   Thereafter, an additional coupling reaction occurs. For example, the pooled generation above The product reacts with the monomers C and D to form the following collection of products:     1.     S-A-C S-A-D     2.     S-B-C S-B-D Even from the simple example above, such a synthetic technique would promptly produce a wide variety of products. It becomes clear that it will produce a huge collection. Such diverse By carefully planning the composition of the child collection and / or Providing parallel synthesis of tags on substrates such as The substrate proves to be useful in a variety of applications, as described above. specific In an embodiment, the present invention efficiently produces substrates for these and other uses. An apparatus and method that can be made are provided. A. General information   FIG. 1 illustrates the apparatus used to synthesize a diverse collection of molecules. You. The device includes a common manifold 212 and tubes 215 at the top (top) and 221 to 229 connected to a plurality of reaction vessels 201 to 209 Including a container 200. The upper common manifold 212 includes tubes 221-229. And 215. The reaction vessels 201 to 209 also have valves 111 to 1 19 and the tubes 241 to 299 selectively supply the monomer addition reagent. It is connected to the servers (reservoirs) 231-239. Delivered under pressure The system (PDS) 265 is provided in both the parent container 200 and the reaction containers 201 to 209. They are connected via tubes 260 and 256, respectively.   In the synthesis reaction, the bead suspension is transferred from the parent container 200 to the reaction tubes 201 to 209. Begins when moved to. Valve 129 is open (all valves are (Ie closed by default), and the bead suspension is placed in the parent container 20. From 0, through tube 215, into the upper common manifold 212. So After that, the bead suspension It is distributed between the reaction vessels 201 to 209 through the tubes 221 to 229. So After that, the reagents selected from the monomer reservoirs 231 to 239 are added to the respective reagents. It enters into each reaction container 201-209 through the tubes 241-249. Next Then, the coupling reaction is included therein in the reaction vessels 201-209. It happens on the beads.   FIG. 1 shows a pressurized container connected to a parent container 200 via a tube 260. 2 shows a delivery system 265. The delivery system 265, under pressure, When the valve 10 and vent valve 90 are open, pressure is applied through the tube 260. Deliver the coated reagent from the delivery system 265 to the parent container 200. .   The pressurized delivery system (PDS) 265 also includes a tube 256, Isolation valve 100, lower manifold valves 101 to 109, lower tube 2 71-279, injection valves 111-119 and tubes 241-2 It is connected to the reaction vessels 201 to 209 through 49. From PDS 265 , Pressure is applied to deliver the reagents to the selected reaction vessels 201-209. The removed reagent is passed through the valve 100 with the opening, and the tube 256 and the bottom marker are removed. Put in Nihold 214. Then press the reagent under pressure into the selected opening. Press the selected valves 271-279 through the selected valves 101-109. Give up. Then pressure is applied The reagents are transferred to the selected reaction vessel 2 through the selected tubes 241-249. Pressed into 01-209.   For example, from a given monomer reservoir 231 to each reaction vessel 201 And activated solution from the PDS 265 under pressure to deliver the reagent. A certain amount of liquid is placed in tube 256, in the lower manifold 214, and in the tube. Push it up to bu 271. Amount from monomer reservoir 231 at appropriate time Inject the Monomer Reagents into Tube 271 into the Flow of Activation Solution that Continues I do. Monomer injected from the reagent reservoir 231 following the injection of the reagent The flow of the solution containing the reagent enters the reaction container 201 through the tube 241 and Participate in the pulling reaction.   The beads in the selected reaction vessels 201 to 209 are optionally mixed with a monomer reservoir. To add tags using the monomer from bars 406-412, The tag monomer reagent under pressure from the servers 406-412 was opened. It enters the common manifold 255 of PDS 256 through valves 4-7. So After that, the tag monomer under pressure and the appropriate activating reagent are added to the open valve. Through 100 and tube 256, enter the lower manifold 214. pressure The monomer-tagged reagent and its appropriate activating reagent selected by Tubes 271-279 and 241-through open valves 101-109 249 and on the beads synthesis of the tag occurs Carry it into the selected reaction vessel 201-209.   After the desired monomer and / or tag addition reaction, the vinyl in the reaction vessels 201-209 is The suspension is returned to the parent container 200 for pooling and mixing. Bead suspension In order to move the reaction vessels 201 to 209 to the parent vessel 200, the bead suspension is Argo from tube 250 through open valves 122 and 101-109. Pressure is applied. The valves 100 and 110 are closed, which The bead suspension under pressure to tubes 221-229 and Through the open valve 129 to the tube 215, Finally, push into the parent container 200. In the parent container 200, the bead suspension is Reposition between reaction vessels 201-209 to further synthesize the desired set of molecules Prepared for mixing, mixed.   In another embodiment, a plurality of three-way valves are provided for each of the reaction vessels 201-209. And the parent container 200. Such a valve is preferably a top valve It is held in a common manifold 212. In this way, a container is It can be separated from the container 200. This re-suspends the bead suspension for further synthesis. It may be particularly advantageous when dispensing. For example, if beads are all reaction vessels 201-209 were first placed for synthesis and then returned to the parent container 200. In the case of a relocation, only certain three-way valves, for example reaction vessels 201, 20 5 and 209 Only the can be opened to receive the bead suspension for further synthesis.   1 also shows a top reaction vessel bracket 290 and a bottom reaction vessel bracket. 295 shows non-concentric agitators 280 and 285 connected to 295. Upper The reaction vessel bracket 290 is not concentric with the bottom reaction vessel bracket 295. Stationary while allowing the agitators 280 and 285 to keep up Have been left. Each non-concentric agitator is a vortex motor (spiral motor -) Together with 300, stirring power is applied to the bottom container bracket 295 and the reaction container 2 Work on the bottom of 01-209. The tube between the brackets is flexible, so This stirring force is such that the contents of each individual reaction vessel 201-209 is stirred in the reaction vessel. This causes the synthetic reaction to be enhanced.   The upper common manifold 212 connects to the tube 215 at one end and Conduit for transferring material between container 200 and upper common manifold 212 Are offered. At the other end, the upper common manifold 212 is the tube 12 It is connected to 6. The tube 216 passes through the valve 121 and the common marker on the top. Nitrogen 212 is provided with pressurized argon. Tube 216 In addition, a common manifold 212 on top drains its contents through valve 120. I am making it available.   The two capacitance sensors 90S and 99S are the same as the parent container 200. Is located near the outer surface of the container to detect the level of liquid in the parent container 200. If the liquid is within the detection envelope of the volume sensor, the volume The sensor switches on (turns on). On the contrary, the capacitance sensor is Cuts off (turns off) when not in the detection envelope.   Sensors 101S-119S detect the presence of liquid in a substantially translucent tube. It is an optical sensor for taking out. In these optical sensors, the column of liquid is a tube. It is on when present in. Optical sensor is off when no liquid is detected It is.   Similarly, the sound wave (or acoustic) sensor 120S is located within its detection envelope. To detect the presence of the liquid. Contains the bead suspension, and the detection envelope of the sonic sensor. The liquid flowing through the tube placed in the rope is the sound wave sensor 120S. Turn on. On the contrary, the sound wave sensor 120S is located inside the detection envelope of the sensor. Off when there is no liquid in the tube placed in. Sound wave sensor -Used for 120S. Because the optical sensor is under certain conditions Is a semipermeable Teflon tube containing an empty semipermeable Teflon tube and bead suspension. This is because it cannot be distinguished from the tube. Furthermore, the sound wave sensor is a sensor Although selected for 120S, it contains an empty tube and a liquid or bead suspension. Any sensor that can recognize the difference between misaligned and filled tubes is used. Can You. Sound wave sensors compare to other types of sensors, such as optical sensors. Since it is expensive, there is only one sonic sensor 120S per synthesizer.   The reaction vessel bank consists of only one or a few valves between the parent vessel and the reaction vessel. Is designed to be In fact, the valve 129 is optional. This design , Is advantageous. Because it is fragile to beads and synthetic polymers. This reduces the possibility of damaging valve mechanical opening and closing movements. . Also, if the beads are large enough, they will be pulled into the valve. It may get stuck and clog the system. In addition, some polymers have Sensitive and adversely affected by the heat generated by the valve during operation May be done. Therefore, the bead suspension must travel through it It is desirable to reduce the number of valves where there are none. If valve 129 is included If not, the technique of applying pressure ensures that the liquid flows between the parent vessel and the reaction vessel bank. Can be used to prevent   As noted previously, the valves used in this embodiment have their differential It is closed in the default state (default state). Specific command for opening (instruction ), The valve always remains in the default closed state. B. Mechanical components   Mechanically speaking, an automated synthesizer roughly has three subsystems. The stem, reaction vessel bank, parent vessel and pressurized delivery system. Can be kicked. As mentioned above, the coupling synthesis is performed in the reaction vessel bank inside the reaction vessel. Happens in. The parent vessel holds beads from all reaction vessels, pools and Mix. The delivery system must be suitable solvent and / or suitable reagent / solvent concentration The reagent solution in 1. Ensure delivery in the proper process. In addition, the entire system It has been sealed for a while. Applying pressure when necessary is an inert gas, for example For example, it is made using argon. Argon is due to its convenience and low chemical reactivity. , A preferred pressurized gas.   To facilitate discussion, here is a specific example of an automated synthesizer. Please refer to. Particular examples used throughout this disclosure include polypeptide Including synthesis on a set of beads. The beads are coupled to each subsequent amino acid For the identification of the reaction, using four nucleotide monomers, A, T, C and G A tag is added.   However, an automated synthesizer is described in the specific example above. Is not limited to the synthesis of specific polymers, but also tagged polymers. -It should be recognized that synthesis is not limited. Through this disclosure , Using a reaction vessel bank having 9 reaction vessels, Although reference is made to the tagging of beads using the nucleotides described above, The number of reaction vessels that can be included in a vessel bank has no inherent upper or lower limit. In fact, the device The modular configuration of allows easy addition of additional reaction vessel banks. Change In addition, many other molecules and tags can be synthesized on the beads, and the tags Can be completely excluded. 1. Pressured delivery system   Specially prepared for synthesizing a polypeptide according to a particular example, under pressure A detailed description of the delivery system 265 is provided in three parts, the polypeptide combination. Reservoir for use in preparation, reservoir and device for attaching tags to beads Divided into livery valves. a. Reservoir for use in polypeptide synthesis   In addition to the nine amino acid monomers used to synthesize the specific example peptides, Several other additional "common" reagents are used in the synthesis. Typical pepti In synthesis, for example, the following reagents can be used: b. Reservoir used to attach tags to beads   In a particular example, the beads are optionally tagged. For peptide synthesis reaction Where the beads are, in one embodiment, from the groups A, T, C and G. A tag is attached using a nucleic acid containing cleotide. Added to four nucleotide reagents Thus, the following solvents and reagents are used during tag synthesis.   These nucleotide reagents synthesize tags using automated synthesizers. Contained in a reservoir having a sufficient volume and quantity of   FIG. 2 shows, for example, a representative reservoir for containing the MeCN solution of Table 3. 400 is shown. Connect two reservoirs to each reservoir listed in Tables 2 and 3. There is a tube. As shown in FIG. 2, the tube 450 is used to pressure the reservoir. Pressure to push the contents of the reservoir up to the second tube 452. It contains argon under pressure. In some embodiments, the reagent reservoir But there is always pressure. In another embodiment, the argon tube is , To the local on / off valve Controlled so that the reagent reservoir is pressurized only when the contents of the reservoir are needed. Apply force. c. Delivery valve   FIG. 3 shows a representative three-way solenoid valve in more detail. The valve is For example, Model 2-110-900 from General Va1ve Corp. of Fairfield, New Jersey. Can be In addition, the three-way valve of FIG. 3 may, for example, remove reagents from reservoir 412. It shows the valve 4 to be livery. The three-way valve is a Used in Livery System 265. The three-sided solenoid used in the embodiment The rhenoid valve includes a first port 454 and a second port 456. Three-way The Renoid valve also has a channel 458 through its body, Communication with the first port 454 and the second port 456 to ensure that the liquid is always first. And allows free flow between the second ports. Common manihall The second port 456 may be formed, for example, by another three-way valve to form the port 462. It is connected to the first port 454. The other three-way valve is, for example, the valve of FIG. It can be Bu 5. The solenoid in the valve responds to the control signal on wire 190. And optionally connect the third port 460 to the channel 458. Wear. The third port 460 of the valve 4 is connected to the reservoir 412 of FIG. You.   Reagent from reservoir 412 into manifold 462 A line 464 carrying a reagent under pressure to control the injection of 4 to a third port 460. When the solenoid opens, And the third port 460 can communicate with the channel 458, thereby The reagent pressurized from the port 460 of the And into the common manifold 462.   FIG. 4 shows a typical on / off two-way solenoid valve 10. This ba Lube is, for example, a Model 2-17 from General Valve Corp. of Fairfield, New Jersey. -900, optionally liquid or gas in the channel between its two ports It is possible to flush with. As shown in FIG. 4, the valve 10 has two ports. 468 and 470. The valve 10 also has a channel through its body. Has a channel in communication with the first port 468 and the second port 470. , Liquid or gas is allowed to flow between the two ports. Valve 10 A solenoid within is selectively responsive to a control signal on wire 472 to selectively The port 468 can be in communication with the second port 470. One of valve 10 When two ports are connected to a tube carrying a gas or liquid under pressure Valve 10 allows flow from that port to the other ports of valve 10. Can be used to inhibit.   FIG. 5 illustrates a pressurized delivery system according to one aspect of the present invention. 265 is shown in more detail. Figure 5 includes 24 valves 0-23, through which a common manifold is formed Show. The 2-way and 3-way valves have a common manifold through which the reagents flow. A chain by connecting their first and second ports to each other to form Can be connected to each other. Three-way valves 1-7, 9-12 and 14-22 are, for example, 3 may be substantially similar to valve 4. Two-way valves 0, 8, 13, 23 are examples For example, it may be substantially similar to valve 10 of FIG. 3 common manifolds Through-channels for one-way valves and on / off valves, and adjacent valves Including connecting tube between. As mentioned above, the third port of the 3-way valve is It is controlled by the solenoid in the valve. The third port of each 3-way valve is a reagent or Connects the reagent or solvent to the tube from the solvent reservoir, PDS265 To and from. Or the third port of the 3-way valve Outlet for delivering reagents to, for example, valve 100 of a reaction vessel bank Can serve as a port. Two-way valves are mainly isolated valves or argon Used as a supply valve.   As shown in FIG. 5, the valve 24 includes three separate valves to save space. It is physically located in the bank. The delivery system of Figure 5 also includes a valve Includes tubes 480 to connect the left bank of the with the central bank. tube 482 connects the right bank to the central bank. Table 4 shows the delivery system Used in the system The valves are listed and, in the exemplary embodiment, the type of valve used and It specifies the reagent reservoir controlled by each valve.   For example, the valve 22 has a 60/1000 inch through channel. Shown with FWO60 valve or Fast Washout (FWO) 3-way valve Was. In addition, the valve 22 was pressurized with DMF, as shown in Table 4. Control reagents from reservoir 438. As a further example, valve 23 is Pressure from the gon source (not shown) to the common manifold of the PDS265 It is an on / off valve that controls the flow of the generated argon.   FIG. 5 shows pressure applied to the common manifold of PDS 265 from one end. Tube 484 is shown connected to valve 0 for this purpose. The other tube 486 , PDS265 with argon connected to valve 23 and from the other end Applying pressure to a common manifold.   As an illustration, pressure-delivered delivery during the peptide synthesis deprotection cycle. -The operation of the system 265 is described below. DMF for deprotection of polypeptides The solution of 10% piperidine in the reaction vessel (RV) is delivered to the reaction vessel in the bank. Read. Table 4 shows that the valve 18 controls the flow of piperidine from the reservoir 430. It shows that it is possible. As a result, the valve 18 is under pressure. Piperidine from the stored reservoir 430 in the common manifold of PDS265 It must be open to allow it to flow to. Solution to RV bank valve 1 To enter 1, the isolation valve 8 is closed and the isolation valve 13 is opened, RV Bankva with open piperidine under pressure Put it in Rube 11.   As shown in FIG. 5 and Table 4, the RV bank valve 11 and the parent container valve 1 The 0 is located in the center of the valve chain. This arrangement is advantageously with these valves Minimize the length of the manifold compartment between a given reagent valve. As a result Less reagent volume is required to fill this manifold compartment. Separation bar The valves 8 and 13 allow the reagent from one end of the manifold to flow to the RV bank valve 11 Or overshoot the parent container valve 10 and to other parts of the common manifold. It can be closed to prevent unnecessary entry.   Table 4 also shows 3-way with through channels of 30/1000 inch diameter Valves 4-7 and 9-12 are shown. In contrast, Maniho The remaining valves in the valve are pierced chambers with a diameter of 60/1000 inches. Has a flannel. Reduced cross section of channel in each part of manifold Further reduce the capacity of. As a result, less reagent fills the manifold Required.   For example, nucleotides A, T, C and G are relatively expensive. Hence for It is desirable to keep the amount of reagents present to the minimum necessary. Nucleotide valve 4-7 Is located in close proximity to the RV bank outlet valve 11 and includes a nucleotide valve and an RV The distance between the bank outlet valves 11 is kept short, and the required amount of reagent is kept small. Any nucleotide valve The cross section of the manifold along the path from the valve to the RV bank outlet valve 11 is also Keep small to further reduce the amount of nucleotide reagent present in the nihold. ing. In fact, the tube 480 of FIG. 5, as well as the nucleotide and isolation valves The portion of the manifold between the 8 and 8 has a reduced cross section of 30/1000 inches. I do. C.   Reaction vessel bank   FIG. 6 illustrates a simplified reaction vessel bank 500 according to one aspect of the invention. doing. For ease of explanation, the tubing through which the solution flows is partially I omitted it. The reaction vessel bank 500 includes an upper bracket 502 and two side walls. Includes brackets 504 and 506 and two bases 508 and 510. Upper The reaction vessel bracket 290 is attached to the side brackets 504 and 506. Have been. The vortex motor 300 is installed in a plurality of reaction vessels 201 to 209. In order to supply the stirring force via the drive belt 521, the upper reaction vessel bracket Attached to the hood 290. The bottoms of the reaction vessels 201 to 209 are It is attached to the container bracket 295. Brackets 502, 504, 5 06, 290, and bases 508 and 510 are made of any suitable material. In order to facilitate mechanization, strength and weight saving, aluminum is used in this embodiment. It is used to make the brackets.   The bottom reaction bracket 295 is the upper reaction bracket 290 by the two non-concentric shafts 280 in the shaft housing 520. Tied. The non-concentric shaft 280 is in the upper reaction vessel bracket 290. Is rotatably mounted through an opening (not shown) in the. Non-concentric shaft The 280 is functionally connected to the vortex motor 300 via a belt 521. Have been. As described below, the non-concentric shaft 280 has a vortex The rotational force supplied by the rotor 300 to the bottom reaction vessel bracket 295. Change to the stirring force to move as you draw. The movement of this circle is It exerts a spiral effect on the contents of 209. Each of the reaction vessels 201 to 209 has its own Attached to the bottom reaction vessel bracket 295 at the bottom of each, All reaction vessels are uniformly and simultaneously agitated.   FIG. 6 also shows the bottom bracket 522. Bottom bracket 522 Are attached to side brackets 504 and 506, and also aluminum It can be made from any suitable material, including um. Multiple amino acid reservoirs 231 ~ 239 are mounted on the bottom bracket 522 directly below. Amino acid Liza The amino acid reagents in the peptides 231 to 239 are specific examples of polypeptide sets. Used as a building block to synthesize. In this embodiment, the polypeptide Use 9 different amino acid monomers per bank for set synthesis It is being planned.   FIG. 6 shows a separation valve bracket mounted on side brackets 504 and 506. 526 is shown. The isolation valve bracket 526 includes a plurality of lower manifolds. A channel 530 is included for mounting the shield valves 101-109. Figure 6, each lower manifold valve 101-109 is In an embodiment, it is fixed within channel 530. However, the lower Maniho The field valves 101-109 can be separated by using commercially available mounting equipment or other mounting methods. It may be fixed to the racket 526. The lower manifold valves 101-109 are A three-way solenoid valve, and with the three-way valve discussed above in connection with FIG. Is the same. Since it is used in the reaction vessel bank 500, the lower manifold bar The through channels of the valves 101-109 are connected to each other to form a common lower marker. Nifold 214 is formed through which a delivery system under pressure is applied. The solution from 265 flows.   Three two-way valves 100, 110 and 122 are also shown in FIG. 9 Bars The valves 101 to 109 are the reaction vessels 201 to 20 of the lower manifolds 214 to 9 Control the flow of solution to 9. At the first end of the common lower manifold 214 The separation valve 110 is opened to the waste liquid line (not shown in FIG. 6). . The isolation valve 100 at the second end of the common lower manifold 214 is selected Block the flow from the PDS 265 to the rest of the common lower manifold 214 Or it is possible. Optional isolation valve 1 22 supplies local argon pressure to various parts of the reaction vessel bank 500. Aids in the delivery of solutions to and from.   In another embodiment, 11 isolation valves 100-110 and 122 are Valve block, eg model P / N601374 (ABI from California, Foster City Be prepared for inside. The blocks are pre-assembled and, as a result, the assembly Is simplified. The 11 valves in the 11-valve block are substantially as described above. Works like   Injection valve bracket made of suitable material, e.g. aluminum 532 is attached to side brackets 504 and 506. plural The injection valves 111 to 119 are injection valve brackets. It is installed through the opening of 532. FIG. 6 shows the amino acid reservoir 23. A total of 9 injections that control the injection of amino acids from 1-239. The operation valves 111 to 119 are shown. Injection valve 111- 119 is a 3-way solenoid valve, and the 3-way discussed above with respect to FIG. It is the same as the valve. The first port of each injection valve is the reaction vessel 2 01-209, while the second port of each injection valve is One of the manifold valves 101 to 109 is connected to the third port. Linking Is an appropriately sized tube, for example 1/16 as used in this embodiment. Made using inch teflon tube You. As is clear from the above, the penetration of each injection valve 111-119 The channels are the reaction vessels 201 to 209 and the lower manifold valve 101. Allows the solution to flow freely between the third port of ~ 109. Each inn The third port of the injection valves 111 to 119 is the amino acid reservoir 23. 1 to 239 and optionally lower manifold valves 101 to 109 Amino acid is injected into the flow of the solution flowing between the reaction vessel and the reaction vessels 201 to 209. To inhibit or enable that.   FIG. 6 also shows the upper common manifold 212. Common upper The nidle 212 includes a common manifold 212 on the upper side and nine reaction vessels 201 to 201. Includes nine manifold ports 542 for connecting to 209. In this embodiment The 1/8 inch flexible Teflon tube is used for the manifold port 542. Is connected to the upper ends of the reaction vessels 201 to 209. Common upper Nihold 212 also collects beads from the individual reaction vessels 201-209 there. Connects to a parent container (not shown in Figure 6) that is pooled and mixed with each other A first end port 544 for The second end port 546 is 3-way valve pressurization / exhaust valve on common manifold 212 (not shown in FIG. 6) Connect using. The pressure / drain valve and the second end port 546 are Of the argon, the solution, the reagents, etc., under which pressure is applied. It may be supplied to the common manifold 212. Alternatively, the pressurization / drain valve and the second end port 546 can be routed further. Argon, solutions, reagents, etc., provided through and under pressure through the upper common Can be supplied from manifold 212 to a suitable reservoir   Another arrangement for reservoirs 231-239 is shown in FIG. 6A. single Groups of reservoirs, eg, instead of using reservoirs 231-239 , A plurality of reservoir groups, eg 231a-239a, 231b-23 9b etc. may be provided. The reservoir is inside a rotatable carousel 1000. Is held. The reservoir is such that the reagent retained in the reservoir has an upper surface 1 Upper surface 1002 of carousel 1000 so that it can be accessed from It is open at. The carousel 1000 is held in a pressure vessel (not shown). Thus, each of the reservoirs is under the same pressure in the pressure vessel. Reservoir In order to transfer the reagents in the reaction vessels 201 to 209, tubes 241-249 are provided. A plurality of tubes connected to each other are arranged in the pressure vessel. The pressure vessel Is a selected group of reservoirs, eg, reservoirs 231a-23 It is located in the reservoir of 9a. The tube can be used as a reservoir By moving towards or by moving the carousel 1000 towards the tube By doing so, it can be placed in the reservoir. The carousel 1000 The tube is rotated to align it with the selected group of reservoirs. Pressure The pressure inside the vessel has a pressure gradient between the reservoir and the tubes 241 to 249. It is supposed to be. And when the tube was placed in the reservoir When the valves 111 to 119 are opened, as described above, the reaction vessels 201 to 20 A pressure gradient causes the reagent in the reservoir to be delivered to the tube 241 for delivery to ~ Carry in 249. In this way, the carousel 1000 has various reagents. To allow selective selection from different reagents, e.g. different construction blocks. Synths by allowing a set of Provides flexibility for sizers.   FIG. 7 shows amino acid reservoirs 231-239, injection valve 111. -119, lower manifold valves 101-109 and reaction vessels 201-20 The interconnection between 9 is shown in more detail. Lower manifold valve, eg The valve 101 (which is a valve on three sides) has a common lower manifold 21. 4 to connect the solution to the first port of the lower manifold valve 101 and the second port of the manifold valve 101. It allows free flow between the two ports. Lower manifold bar The third port of the valve 101 is a 3-way injection via a tube 271. It is connected to either the first or second port of valve 111. First or first The other port of either of the two ports is attached to one end of the reaction vessel 201. Are connected via a probe 241. The other end of the reaction vessel 201 is the upper common Maniho The tube 221 to the manifold port 542 (not shown in FIG. 7) of the valve 212. Are connected via Tubes 271, 241, and 221 are chemically resistant Made from other substances, such as Teflon. In fact, this embodiment is a semi-transparent Teflon product. Translucent P with various cross-sections everywhere due to poor reactivity and optical properties Use TFE and FEP Teflon tubes.   The amino acid reservoir 231 is tubed with an inert gas such as argon. Pressure is applied through the valve 562. Pressure in amino acid reservoir 231 The sprayed amino acid solution passes through the tube 560 and is injected in three directions. Enter the third port of valve 111. Injection when receiving appropriate command The valve 111 is opened so that the amino acid solution under pressure is injected. It is possible to enter the channel that penetrates the lube 111.   FIG. 7 also shows two optical sensors 111S and 101S. Optical cell Sensors 111S and 101S are substantially translucent Teflon tube 271 and The presence or absence of the liquid in 241 is detected. As shown in FIG. The server 111S is located below the injection valve 111, and the optical sensor The sensor 101S is arranged below the reaction container 201. Optical sensor 111S and And data from 101S are controlled for use in controlling various stages of the synthetic reaction. Sent to your computer (not shown in FIG. 7).   FIG. 8 shows the non-concentric shaker 280 of FIG. 1 in more detail. Non-concentric swing The mixer 280 has two cylindrical shafts 56 connected to a cylindrical knuckle 566. 4 and 568. The shaft 564 has a central axis and a length of the tubular knuckle 566. Collinear in the direction and of the two planar surfaces of the tubular knuckle 566. Are linked together. The shaft 568 is another planar shape of the tubular knuckle 566. Is connected to the surface of and forms an offset from the central axis of the cylindrical knuckle 566. I have. In one embodiment, shafts 564 and 568 and tubular knucks. Le 566 is machined from a single piece of metal stock.   A cylindrical shaft 564 has a fixed rotation support, such as a roller bearing. Tubular shaft 564 when torqued to rotate in The cylindrical shaft 568 forming an offset with respect to the rotation axis of the It moves in a circular path around the rotation axis of the shaft 564. 1 or more non-concentric shaker 2 80 is operably connected, for example, by a belt-and-pulley arrangement, A plurality of non-concentric shakers 280 are allowed to move in unison. This embodiment , The shafts 568 of the two non-concentric shakers 280 have a single bracket. When the shaft 564 is rotated, the bracket is connected in a circular path. It is designed to move. Furthermore, the shaft 568 and the vortex motor 300 So that the bottom of each reaction vessel moves in a circular path at about 1500 revolutions per minute. Designed to Have been. In order to avoid damage to the beads, the circular path of this embodiment is More preferably, about 3. Limited to a radius of 5 mm.   FIG. 9 shows the reaction vessels 201 to 209 and the vortex motor 300 of FIG. The upper portion of the containing reaction vessel bank 500 is shown in more detail. Discuss with respect to FIG. As described above, the reaction vessel bank 500 includes a plurality of reaction vessel banks 500 connected to the upper bracket 290. Of the reaction vessels 201 to 209. The upper bracket 290 is used for the reaction vessel 201. ˜209 have multiple openings 630 where they connect. Above opening (shown In the Teflon tube from (No.), the liquid is Teflon tube and reaction tube 201- 209 at the openings 630 in a manner that allows them to flow freely between them. It is connected to the upper ends of the respective reaction vessels 201 to 209.   The lower ends of the reaction vessels 201 to 209 are connected to the lower reaction vessel bracket 295. ing. 10A and 10B show the bottom of the bottom reaction vessel bracket 295 of FIG. It shows the situation in more detail. FIG. 10A illustrates the lower reaction vessel bracket 295. It is a part of the bottom of the close-up.   FIG. 10B illustrates a plurality of channels 650 in the lower reaction vessel bracket 295. Is shown. Flexible and substantially translucent Teflon tube (for ease of explanation) 10B is omitted from FIG. 10B, the lower end of each reaction vessel 201 to 209 (this is omitted). (Also omitted from FIG. 10B for ease of explanation) Fits within the flannel 650 You. A groove 652 for mounting an optical sensor is associated with each channel 650. ing. The groove 652 is clearly illustrated in FIG. 10A.   FIG. 10B shows that the optical sensor is firmly attached to the lower reaction vessel bracket 295. A plurality of mounting holes 654 are shown for defining. Also reduce weight A plurality of optional holes 656 for are shown in FIG. 10B. As discussed earlier In addition, the lower reaction vessel bracket 295 accommodates the movement of the non-concentric shaker in the circular path. Following, stir the contents of the reaction vessel. Optional holes 656 are brackets Must be machined over the entire lower reaction vessel bracket 295 to reduce the weight of the Can reduce the amount of force required to move the bracket. Wear.   The through holes 658 near each end of the lower reaction vessel bracket 295 are not A heart shaker 280 is connected to the lower reaction vessel bracket 295. Reaction volume The lower ends of the vessels 201 to 209 are provided with a penetrating chamber in the lower reaction vessel bracket 295. The flexible Teflon tubes 241-249 to fit into the channel 650. And then follow the circular movement of the lower reaction vessel bracket 295. Downward reaction The vessel bracket 295 moves in a circular path, so all the reactions in the reaction vessel bank are The contents in containers 201-209 are agitated in a side-by-side fashion.   FIG. 9 also shows any non-concentric shaker for fitting around the non-concentric shaker 280. Showing the mill housing 520 You. The optional non-concentric shaker housing 520 is not contained within a hollow tubular housing. Human user enclosing a concentric shaft and when the non-concentric shaft is moving The possibility of personal injury and damage to the equipment.   This embodiment uses a stepping motor to supply the rotational force to a non-concentric shaker. Controller (model RD122, Semif Co, California Fremont rp. (Made by) and stepper motor (model PX245-01AA, Califor) nia Torrance Oriental Motor U. S. A. Corp. Product) is used. As shown in FIG. 9A As shown, the three pulleys 670, 672 and 674 are vortex motors. -Two non-concentric in cooperation with 300 and two drive belts 521 and 676 Of the shaker 280 in an optional non-concentric shaker housing 520 Rotate. This embodiment is a stepper motor and stepping controller. , But the rotational force includes other electric motors or pneumatic motors , And may be powered by any other suitable type of motor. Furthermore, Volte The power supplied by the xox motor 300 is sprocket, pulley and belt. Transmission to the non-concentric shaker 280 by any suitable transmission means including a belt, gear, etc. Can be reached.   FIG. 11A shows a detection of the presence or absence of liquid in a substantially translucent Teflon tube. 9 illustrates an exemplary optical sensor 680 for use in export. Optical sensor 68 0 is the same as the optical sensors 101S to 109S of this embodiment. light Sensor 680 (model EE-SX671, Omro from Illinois, Schaumburg) n, Inc. Made to house the optical transmitter and collector respectively. And includes two forked ends 682 and 684. The optical sensor 680 also , An appropriate electrical circuit configuration for transmitting the sensor data to the control computer Book for housing and mounting optical sensor 680 to bracket It has a body 686.   FIG. 11B shows the forked ends 682 and 684 of the optical sensor 680 in greater detail. It is shown in detail. In the direction of arrow 688, the light is substantially square-shaped (FIG. 11B). A substantially rectangular transmitter 685 for transmission (not shown) Located on the inner surface of the forked end 682. The controller is Located on the inner surface of the hooked end 684, and is also rectangular. Practically half In order to detect the presence of liquid in the transparent Teflon tube, the Teflon tube It is mounted through the gap between the two forked ends 682 and 684. When the liquid is in a substantially translucent Teflon tube, the light-collecting part triggers (tries). Gard) shows detection of fluid in Teflon tubes.   In fact, the substantially translucent Teflon material, when empty, is the optical sensor 680. It turns out that you can't trigger. Substantially translucent chew A common optical sensor is advantageously used to detect the presence of liquid in the chamber. To this end, a new optical array block is used. FIG. 11C shows an optical array block. 690 is shown in more detail. The optical array block 690 is essentially a transformer. An opaque material that blocks any light emitted by the smitter 685 Made from. The optical array block 690 is a block 690 which is an optical sensor 68. Two retaining walls 692 for frictionally engaging one of the forked ends of 0 and 694 on the first surface 696 and forked optical alignment block 690 Holds tightly between the edges. In one embodiment, retaining wall 692 and And 694 are designed to engage the light collector fork end 684 of FIG. 11B.   The optics array block 690 also includes a chamber formed in the second block surface 700. Channel 698. The second block surface 700 is similar to the first surface 696 above. The opposite surface. The axis of channel 698 is orthogonal to retaining walls 692 and 694 doing. Channel 698 can hold the Teflon tube snugly It is sized. As a result, the Teflon tube is channel 698. The optical array block 700 is housed within and has forked ends 682 and 6 When installed in the gap between 84, the transmitter stream above Arranged at right angles to the top 685.   FIG. 11D shows opening 70 located along the center line of channel 698. 2 is shown. The opening 702 is A small amount of light is transmitted along the hole between the first surface 696 and the second surface 700 along the optical array. It is possible to transmit through block 690. Substantially translucent tefro Tube, for example tubes 241-249 or 271-279, When fitted snugly within 698, the axis of opening 702 is Run through the center of the tube. The shape and size of the opening 702 depends on the optical characteristics of the tube. It is a function of sex.   Optical array block 690 is provided with forked ends 6 in the manner shown in FIG. 11A. When mounted between 82 and 684, the transformer in the forked end 682 is Light from the smitter 685 travels through the substantially translucent tube 704. . Most of the light passes through the tube 704 before it reaches the optical array block 690. Blocked by. Some of the light that passes through the tube 704 is opened. Mouth 702 (hidden from view in FIG. 11A) is reached, and opening 702 It travels along the hole to reach the light collector within the forked end 684.   The axis of the opening 702 runs through the center of the substantially translucent tube 704, The light that passes through the center of the tube is part of the light collection in the forked end 684. Reach the minute. Light that travels through the empty tube 704 is diffracted so that the sensor Insufficient amount of light to reach the light collector. The liquid is translucent Teflon When present in the tube 704, the light passing through the filled tube is of It is focused by the liquid inside. The focused liquid is directed from the direction of the fork-shaped end 682. It enters into the opening 702 and induces the light collecting portion in the forked end 684. Liquid present If not, the focusing effect is relatively small. Therefore, less light is available in the opening 70. Enter 2. In fact, when there is no liquid in the Teflon tube 704, the opening 702 is The light passing through is insufficient to trigger the light collection in the forked end 684. is there.   As discussed above, the optical array block 690 replaces the Teflon tube 704. The size is set so that it can be caught exactly. The optical array block 690 , Shown in Figure 11A when mounted between forked ends 682 and 684. As described above, the Teflon tube is formed by the optical center 680 and the block 690. It is completely grasped. By fixing the optical sensor 680 to the bracket The Teflon tube 704 is fixed to the bracket. This way Teflon tubes 241-2 extending from the bottom of the reaction vessels 201-209 of the embodiment 49 is fixed to the bottom reaction vessel bracket 295.   As shown in FIG. 12, a reaction vessel, for example, the reaction vessel 20 of the embodiment of the present invention. 1 consists of a segment of FEP Teflon tube 710. Tube 710 , 1/4 inch outer diameter and 0. It has an inner diameter of 19 inches. More details below As described above, the diameter of the tube 710 is such that two or more reagents are simultaneously stored in the reaction vessel 201. For use when mixing Alternatively, it could be made quite large. Tube 710 is a flexible Teflon The tube 271 is sealed and connected. Flexible Teflon tube 271 Is fixed to the reaction vessel bracket 295 below the reaction vessel bank. Below When the reaction vessel bracket 295 of is moved in a circular motion, the flexible Teflon tube The bottom of the tube 271 is used to stir the contents in the tube 710. Follow the circular movement represented by the racket 295.   In this embodiment, the tube 271 has an outer diameter of 1/8 inch and a 1/16 Has an inner diameter of inches. Figure 12 shows that tubes of different cross sections are sealed to each other. First coupler 714, second mutual connector for connecting in a closed state 7 shows a tube connector including 716 and a third coupler 718. Up The tube connectors noted are from Norton, Inc. Available from (Ohio, Akron). Ju Tube 710 to prevent the substrate in tube 710 from entering flexible tube 712. A frit or filter 1102 located at the bottom edge of the There is a hiding). The frit is, for example, a 2 micron titanium frit obtain.   At the other end of tube 710, a fourth coupler 720, a fifth interconnector -721 and the sixth coupler 722 are sealed, the tube 710 Are connected to the tube 221. The tube 221 of this embodiment is the tube 7 Since it has a different cross-sectional area than 10, The rails 720 and 722 and the mutual connector 721 are essential. Tube 2 21 is a manifold port 542 of the upper manifold 212 (shown in FIG. 6). Sazu) is connected.   The flexible O-ring 724 is located in the hole 726 in the bracket 290 (shown in cutaway (Shown in 12). The O-ring 724 is a flexible coupler. 722, thereby flexibly attaching the reaction vessel 201 to the bracket 290. It is fixed. If the bottom edge of the reaction vessel 201 is shaken, the O-ring 724 , Serves as a pivot point, and relatively moves the top of the reaction vessel 201. Hold it so that it does not stir and enhance the stirring effect.   As shown in Figures 12A and 12B, the reaction vessel in one particular embodiment. 201 is a temperature control for controlling the temperature of the reaction vessels 201 to 209. A jacket 1100 may be provided. Temperature control jacket 1 in this embodiment 100 for connecting reaction vessel 201 to lines 221 and 241 (not shown) With fitting 1104. Inlet / outlet ports 1106, 1108 provide heat transfer It is provided to supply the conductive liquid to the reaction vessel 201. Thermally conductive liquid is a reaction vessel It can be used to heat or cool 201. In this embodiment, the reaction volume The vessel 201 is formed from a Teflon tube 1110, preferably a quarter-inch tube. It has an outside diameter of 1 and a wall thickness of 1/32 inch. Outside the tube 1110 There is a tube 1112 on the side, It is preferably composed of Teflon and has an outer diameter of 3/4 inch and 1 / It has a wall thickness of 32 inches. The outer tube 1112 has a fitting 1104. Overlying and forming an annular space 1114 for containing a thermally conductive liquid. I have. O-rings 1116, 1118 are fitted with outer tube 1112 and fittings. Located between the lugs 1104 to form a tight seal of liquid. like this In order to heat or cool the reaction vessel 210, the heat conductive liquid is supplied to the port 11 06, 1108, and can be introduced into the annular space 1114. it can. The ports 1106, 1108 are also provided with a thermally conductive liquid in an annular space 1114. Through which it is possible to circulate continuously.   Of a reaction vessel 1120 having a temperature control jacket 1122 integrally formed Another embodiment is shown in Figures 12C and 12D. The reaction vessel 1120 is substantially It functions similarly to the reaction vessel 201, except that the reaction vessel 1120 is preferably glass. Made in. The temperature control jacket 1122 is also preferably made of glass. . In such a configuration, the reaction vessel 1120 and the temperature control jacket 1122 are integrated. It can be formed as a unit of. The reaction container 1120 is a frit 11 24 and connector 11 for connection to lines 221 and 241 (not shown) 26 and 1128. Inlet / outlet ports 1130, 1132 are jackets Annular space 1 between 1122 and reaction vessel 1120 A thermally conductive liquid is provided for circulation through 134. Hose barb  barb) 1136, 1138 are conveniently the inlet / outlet ports 1130, 113 Provided for connection to two suitable liquid sources. In this way, the thermally conductive liquid Circulates through the annular space 1134 to heat or cool the reaction vessel 1120. Can be looped. D.   Parent container   FIG. 13 shows the parent container of this embodiment in more detail. The parent container 200 is an example For example, the volume is about 30 mL. Tube 260 delivers reagent or argon. Lee system PDS265 (not shown in FIG. 13) and from there. For movement, it is connected to the bottom end of the parent container 200 in a sealed state. . The frit or filter 746 prevents the substrate from entering the tube 260. Therefore, it is fixed near the bottom of the parent container 200. To add and remove substances There is a removable cap 743 fixed to the upper end of the parent container 200. cap The tube 215 fixed through 743 is used for the parent container 200 and the reaction container van. Transfer material to and from a common manifold 212 (not shown in FIG. 13) within the chamber. You. The tube 215 extends through the cap 743. Pressured al The gon / drain line 275 also extends through the cap 743. Any rinse Line 281 contains the solvent under pressure to rinse the inner wall of parent container 5. Delivery to the side wall Is connected to a solvent source for   FIG. 13 shows two capacitive sensors 9 mounted near the outside of the parent container 200. 0S and 99S (Model 18-08, Electro of Illinois, Hoffman Estates) matic Control Corp. Manufactured). Each capacitance sensor 90S or 95S, The presence of liquid is detected in the vicinity, and the sensor data is Via 756 and 758 to a control computer (not shown). Content The quantity sensor 99S is used to detect the level of reagent added to the parent container. It is. Volume sensor 90S detects the level of bead suspension for redistribution This is because. Both sensor levels depend on the amount of beads and the number of reaction vessels used. Therefore it can be adjusted. Capacitive sensor data can be used in various cycles of the synthesis process. Used by the software to control the. E. FIG. Control system   1. Control computer   An automated synthesizer uses a control computer to synthesize During the various cycles of the process, data is collected from the sensors and valves and And control the spiral motor. When used with an automated synthesizer, Black computer, mini computer, workstation, mainframe Any computer, including those commonly known as Used to process data and issue commands to control valves and spiral motors Can be done.   In addition, the sensor data from the synthesizer's sensor uses the normal data collection method. And can be collected by many commercially available data collection devices. Similarly, In response to the appropriate command from the computer, a commercially available I / O controller Thus, the valve and spiral motor can be controlled.   In one embodiment, an IBM-compatible microcomputer (per computer Control computer, also known as Sonar computer or PC) (Model Gateway 2000 4DX2-50V, South Dakota, S. ー City's Gateway 2000 Inc. Than). There are multiple expansion slots in the PC. This allows the addition of various expansion boards. Bus supply of these boards to PC It connects to the bus resource and the PC connects to the circuit components on the card. Allows you to contact and perform electronic functions. Some expansion boards also have an external PC To allow communication with the devices and circuit components of For example, with a modem board Then, a commonly known board is connected to an expansion slot on the PC, and the PC is a modem. Allows you to contact another computer that has Personal computer Using an expansion board with a motor is a matter of common engineering knowledge.   In one embodiment, the automated synthesizer is a multi-channel digital I / O board (Model PCDIO120-P, Industrial, San Diego, CA)  (Via Computer Source) and contact the PC. For details of PCDIO120-P board, see In Product Manual No. available from dustrial Computer Source. 00431-050-20A Are described in detail in.   Each PCDIO120-P has a 120-channel bus in five 24-channel groups. Equipped with buffered input / output (I / O). Each 24-channel group is programmed Programmable peripheral interface (PPI) 8255A chip. Channels are selectable in sets of 8 for either input or output You.   FIG. 14 shows a simplified diagram of part of the control hardware. Compu The monitor 760, for example a PC with multiple expansion slots, is a display monitor. Connect to the keyboard 762 and keyboard 764. PCDP120-P I / O board 76 6 is inserted into one of the expansion slots and the PC communicates with 5 controller circuits 768 To be able to. Each system Controller circuit 768 communicates with I / O board 766 via conductor channel 770. Real In an embodiment, the conductor channel 770 is capable of using 24 I / O channels. 50-conductor ribbons with.   2. Controller circuit   The output signal from the I / O board 766, typically 15 mA source current and sink current. A flow of 24 mA is unsuitable for operating solenoid valves. Therefore, control The control device circuit 768 outputs the output signal from the I / O board 766 to the solenoid valve. It is converted into a power signal having a power suitable for actual operation. Controller circuit 768 Receives the 24 output signals from the I / O board 766, and then the various combiner It has the ability to output 24 power signals 771 to control the device. Each controller times The 24 power lines 771 on line 768 are shown in Figure 14.   Each controller circuit 768 also includes up to 24 sensor lines (one for each sensor). A) a central physical location where sensor data from can be collected. Up to 24 Data from different sensors is output to sensor port 772 on each controller circuit 768. Can be better received.   In this way, each controller circuit 768 can have up to 48 I / O channels on the I / O board 766. You can enable channels, 24 inputs and 24 outputs. Figure 15 shows 5 shows an exemplary diagram of a controller circuit 768 according to one aspect of the present invention. 50 -The pin header 781 connects the controller circuit 768 to the I / O. Connect to substrate 766 (not shown in FIG. 15). Header 781 is the input header Yes, connected to sensor port 772 via bus 780. Sensor port 772 , Header or controller circuit 768 to connect with up to 24 input devices such as sensors Including a connector for. Each conductor on bus 780 is input from one sensor Carry a signal to Da 781. FIG. 15 also shows the LED indicator signal from the sensor port 772. Optional LED bus 784 to carry to any bank of light emitting diodes (LEDs) Is shown. Each conductor in any LED bus 784 is an L in bank 786. Carry a signal to the ED.   FIG. 15 also shows the controller circuit 768 on an I / O board 766 (not shown in FIG. 15). Shows another 50-pin header 788 for connecting. The header 788 is the I / O board 76 Output header for receiving the output signal from 6. Up to 8 buses 790 The output signal is transferred from the header 788 to the 8-pole inverter 74LS240 chip 792. Bu The 8-pole Inverter 74LS240 chip 792 is a Te, Dallas, Texas Te xas Instruments, Inc. Is manufactured by. Bus 794 has chips 792-8 Carries inverted buffered output signal to pole latch driver 796. latch Driver chip 796 is manufactured by Micrel, Inc. of San Jose, Calif. To Model MIC59P50 manufactured by Each latch driver The output signal from the 796 is connected to the output port 796 via the bus 800. any LED bus The 802 is an LE from the chip 796 to any bank of light emitting diodes (LEDs) 804. Carry D indicator signal. Each conductor in any LED bus 802 is in bank 804. Carries a signal to one LED. 3. valve   A solenoid valve used in embodiments of the present invention, For example, valves 4-7, 10, 14, 90-91, 100-121, and 129 are commands to open. It is normally closed until ordered. Therefore, all of the synthesizer The default state for all valves is that they are closed. No synthesizer Safety is assured by the fact that the substance cannot flow when it is in compliance. Open When opened, the valve uses electricity to heat up. In addition to the obvious consumption of system power, heat Large valves can adversely affect the chemicals that pass through it. Normally, Hold open valve already open to open renoid valve Strike relays, for example models, because they require more power. D1D20 (Kridom Inc., Long Beach, CA) Crydom, Inc. ) To) to operate the valve. Like D1D20 The strike relay is +12 on the valve for a specified time, typically 100 ms. Supply the bolt and open the solenoid valve from the closed state. Strike The time during which the relay supplies +12 volts is determined by software control And the strike is I / O It is supplied through the panel. After that, the strike relay will reduce the reduced voltage, typically Typically half the estimated voltage, or about 6 volts in an embodiment of the invention. To hold the solenoid valve open. Therefore, operate the valve. Less energy is required to make and less heat is produced.   The present invention provides four separate power supplies. The first power supply is the controller -+ 5 Volts to power a TTL chip as found in circuit 768 Output. The second power supply is used by the stepper motor. + 32V output for use. Third power supply actuates a solenoid valve Give +12 volts to do. An optional fourth power supply is also provided by the sensor. Give +12 volts for use. Another fourth power supply for the valve is Any noise generated by the valves as they open and close , Ensure that it does not interfere with the operation of the sensor.   In its default state, all valves are closed. Further safety standards Then, the synthesizer also provides all the buffers when a control computer malfunction occurs. The solid state watchdog relay to shut off the lube. lay) is prepared. Solid watchdog relay, for example, Brentec International Model SM-WDT5 (Californi Industrial computer in San Diego, A.   Source (available from Industrial Computer Source) is the control computer And the power supply to the valve. The pulse generated by the software is From the control computer to the watchdog relay on one of the I / O channels Transmitted. When there is no pulse, eg CPU failure, latch-up or power failure In the event of a failure, the watchdog relay will shut off the power to the valve and Then close all valves. F. Control software   This section discusses the control software in detail with respect to the flowcharts in Figures 16-24. Argue. These flowcharts show the lifespan of the control computer. Explain the completion of the relevant steps in the process, or synthesis process. The following discussion To facilitate delivery, at all relevant times, a pressurized delivery system (delivery system) stem) valve receives the appropriate command from the control computer and the desired reagent Is to be delivered to a valve in the reactor bank.   Figure 16 shows the control computer so that reactors 201-209 discharge their contents. It is a flow chart explaining a series of instructions issued by. Reaction before discharge The vessels 201-209 contain liquid or bead suspensions. Common manifold (common The argon supply valve 121, which is connected to Open and pressurize the top common manifold 212 with argon. Chosen at the same time Three Mouth valves 101-109 are open and liquid is flowing from reactors 201-209 to the lower manifold. Allows you to enter Do 214. During some coupling reactions, all reaction Only the selected valves 201-209 are used, since the reactors 201-209 are not used. Opens. If the reactor is empty during the synthesis, it is not necessary to drain its contents . Waste valve 110 on bottom manifold 214 opens to allow fluid to exit I do. As a result of the pressure differential, argon pressure forces the fluid from the reactors 201-209 to a lower level. Push out through waste manifold 214 and out of waste valve 110. Liquid to be discharged remains When the sensor 110S that informs that it has not been turned off, the valve is further 2 to 5 All liquids drain from the reactor bank, staying open for 2 seconds Guarantee.   Figure 17 shows the control computer to clean the lower manifold 214. Draw a series of commands issued by Lower manifold 214 at start-up There is a substance in. After that, the separation valve 100 is opened and the pressurized argon is discharged into the PDS.   From 265 it is possible to enter the lower manifold 214. At the same time, discard The material valve 110 opens to expel material from the lower manifold 214. Substance remains The material exits the lower manifold 214 until it disappears. Optical sensor 11 If 0S detects that there is no material in the waste pipe, the valve returns to its default state.   Figure 18 shows the control computer telling you to mix the contents of the parent container 200. Shows a sequence of instructions issued. The system is Lgon is introduced into the parent container from below to mix the contents. Argon bubbles in the parent container Remove the contents of the parent container 200 when climbing from the bottom to the surface of the contents inside the parent container. Turn around. Again, everything that wasn't explicitly commanded to stay open Valve is in default. The valve 100 opens and the pressurized argon is discharged to PD. From S265, it is introduced into the bottom of the parent container and the contents in it are stirred. valve The 90 opens again and evacuates argon from the parent vessel. Valves 100 and 90 have approximately 15 Returns to default after ~ 30 seconds.   Figures 19A and 19B show that the bead suspension was distributed from the parent vessel to the various reactors. Then, draw a series of commands issued by the control computer. The sequence of instructions is the seed Perform a volumetric technique to fill each reactor with the bead suspension. This way With, the reactor is filled with less dependence on the flow rate. Thus, as you can see In addition, the bead suspension consists of the reactor and the manifold port where the bead suspension enters. Evenly distributed between the reactors regardless of the distance between. At the start of the allocation cycle In addition, the reactor bank contains only argon and the parent vessel contains the bead suspension from step 852. No.   At stage 854, the reactor bank is present in the reactor bank prior to the redistribution stage. Partially filled with DMF to replace argon. Isolation valve 100 open, pressurized DMF lowers from pressurized delivery system Allows you to enter. Valves 101-109 open and DMF goes to the reactor. Enable you to climb You. Valve 120 opens to vent argon from the common manifold at the top. Bar Tabs 100, 101-109 and 120 are programmed for a pre-programmed period (respectively After that (which may be about 3 seconds), preferably continuously, returning to its default state. next, Reactors 201-209 are mixed to remove bubbles. Valves 100-109 are about 3 Reopen for seconds. Upon expiration of this pre-programmed period, direct to reactor The level of DMF in each tube 241-249 was close to the common manifold 212 at the top. It becomes.   The reactor bank and parent vessel 200 are then filled with DMF in step 858. You. Valve 100 opened and pressurized DMF from PDS 265 to reactor bank. Allows you to enter. Valves 101-109 open to fill the reactor bank with DMF. Continue to add. The valve 129 was opened and the DMF overflowing from the reactor bank became the parent. Allows entry into container 200. Valve 90 opened again and replaced argon Is discharged from the parent container 200. When the sensor 99S detects DMF within the detection range , Until the level of fluid in parent container 200 rises to the level of the upper capacitive sensor 99S , Continue filling. The reactor bank is then completely filled. The parent container 200 is The second capacitive sensor 99S is charged up to the level of the other. Next, valves 90 and 100 , 101-109 return to their closed states in step 860.   Next, at step 862, the top common manifold DMF is swept. valve 121 opens and the common manifold at the top Pressurized with pressurized argon. Valve 129 open, DMF common on top Allows entry into the parent container from the manifold. Valve 90 opened and replaced Drain argon from the parent vessel. Which allows DMF from the common manifold at the top Then, transfer it to the parent container. The parent container accepts more DMF without overflowing Designed to have sufficient volume for Pre-programmed time, After about 5 seconds, for example, valves 90, 121 and 129 are in a default state at step 864. Return to The pre-programmed time is variable, but the top common manifold It should be equal to or greater than the time it takes to clear the DMF of the card.   The reactor must be pre-filled with DMF prior to the introduction of the bead suspension. That is, an empty reactor leads to an uneven distribution of the bead suspension. reaction When the vessel is empty, it is closest to the manifold mouth where the bead suspension enters from the parent container. The first reactor is filled first. How much is allowed to be overfilled? For some reactors, there may be no bead suspension left.   The present invention uses a novel method of controlling the volume in which a reactor receives a bead suspension. To use. First, in step 866, a small column of argon was added to the common reactor top. Introduce to the top of each tube where it is attached to the manifold. This argon To create a bubble of water, open valve 121 and allow pressurized argon to Possible to enter the hold To Selected valves 101-109 open continuously and some DMF reacts Allows discharge from the vessel to a lower manifold. Valve 110 opens, Drain the displaced DMF from the bottom manifold. About 0. After 3 seconds, a small algo The top of the tube where the column connects the reactor to the common manifold on the top Appears in. Next, valves 101-110 and 121 are checked for default at step 868. Return to the state.   In step 869, the beads suspended in the parent vessel were prepared in preparation for distribution between the reactors. The liquids are mixed. The instructions related to this stage are similar to those discussed with respect to Figure 18. It is. The valves 10 and 90 are then returned to their default state at step 870. this The steps ensure that the uniform bead suspension is evenly distributed among the selected reactors. I testify.   Some of the bead suspension was then introduced into the common manifold at the top in step 871. I do. At this stage, the bead suspension entering the common manifold at the top is the last reactor Manifold port to which a tube, eg, the reaction tube associated with sensor 109S, attaches Most of the argon present in the common manifold at the summit without flowing too much through the It is set according to a pre-programmed time to replace the throat. about 0. A pre-programmed time of 5 seconds was found to be sufficient. Bee Open valve 91 to introduce this portion of the suspension into the common manifold on the top. Pressurize the parent vessel with argon. Open valve 129 to allow bead suspension to top Allows inflow into the common manifold of. Open valve 120, common on top Evacuate the argon present in the manifold. Pre-programmed as discussed above After the expiry of the allotted time, valves 91 and 120 return to their default state at stage 872. You. Most importantly, keep valve 129 open to Prevents the limmer from being damaged by the valve closing action. Embodiments of the present invention And the two-port valve 129 is controlled to remain open in the manner previously discussed. Continue to receive command signals from your computer. However, valve 129 When a command pulse is received from the control computer, it can be opened or closed. It can be a latch valve that toggles in between. Valve 1 If 29 is a latch valve and it is already open, open latch valve 129. No action needs to be taken by the control computer to keep it up.   FIG. 19B is a continuation of FIG. 19A. How much is present on the common manifold 212 at the summit After some argon was replaced, the rest of the bead suspension was collected at step 874. Moved to upper common manifold 212. Valve 91 opens and the parent vessel 200 is flushed with argon. Pressurize with. Valve 129, which was held open, allowed pressurized bead suspension. Allows liquid to enter common manifold 212 at the top. Selected valve 101-10 9 opens to allow DMF in reactor 200 to exit to lower manifold 214. Noh. Valve 110 opens to lower DMF is discharged from the manifold 214. Somewhat dependent on the resistance of the bead suspension , DMF withdraws the selected reaction tubes 201-209 relatively slowly. Was chosen Selected reactors connecting reactors 201-209 with a common manifold 212 at the summit. The displaced DMF from Bue 221-229 is replaced with the bead suspension. Hang The suspension-foam-DMF column slowly descends toward the lower manifold 214. No.   The previously introduced foam pillars also advantageously serve as volume markers, ie The control computer determines when each reactor receives a sufficient amount of bead suspension Provide a way to do. The pillars of foam are the surface characteristics of Teflon and DMF and Teflon. Stays complemented between the DMF column and the suspension column due to the high contact angle between . As discussed above, the optical sensors used in the embodiments of the present invention include: Detects the presence or absence of a liquid column inside a substantially translucent Teflon tube. Can be When DMF is replaced by a large volume of bead suspension that goes downwards , Foam to the reaction tube and to the reactor where it couples the reactor to the lower manifold. Move down to the tube. When the bubble of argon moves downward, the optical detector 101S ~ At the moment detected by 109S, the detector turns off immediately. It was discussed earlier As described above, the sensor data immediately shows the command signal to close each valve 101-109. It is transmitted to the control computer that emits to the seat. All valves 101-109 closed After that, the bead suspension is restarted to be transported, and the acoustic sensor 120S reaches the parent container. Until we detect the absence of fluid in the tubing associated with the common manifold above , Continued at step 878. In step 882, except for valve 129, The valve returns to its default state. As discussed, valve 129 remains open. To avoid damage to the beads and bulb 129.   At this point the synthesizer has suspension in the reactor or how much is in the parent vessel. Or has a bead suspension residue. Ensure all beads are transferred to the reactor A rinsing process including at least one rinsing cycle is used to It is.   The rinse cycle consists of washing the inner wall of the parent container with DMF spray in step 890. To release any bead suspension residue sticking to the wall. I can start. Valve 14 open to allow DMF spray to wash walls To Valve 90 opens to allow displaced argon to drain from the parent vessel. Inside The wall raises the level of DMF in the parent container to the level of the lower capacitive sensor 90S. And continue to spray DMF until the sensor turns on. This sensor Data is received by the control computer, which Immediately order on floor 892 to default all valves except valve 129. Return to the state. The contents of the parent container are then bubbled with argon in the manner previously discussed. And stir.   Alternatively the parent container can be refilled with DMF and then Any beads that mix it and stick to the inner wall or frit of the parent container The suspension residue can also be rinsed by letting it loose. First, the stage At 884, the parent container is refilled with fresh DMF. Refill open valve 10 By allowing the DMF to enter the parent container from a pressurized delivery system. Can be accomplished. Valve 90 will open again and the displaced argon will exit the parent vessel. And enable. The level of DMF in the parent container is determined by the level of the top capacitive sensor 99S. At the top of the bell, the top capacitive sensor 99S turns on. Day of this sensor Data is received by the control computer, which in step 886. Command immediately to reset all valves except valve 129 to default. You. The parent container is then filled with argon bubbles at step 888 in the manner discussed above. It is mixed by introducing it into a container.   The mixture of DMF and bead suspension is then opened in step 894 by opening valve 91. Then pressurize the parent vessel with argon and add the mixture to valve 129 (of the rinse process). (Open all the way through) to the common manifold at the summit To the reactor. Open the selected valve 101-109 to see if the fluid is a reactor. From the lower to the lower Maihord. The valve 110 opens and the bottom manifold DMF is discharged from the holder. The frit on the bottom of the reactor is a Strain and remove. Eventually, the parent container is discharged. Sensor 120 is the top of the parent container Common manifold Turns off when there is no fluid present in the tube bound to. This sensor Data is transmitted to the control computer, and the fluid sent to the parent container is Notify that there is no remaining. Control computer opens valve 91 for another 5-10 seconds Continue to open, pressurize the common manifold on the top, and remove any residual mixture. Also move into the reactor. After 5-10 seconds all valves except valve 129 failed Return to the state. One rinse cycle is complete.   As discussed above, a large number of rinse cycles are used to Ensure that the bead suspension is transferred from the parent vessel to the reactor. 2-3 rinses The cycle proved to be sufficient.   After completing all rinse cycles, turn on all valves, including valve 129. Return to default at step 896. Valve 129 is a bee that includes the rinse process. The suspension stays open throughout the redistribution process to beads and polymers. Note that the damage is minimized. Next, the reactor bank is emptied at step 898. Drain all fluids in the manner discussed in.   From the steps in Figures 19A-19B, the use of an argon bubble allows the parent vessel and reactor to be The bead suspension can be evenly distributed between the reactors regardless of the flow rate in each flow path between Can be recognized. Argon bubble is stable between bead suspension and DMF reagent Visible marker is provided and the sensor indicates the status of the reactor as long as Allows you to make decisions You. As mentioned above, the ability to hold a stable argon bubble is preferred for DMF. Due to its excellent physical properties, that is, a high contact angle between DMF and Teflon.   However, fluids other than DMF should be used when carrying the beads to the reactor. And can be desirable. This is especially true when the alternative fluid is stable argon. It can create problems if you don't have the physical properties that help create bubbles. For example, DNA Acetonitrile (MeCN), used as a solvent for synthesis, is a stable argon gas. Unable to produce foam. Thus, different beads for dispensing the beads into the reactor Method is needed.   19C-19D show the contents of the parent container when stable argon bubbles cannot be produced. An alternative set of instructions issued by the control computer to mix the I will tell. This technique is particularly applicable to only a small number of reactors, eg from 4 to 5 It is useful when   Steps 854a-860a are only open if only a smaller number of valves 101-109 are open. Step 85 of Figure 19 except that only the one corresponding to the selected reactor is open. Same as 4-860. The selected reactor will be placed in the reactor van prior to the redistribution stage. Partially filled with a solvent such as MeCN to replace the argon present in the It is. Thereafter, the selected reactor and parent vessel are filled with MeCN. Parent container 20 The amount of MeCN in 0 depends on the level of the second capacitive sensor 99S (when filling is complete). MeCN) Flow stops. When complete, the selected valve then returns to its closed state.   Next, in step 872a, the bead suspension placed in the parent vessel is loaded into the selected reactor. In preparation for dispensing into, mixed. In step 874a, the bead suspension is then topped Introduced into the common manifold and selected for valves 91, 129 and 101-109 By opening the container, it is distributed to the selected reactor. These valves are Sensor 120S is placed in the tube that connects the parent container to the common manifold on the top. It is held open until it detects the absence of fluid. Argon bubble Are not manufactured, the sensors 101S-109S are not used. Small number of reactors Only accept the beads, so it takes beads to reach each of the reactors The times are almost equal, thereby generally equal for each of the reactors Guarantee distribution.   In step 890a, the rinse process as previously discussed in Figures 19A-19B is used. Used to ensure that all beads are transferred to the reactor. Rinse process Once completed, all valves, including valve 129, are removed in step 896a. Return to line status. Next, the reactor bank, in step 898a, drains all fluid. Is done.   FIG. 20 shows a control computer for filling the reactor with the desired reagents from the delivery system. 3 is a flow chart showing a series of commands issued by a user. Regarding Figure 20 The steps discussed are also outlined in Figures 21A-21D. You. This process begins at step 900 where the lower manifold is inert argon. Suppose that it is satisfied only at. Figure 21A shows a reactor van with an empty manifold. The relevant parts of the graph are shown in the figure. The lower manifold is tested first in stage 902. Filled with medicine. Valve 100 will open to allow reagents to enter the lower manifold. Allowed, and valve 110 opened to replace the lower manifold. The generated argon is discharged. When the reagent is detected by sensor 110S, all The valve returns to default in step 903.   In some examples, the tube connecting the parent container and the common manifold on the top is Sensors 101S-109S should be operated inadvertently or prematurely while Can be For example, by a stray drop of reagent before it actually reaches it The sensor can be activated. This makes it insufficient to deliver to the reactor Amounts of reagents may be present in the tube.   To reduce or eliminate problems with premature sensor activation, The control computer, in step 904, prefills the tubes. Optionally programmed to open valves 101-109 for a set amount of time Can be In general, the tubes are detected before being detected by sensors 101S-109S. Is set to approximately 75% in advance. This as described The stage does not rely on the use of sensors 101S-109S. Hide And pre-filling at least a sufficient amount of sample to be injected into the reactor. Even if the drug is in the tube and the sensor activates prematurely, the mixing is sufficient. Guaranteed to do in minutes. In step 905, the predefined time After finishing in preparation to fill the valve, the valve returns to its default state.   In step 906, the tube connecting the reactor and the lower manifold was , Sensor filled with reagent. The filling process saves time It can be done in parallel or sequentially. Valve 100 opens and pressurizes Allows pressurized argon from the delivery system to enter the lower manifold. Selection Flaked valves 101-109 open continuously or in parallel to allow reagents to Allows entry into the tubing associated with the lower manifold. Chu When the reagent level in the chamber reaches the upper photosensors 101S-109S, the photosensors When turned on, the control computer immediately closes the associated valve 101-109. When all sensors 101S-109S are on, all valves are in step 908. Return to the closed state. FIG. 21B schematically shows the results after this filling step is completed. explain.   Next, clean the bottom manifold in step 910 as previously discussed. . Figure 21C shows the relevant portion of the reactor bank with a clean manifold and And a tube between the top optical sensor, eg 101S, and the valve, eg 101. Shown is a column of reagents inside the lobe portion. After the bottom manifold has been cleaned, Return all valves to default on floor 912. Then in the tube The reagents are pushed through the frit into the reactor.   Pressurized delivery by opening the argon valve 122 to push the reagents into the reactor. Argon from the system allows to pressurize the lower manifold. Discharge bar Lube 120 opens again.   In step 913, start the spiral (stirring) motor to determine the predetermined Start stirring the reactor bank for a certain amount of time. Time depends on the contents in the reactor Long enough to allow to be thoroughly mixed. Generally, about 4 hours is appropriate Was found to be present, but can vary depending on the type of synthesis.   During the mixing period described above, at steps 914, valves 101-109 were pre-loaded. Open for the programmed time and allow a small amount of reagent to flow into the reactor. Usually during the synthesis process , The reactor is filled and discharged. Each time the reactor is drained, The beads are allowed to dry and aggregate together to form a "bead cake". You. Step 914 fluidizes the reactor contents and dissolves the bead cake. Bee When it is at or near the bottom of the reactor, the swirl is most effective, so a small amount Should only be injected. Otherwise, the bead cake is at the top of the reactor It may take more time to float and melt it. Stage 915 odor And return the valve to the default state after the predetermined amount of time has expired.   At step 916, the remaining portion of the reagent is removed by opening valves 101-109. Then push it into the reactor. Each container has its associated sensor 101S-109S Is detected and the control computer is filled until the valve is closed. When all sensors 101S-109S are off, all reactors are full. mixture To improve, the container bank is swirled as soon as it is filled. Written earlier As shown, the reactor opens all valves 101-109 at once or sequentially. Be programmed to fill containers in parallel or in series by obtain.   Alternatively, the reactor can be filled without relying on sensors 101S-109S. You. For example, valves 101-109 are sufficient to fill the container to the desired level. Where it can be opened for a pre-programmed time. 0. 5 seconds to 1 second Was found to be sufficient. However, this time depends on the number of containers used. It can change depending on That is, the larger the number, the longer the time required. Figure 21D , Shows a diagram of the reactor after being filled.   The volume of reagent to be pushed into the reactor also depends on valves 101-109 and sensor 101S. Can be easily changed by changing the length or diameter of the tubing between ~ 109S Note that This change couples the reactor to an injection valve, such as valve 111. Tubes with different lengths or cross sections Can be easily accomplished by replacing with a tube.   Increasing the diameter of the reactor itself is also advantageous in some instances. Can be For example, it may be desirable to mix the beads with two or more reagents at the same time. You. Such mixing is followed by the steps described in FIGS. Can be introduced into the reactor. Next, as shown in FIGS. The second reagent is introduced into the reactor by repeating the steps. However, In doing so, prior to the introduction of the second reagent, tubes 241-249 and 271 The remaining argon at ~ 279 allowed the argon bubbles to bubble with the bead suspension and the second sample. Will be placed between the drugs. React to remove argon bubbles from the reactor The inner diameter of the vessel can be made larger to reduce the height of the bead suspension in the reactor. And thus facilitate the escape of argon bubbles through the bead suspension. You. After removal of the argon bubbles, the bead suspension and the second reagent are as described above. They are mixed with each other by swirling them.   Figure 22 shows the control computer to inject the activated amino acid reagent into the reactor. 3 is a flow chart showing a series of commands issued by a computer. 23A to 23C Outlines the relevant steps of the amino acid injection process. Again, at the start The lower manifold is cleaned, i.e. it contains only argon. Is assumed. Figure 23A Is the starting reactor, lower manifold, valves, sensors and related The diagram of the made tube is shown. Diagram of alternative arrangements for reservoirs 231-239 6A.   Implements a rotatable carousel for holding multiple groups of reservoirs For the embodiment, the carousel is rotated to store the selected group in step 917. Adjust the container and tube. Once the appropriate reservoir is selected, step 918 is reached. The manifold is an amino acid activating reagent, such as 0. 2 M HBTU and 0. 6M Fill with a solution of DIEA in DMF to DCM 3: 1. Manifold, Figure 20 The procedure described above, i.e. valve 100 until sensor 110S is turned on. And 110 are filled in such a way. Then in step 920, everything Valve closes.   The activating reagent is then placed in the tube connecting the reactor and the lower manifold. enter. Valves 100 and 120 are opened and in step 922 a pressurized activation test is performed. Allow the drug to enter the lower manifold. The selected one of valves 101-109 , Lower until the lower optical sensors 111S-119S detect the presence of fluid. Stage 924 At, all valves close again. Figure 23B shows that after this initial filling step, The presence of fluid in the relevant parts of the reactor is shown graphically.   To complete the injection, reopen valves 100 and 120 and go to step 926. The pressurized activation reagent Put in the lower manifold. The selected one of valves 101-109 opens in parallel, Allows a column of pressurized activating reagent to advance up the tube described above. You. At the same time, the relevant ones of valves 111-119 are opened and amino acids are pressurized. The activating reagent is poured into the column that goes up and mixed with the activating reagent. Figure 23C shows This injection step is shown diagrammatically.   When each sensor 101S-109S detects the presence of fluid, the control computer Close the valves 101-109 associated with the sensor of. In stage 928, all When valves 101-109 are closed, all valves in the reactor bank are returned to their default state. You. The mixture of amino acid and activating reagent is preferably placed in the tube described above for about 2 minutes. Allowing to intervene to ensure proper activation in step 930.   Then, in step 932, the bottom manipu is used in the manner discussed above with respect to FIG. Ludo is cleaned. Next, the valves 101-109 and the upper sensors 101S-109S The columns of the mixture between are in the manner discussed above with respect to Figure 20 in steps 913-916. , Push it into the reaction tube.   Figure 24 shows a control computer for transferring the bead suspension in the reactor bank to the parent vessel. Indicates the sequence of commands issued by the server. At the start, the reactor was discharged , And the lower manifold is assumed to be filled with argon. Reactor First, it was filled with solvent in step 936 in the manner discussed above with respect to FIG. You. Then all valves return to their default state.   The bead and reagent mixture is then vortexed to form a suspension at step 938. You. Alternatively, the mixture of beads is vortexed at the same time the reactor is full. Can be Such a process improves the speed of suspension and causes the beak at the bottom of the container. Help prevent the cells from agglomerating with each other. The contents of the reaction tube are step 94. At 0 transferred to parent container. Valve 122 opens to allow lower manifold Pressurize with gon. Valve 129 opens and the bead suspension is removed from the reactor bank into the parent container. To be able to move to. A selection of valves 101-109, preferably continuous And then opened, allowing argon to move the contents of each reactor toward the common manifold at the top. Allows to be blown upwards and into the parent container. The contents of the reactor are , Continuously transferred to the parent container. The above movement also simultaneously opens valves 101-109. It can be done in parallel by opening it, but continuous movement is It makes it possible to keep the argon pressure high and is therefore preferred. In addition, preferred Preferably, each of valves 101-109 is held open for about 4 seconds to Ensure that substantially all of the contents of the obtained reactor are transferred to the parent vessel. Valves 90, 122 and 129 are held open during this process.   After all reactor contents have been transferred to the parent vessel, the reactor is rinsed and another transfer Process can happen. Repeat the above steps to rinse the reactor Start at 936 by refilling the reactor with DMF. Stage 937 Valve 129 remains open during the line cycle, as shown in To prevent damage to the beads and bulb 129. Parent container is preferred Has a volume of at least three movements. At the end of recombination, preferably the parent The fluid level in the container has reached below the lower capacitive sensor 90S, The parent container is drained by opening valves 110 and 91 until turned off. You. Three rinse cycles have been found to be sufficient.   Then all valves, including valve 129, were defaulted at step 942. return. The contents of the parent container were swirled in step 944 and discussed in relation to Figure 20. Mix beads from different containers in the same manner. The beads are removed from the parent container If so, the parent container preferably has a lower volume of fluid in the parent container. Valve 10 and until the sensor is below 90S and turns it off. By opening 91 it is discharged in stage 946. All valves continue And return to default at step 948. The mixture containing the beads is then used Therefore, it can be removed from the parent container.   Alternatively, the beads are re-loaded into the reactor in the manner discussed in connection with Figures 21A-21B. Can be distributed. After redistribution, all valves return to their default state.   G. FIG. Overall software diagram   Figure 25 shows the source code included here as Appendix I It is a flowchart of. Module 950 represents a user. Command in Tarpreter 952 receives text commands from the user. Alternatively, The user operates a synthesizer using the menu system 953. You can enter a command to skip. Received by menu system 953 The command that has been created can be used by the command interpreter 952. Support routine in the support routine module 962. Call him. The command interpreter 952 can also be written by the user. Interpret commands entered by or otherwise. Then the interpreted man Code calls and executes the support routine in the support routine module 962. You. In addition, the interpreted command is printed by the display formatter 954. It is formatted and displayed on the display 762.   Module 958 includes multiple macro files. One macro file is , The actual steps that must be taken to perform synthesis or build a library, for example. Define the order of At its most basic level, macro files A separate set of commands to control the valve and read sensor information. Including the macro where it is included. The macro should drain the reaction vessels 201-209. Other basic macros can be used to perform even higher level functions.   Command interpreter 9 from macro file 958 The macro received by the 52 is the synthesizer library controller 96 Passed through 0. Synthesizer Library Controller 960 Call a macro for composition. For example, one macro can be set up before composition begins. Can identify the initial global variable to be loaded.   FIG. 25 shows a support routine module for operating various support subroutines. Joule 962 is shown. One such subroutine is autofill, This is a service to automatically fill the reaction vessel until all sensors are on. Brutin. The support routine 962 is a command interpreter 95. 2 or receive input from menu system 953. Option list 9 64 includes pointers to functions and the like. FIG. 26 shows the option list 9 64 inputs and outputs, and command interpreter 95 2 and the support routine module 962.   An initialization file to hold global variables and settings. There is also IL 966. The initialization file 966 may open, for example, a closed valve. Therefore, a value indicating the amount of time to supply the strike voltage to the valve is held. Looka Uptable file 968 is, for example, a selected reaction vessel in which the monomer is appropriate. Synthesizer command library controller 9 to allow entry Collaborate with 60. The lookup table file 968 is For example, synthesize each reaction vessel list, its corresponding tag monomer, and desired polymer. It may include a list of the monomers needed to do so.   FIG. 25 also shows a log file 970. The log file 970 is Deinterpreter 952 and synthesizer library controller 96 Receives input from 0. The log file 970 is an operation file for diagnostic purposes. Includes commercial data. The entry in the log file 970 is, for example, Contains information related to the called macro.   The associated file 972 is a list of each reactor and its associated valves and sensors. Including The related file 972 includes each reaction vessel and its associated valves and sensors. To simplify the task of addressing synthesizers, synthesizer library Cooperate with both the Troller 960 and the Support Routine 962.   The digital command output by the support routine 962 is Enter the Larel driver 974. The parallel driver 974 may be a PCD, for example. It may be an IO120-PI / O board 766. The parallel driver 974 To drive the solenoid valve, the valve controller is connected through its I / O channel. The troll signal 976 is output. As mentioned above, valve control The signal is further processed by controller circuit 768. Parallel dry Bar 974 is a stirring stepper motor Step motor controller signal 978 to control To put. Synthesizer optical sensor, ultrasonic sensor and capacitance sensor Sensor input 980 from the Parallel driver 97 for processing by support routine 962 via Received by 4.   FIG. 27 shows the data structure required to control the valve. Valve index The VINDEX array 986 receives the number of valves 984 as an input and V It has a pointer to the ALVES array 988. Of the VALVES array 988 Each element contains a pointer to a data byte 990. Each data byte 9 90 includes 8 pits of valve data information. Valve data for a given valve The bit containing the data information is the address of the data byte 990 and the data byte. It can be accessed by a shift value indicating the relative position of that bit in 990. Day Each bit of the Tabite 990 can be manipulated to properly open or close the valve. . The valve data information in each bit corresponds through the output port 992. Control the valve.   FIG. 28 is required to receive sensor information from the input port 994. Shows a simple data structure. Information indicating the binary state of each sensor is data byte 996. It is stored in 1 bit. Access the information in data byte 996 In order to access the SENSOR array 998, the sensor number is used to You. SENSOR array 9 Each element of 98 contains a pointer to the appropriate data byte. Each database Byte 996 includes 8 bits of sensor data information. A bar for a given valve The bit containing the loop data information is the address of the data byte 996 and the data Access by a shift value that indicates the relative position of that bit within tabite 996. You. H. Windows interface   The control software is Windows interface or workspace May be included. In general, Windows interface is On screen display Rectangular graphical user interface (GUI) with one or more windows for ). Additional Windows targets are: Various sizes and formers if desired Display (eg tilted or cascaded). Window At the top, There is a menu bar with multiple user command selections, Each of them Is Additional submenus, And software for use with the application subject Wear tools may be included. Windows is also Display and manipulate screen objects Includes an area for making. This area is Control computer system memory -A workspace or view for the user to interact with the Port.   Windows interface is Select the screen target you are interested in Includes a screen cursor (Cursor) or pointer for activating the beak. Mau Pos like Su In response to the user motion signal from the inching device, Curser Desired Float across the screen to screen position (ie move freely) . During or after the cursor movement, The user Select the target as is conventionally known And can generate user event signals to operate Push and drag). For example, The window is Screen components It can be closed by clicking (selecting) it, Resizeable or black Can be dealt with. Kistro with keyboard accelerator or hotkeys Is the equivalent Perform these and other user actions through the keyboard Be prepared for That is, Windows interface is Control computer Provides a more intuitive approach to interaction with data.   The basis of the Windows interface is Message or event It is driven by a digital camera. This model is probably The operation, In Figure 25 As illustrated by the command interpreter, Used traditionally Best by contrasting the operation of a modal or sequential configuration Described in. In this way, The reader, Event driven system additions Will understand the great flexibility and complexity.   The modal program is Well-defined start, A series of pieces with middle and end It includes another operating block or mode. That is, The program is Pretty hard to operate Fixed sea Chasing Kens, Each step is The program must complete before proceeding to the next step It is necessary.   The modal program is It is relatively easy to design and implement, Generally acceptable Not easy. The design certainly ensures that you have all the necessary information, but Only Then The condition is that the user operates in the format instructed by the program. Especially, Since the program is made according to a predetermined set of modes, You The You can move from one mode to another without first completing the required mode. I can't go into mode. The user deviates from this sequence Simply not allowed. This inflexibility of modal programming is Work in the real world Not enough to do.   On the other hand, The event driven configuration is Avoid pre-selected sequences, Instead Select an event loop. The event loop is User and system events Is a centralized mechanism for processing messages about. that is, many kinds An event queue for retrieving and sending messages to your window class And mechanism.   The message is How many applications does the operating system And hardware events such as mouse switch-on or keyboard Whether to manage the pressing of the This is Win on MS-Windows Converted to a message by the dows event handler. Programming From the perspective, The message is simply, A data structure that contains information about a particular event. This message The structure is Message identity that serves as a symbolic constant for a particular event May include a fire. For example, The message from the window target is Hui Creation of the window, Closure, It may include information about moves and resize. Additional Bent data It can be used as a message parameter. For a given parameter The exact interpretation is Varies with each event type represented. The input message is , Collected in the system wide queue, It will be directed to the appropriate window. these The message is Timer and screen paint (screen refresh) menu With the sage, Passed to the target application of interest.   Retrieve messages from system queues, And put them in a suitable application Yon (This is next To handle any message that arrives) One mechanism is provided. Each window is All wins of that type Belongs to a particular window type that defines some common features of dough. For each type Is A window that handles all messages sent to that type of window It is accompanied by the louze function. Windows is a message that belongs to a particular application. If you can place a sage, Has application queue available. When the application is ready to receive input, that is Just read the waiting message. If you can't find anything , Or if the message exists for another application of higher priority If you do Windows passes control to other applications.   Event-based systems such as Microsoft (TM) Windows The general mechanism for retrieving and sending messages in For example, Petzold, C, "Programming Windows" Second Edition, micro Soft press, 1990 and Custer, H, "Inside Windows NT" Microsoft Press, See 1993. For more information, State of Washington, Redmon Microsoft's Windows software available from Microsoft Seen during air development. Each of the above disclosures By quoting Incorporated herein for all purposes.   Figure 33 shows 3 illustrates a GUI deployed on a control computer. GUI , It includes a rectangular window 1301 with a workspace 1303. Wind At the top of c Menu bar with user command selections 1306-1313 There is 1305. Each of these command selections Controls the operation of the synthesizer Includes an additional subunit that contains commands to save. These command selections are Ma By invoking the appropriate command with us, Do the synthesis automatically or manually Give the user the flexibility to do it dynamically.   The GUI is User friendly environment, You In other words, with the intention of minimizing the effort required to program a synthesizer. Designed. For example, Dialog box target or a set of dialog boxes The target Accompany each command. When the command is triggered, Appropriate dialog The target is displayed in the workspace, Interaction so that users can enter the necessary information Purposely promote. The Help command 1313 Users across this process Provide information to help Using the diamond target, The user Traditional textile You can program the synthesizer without using strike commands.   Figure 34 shows Shows submenu 1320 associated with Macros option . The user Mouse over the submenu item 1321 or 1322 By putting in Each can issue a Learn or Run command. Windows In the environment, The macro is One of the separate commands to control the synthesizer A set is an object that includes, for example, the commands described in connection with FIGS. 16-24. Le Using the arn command 1321, The user defines a macro to perform a particular function. Can be justified. The submenu is also Assigned to trigger available commands Keystrokes can be identified. For example, "Alt + L" Run the Learn command Used for.   Once the macro is "learned", A learned macro or something predefined To run another macro of Run command 1322 may be selected. Fig. 35 Is Run piece 7 illustrates an example of a dialog box target that is displayed when a command is invoked. This da The target of the dialog box is Area 1325 (list of available macros) Combination box). To select a macro The user Select  Put the name of the macro in the Macro space 1324. Alternatively, The user Ma By switching on and dragging the us, Desired in space 1325 You can scroll until the macro is selected. Then the user Macro is space Enter the number of times to be repeated in 1326. Finally to run the macro, The user Mouse over RunMacro button 1327 or RunSMacro button 1328 Switch on. The RunSMacro command Execute macro functions sequentially, That is, the system is instructed to run one reaction vessel at a time. Cancel selection 13 When 29 is selected, It will exit the dialog box object 1326. Help selection 1330 Information about another choice in the dialog box target provide.   Figure 36 shows When the Groups option 1310 on the menu bar is selected A displayed sub-menu 1335 is shown. The submenu 1330 is Specific reaction Defi used to define a set of valves associated with a group of vessels Includes ne command 1331. Once defined, The valve group is Memo as a group target Stored during Lee. Computer memory is Can include many group objects Can, Each defines a unique valve group.   Submenu 1330 also Fill the selected reaction vessel for a specified period and Includes an Open / Pulse command 1332 for discharging the liquid. Connect with Open / Pulse command When the dialog object that was kicked is activated, This is displayed. This dialog pair The elephant box (somewhat similar to the one shown in Figure 35) Then selected It contains a combination box that lists the available group objects in place. The user selects the desired group, Assigned space given by the dialog object During the pace, Enter the desired duration for pulsing the selected valve. In the reaction vessel To fill The user switches on the Open button, Valve opened Indicates what should be done. next, The user Pulse or SPulse (reaction vessels are filled sequentially The filling process is started by switching on the button (for filling). To drain the reaction vessel, The user Close button, Or Pulse or Switch on the SPulse button.   Use the Fill / Drain command 1333 The user Automatically using sensor The container can be filled or drained. When the Fill / Drain command is selected, that is, Displays a dialog box similar to the Open / Pulse command dialog box target. User -Is Select a group of valves, And AutoFill or SAut to perform the filling function o Fill button, Or AutoDrain or SAutoDrain button to empty the reaction vessel Press the switch. In some embodiments, After triggering the sensor Delay closing or opening the valve In order to A Time Delay option may be provided. This feature Sensor activated In situations where the reaction vessel has not fully reached its desired level when Useful for. By setting a delay option with a specific delay period, The reaction vessel can be appropriately filled. As you can see, Connect to the Group menu The command I had was Flexibility in choosing the combination of reaction vessels to use during synthesis Giving the user sex.   Figure 37 shows Variables A sub that contains options available for menu selection. A menu 1340 is shown. The create command 1341 is Performed in a macro Variable that is defined. Variables are typically For example, a value, for example a composite of one time Used in the context of changing from one composition to another composition. Make a macro for each time value Instead, Variables are simply defined in time. Use set command 1342 Before each synthesis cycle, The variable is set to the desired value.   FIG. 38 shows Shows submenu 1345 associated with Diagnostics option . The submenu 1345 is Synthesizer user tight control and diagnostic purposes To give access to information about synthesizers for Valves1346 , Includes Sensors 1347 and Mix 1348 commands. When the Mix command 1348 is selected Come It starts the stirring motor, For a period specified by the user, Reaction vessel Mix.   In FIG. 39, When the Valves command is selected, The Valve Diagnostic dialog box target 1390 is displayed. The user Via the dialog box target 1390, Entry corresponding to desired valve You can control the operation of any valve in the synthesizer by selecting. Was For example, Valve 100 in Bank 1 Switch on the cursor on box 1391 It is opened by clicking (clicking). For convenience, All valves Clos It can be closed by selecting the e All command 1397. Cancel command 1 398 is The dialog box target 1390 is closed.   Figure 40 shows The Sensor Diagnostics dialog box corresponding to the sensor command. The target object 1391 is shown. As shown Dialog box target 1391 is , It includes a box 1396 associated with each sensor. Box 1396 Correspondence Notify the user of the sensor status. For example, If the box is in it If you have an "X", This indicates that the sensor it is related to is on . vice versa, Empty box Indicates that the sensor is off.   Figure 41 shows Submenu 13 displayed when the File option is selected 50 is shown. The New command 1351 A new composition set for performing composition Make up file, Modify command 1352 Pre-existing for editing Allows the user to select a composite file. Complete the synthesis setup file Once done, User can also save 1354 to save the file in memory Is Sa Invokes the ve As command 1355. The Print command 1357 Selected composition Print the file. The print Setup command 1358 is I want a printer The mode of For example, print a file in overview mode. Exit coma Hand 1359 is Triggered to leave the File option.   In some examples, For example, before each synthesis, Or a new valve group is selected When User must invoke Load CFG File Command 1356 May want to form a system with. the system, Which one to use In order to let you know if the valve is suitable, Read the appropriate file. actually, The CFG file is Make a map of the valve, I.e., the valve for each selected reaction volume Connect with a vessel.   42 to 44, Create a synthetic setup file, Used in modifying Indicates the target of the dialog. In FIG. 42, Set Associate dialog box Target 1360 is Checkmark in the appropriate box contained in space 1361 By adding Allows the user to select the desired reaction vessel. Flight For convenience, To easily select or discard all reaction vessels, Check All button 1362 and an Uncheck AlL button 1363. Check Bank button 1 364a-1364d, The user can Allows you to choose. When the Cancel button 1368 is selected, That Aborts the synthesis setup process. As mentioned above, Help button 13 69 is Provide information to help the user through the process. Synthetic set To continue the up process, User can set Associate dialog box Close and turn on the OK button 1365, And Synthesis Setup dialo Display the box target.   Figure 43 shows Synthesis Setup dialog box shows target 1370, this Allows users to start Macro 1371 for synthesis, Loop Macro 1372 and And End Macro 1373 are defined. Start Macro and End Macro are For example, each Includes commands for washing the reaction vessel before or after the synthesis process. Loop Macro Is Contains commands to perform compositing. These commands Selected reaction Injecting the amino acid building block and the oligonucleotide into a container, Reaction vessel Pooling the contents of the And mix them to form a bead suspension To do, And including dispensing the bead suspension into the selected reaction vessels. This shot, The pooling and distribution process is Complete a synthesis cycle. The synthesis cycle is Nu repeated according to the value put in the meber Synthesis Steps parameter 1374 . Once Macros is defined, User can create Synthesis Setup dialog box Save the contents of the target Set by turning on the OK button 1375. Continue the upgrade process. also, The user can press the Cancel button 1376. The process can be stopped by putting a check.   FIG. Amino & Oligo Setup dialog box target 1380 is shown. As shown Box Contains entry 1381 associated with each reaction vessel . The user Enter the desired amino acid code and nucleotide code in the entry You. For example, If the entry for reaction vessel 1 (RV01) is "VATG" If you include "CCGA", This is a synthesizer with amino acid V and oligonucleosides Ctide ATGCCGA is injected into reaction vessel 1. After the appropriate code is entered To The user Switch on the OK button 1382, Setup process Complete. To stop the process Cancel button 1383 is provided . As you can see The synthetic library is Easy to use with this synthetic setup procedure Can be made.   Once the synthesis setup is complete, User can start the synthesis process You. In FIG. 45, The user first Select Synthesus Option 1308 , Then select Go command 1391 to start the process. Alternatively, User -Is A single marker for performing functions similar to those stored in the composite file. You can build black.   In FIG. 46, The GUI is During composition or macro execution Status screen 1400 is displayed. As shown The status screen is Two separate Area 1401 and 1402. The first area 1401 is For synthesis Display the information that you want. For example Start Macr o, The names of the Loop Macro and End Macro are listed. in addition, Loop Number Rye 1408 Inform the user about the number of loops remaining in synthesis.   The second area 1402 is Show the filename of the currently running Macro I do. As mentioned above, Macros are nested with other macros, Higher level The function can be achieved. The status screen is 10 of the called macros Designed to list up to nested levels 1409a-1409j Can be To display additional status or configuration information, Command Line 1410, Timing line 1411, Message line 1412 and RV LA An in 1413 can be provided. For example, Command line is Which ma It may indicate whether a black command is being executed. The Message line is if it applies , Display a text message as pre-programmed by the user. The Timing line is Display the amount of time remaining for the valve to be opened or closed. R The V line is Inform the user which reaction vessel will be used. Time to Co mplete bar 1414 Use the percentage of time remaining before the synthesis is complete. Inform the user.   At any point during composition or macro execution, The user Pre-designated fan By pressing the operation key, that is, F12, Switch to User Abort mode sell. User Abort mode is Macros that are not defined as part of composition Synthesis Pause and execute processes without having to stop them Enable users to do When activated, System temporarily suspends execution Then And as shown in FIG. 47, the User Abort dialog box target 142 Display 0. at this point, User can insert arbitrary macro for execution . this is, Type the name of the desired macro in the Select Macro box 1421, Then press the Run Macro button 1422 to switch on the mouse. Can be.   The User Abort dialog box also targets Equipped with an Ignore button 1424, This This is a synthetic process as if the system had not entered User Abort mode. Allow users to continue The skip option 1423 Synthesis Tells the system to first skip the current command in the macro before continuing I do. The Retry button 1425 Causes the system to run the current macro from the beginning. Abort Button 1426 is Allows the user to cancel the composition.   Figure 48 shows 14 illustrates a submenu 1430 associated with the Edit command. Edit frame And Seed by selecting the appropriate item listed in the submenu. Users can easily access various objects, Allows you to change. Associat When the e command 1431 is selected, Set Associate stored in memory A dialog box listing the targets displays the targets. To choose the desired target Than, The Set Associate dialog box Target is displayed. You. at this point, User edits the contents of the dialog box target with the mouse it can. When finished, The user switches on the OK button, Save changes You. Similarly, The user By selecting the desired target, Group, Macro, Va You can edit riable and Code targets. next, The selected target is Place in editor And Allows the user to change the text.   As described above, The sensor is Unintentionally triggered by the presence of a single drop It may be. this is, In particular, the solution used during the synthesis cycle has a high concentration of air bubbles. When included in It can be a problem. Avoid unintentional triggering of the sensor or To mitigate Their sensitivity can be programmed.   The sensor is It is programmed using the Machine Config command 1456. This The command Some entries, Fill%, Drain%, TimeOut% and PtsAve Display the target box of the Config dialog including. Fill% is When is the reaction container To decide if it ’s a cup, Specify the percentage that the sensor remains on. Set. For example, If 80% is entered, The sensor is Time it is read Must be 80% on. Drain% is Decide when the reaction vessel is empty In order to Specifies the percentage that the sensor remains off. For example, if When 20% is entered, The sensor is 80% off the time it is read No. Timeout is Sensor is switched on or off Specifies the delay time before being turned off. PtsAve is When the sensor is on or off Specifies the number of points or readings used to determine the percentage between. You The user You can specify the sensor or sensors to be read. After that, The user Switch on the OK button, This is a system Automatically configure the system.   Figure 49 shows Event-driven by the control software shown in FIGS. It is a flow chart which shows construction. As shown Of the synthesizer GUI The I / O Object module 1450, which is the heart, Various target modules that make up the GUI Facilitate communication between the two. When the control software is started, Module 14 60 is Load the configuration file into the I / O Object module. These files Is Variable Objects module 1456, Lookup Table Objects Module 1 457, Macro Objects module 1458, Group Objects Module 1459 , Valve Object Array Module 1461 and Sensor Object Array Module Used to construct 1462.   The Lookup Table Objects module Valves and sensors their corresponding Map it to a reaction vessel, Each individual valve and sensor associated with a particular reaction vessel. This file is used to eliminate the need to manually address the server. Macro Ob The jects module Store a list of defined macros. Variable Object s module Store the defined variables, The defined group object is Group Objec ts Stored in the module.   The Valve Object Array module Identify each individual valve in the system Store the array. To open and close the valve, The I / O Object module Opponent Scan the Valve Object Array module until is found. Find a partner When, The Valve Object Array module Emits a signal to control that particular valve . To control the valve group, The I / O module is Together with the Group Objects module Similarly, scan the Valve Object Array, It selects the appropriate control signal I will send it to Lube.   The Sensor Object Array module Identify each individual sensor in the system Store the array. The I / O module is Sensor Object A until a partner is found By scanning the rray module, I can read the sensor, That To read a particular sensor. To read the sensors, I / O and Group O The bjects module Scan the Sensor Object Array module, Read Choose an appropriate sensor.   The GUI also Dialog Box Objects Module 1453 and Synthesis Object Includes module 1454. The Dialog Box Objects module A command Contains the dialog box target that will be displayed when invoked. Synthesis Object The module is Store the current synthesis setup file. Run another synthesis To do The GUI is Synthe desired synthesis setup file from memory sis Read into Object module. vice versa, The contents in the Synthesis Object module are , Written in memory, Save the synthesis setup file.   The Main Window Object (MWO) module 1452 GUI on screen Communicates with the View Object module 1455, which displays the main window (Fig. 33) of I do. In response to a user command, The Message is sent to MWO. This Messe The page is Interpreted to determine the appropriate command to execute. For example, if When the Macro command is received, MWO is for I / O target, Dialog Box Objects Module to Macro Dialog Box Target and Macros Objects Module To receive a list of macros from. The View Object module This feeling Information from the I / O module, Display it on the GUI.   In one embodiment, The I / O module is If the computer is in error When, Contains a Watchdog routine to stop the synthesizer. For example, I / O target Jules Programmed to send one pulse to the synthesizer every 2 seconds be able to. if, with some reason, The synthesizer receives these pulses If you don't It is thought that the control computer malfunctioned, The power is turned off.   Example   The example below Further exemplifies the present invention, Is intended to limit the invention. There is no.   Example 1 Library creation and screening   In this example, How to combine peptide synthesis products on resin beads But, Oligos on beads that correspond to each amino coupling step in the synthesis It shows whether it can be explicitly identified by attaching a nucleotide identification tag. Each Is Telling you which amino acid monomer was attached at a particular step in the synthesis, The entire sequence of peptides on any bead is obtained by reading the tag on that bead. Can be inferred. Beads collection Fluorescence activated cell sorting (FACS) equipment Using Screened for binding to fluorescently labeled anti-peptide antibody Can be These beads, to which the antibody binds strongly, By FACS Can be separated, Oligonucleotides attached to individual sorted beads The otid identification tag can be amplified by PCR. The sequence of the amplified DNA was determined Is We demonstrate the identification of peptide sequences that bind to antibodies with high affinity. High capacity ( capacity), Information storage based on oligonucleotide code, Amplification methodology and fluorescence By combining the classification based on The method is Natural and non-natural chemical structures Identify the identity of each member of a vast library of molecules synthesized from building blocks To do And individual with high affinity ligands for biological receptors It provides a means for the rapid and efficient separation of beads.   In this example, Single-stranded oligonucleotide L- and D-amino acid construction Combined peptide synthesis using blocks and 10 μm diameter polystyrene beads Code Used for The oligonucleotide tag is Have high information content, Extremely high Can be detected and decoded for sensitivity, And using this method, It is stable to the reagents used for tide synthesis. Peptides and nucleotides are Each bead has both a single peptide sequence and a unique oligonucleotide identification tag To have many copies of Assembled in parallel alternating composition. Orient Gonucleotide is Have both a common 5'and 3'-PRR priming site, Thus the beads can serve as templates for PCR. Enko The synthetic library About 8. 2 x 10^FiveAnti-dinorph containing a hepta-peptide In B monoclonal antibody D32.39 (Barrett and Goldstin, 198 5. See Neuropeptize 6: 113-120. This is cited in the present invention by reference. Individual beads that bind strongly to the antibody are selected for binding to Screened using a fluorescence activated cell sorting (FACS) instrument. Classification After PCR amplification of the oligonucleotide tags on the beads, the DNA was Sequenced to determine the identity of the Tide ligand.   A. Reagents and general methods   The monodisperse 10 μm diameter bead material used in this work was manufactured by Pharmacia (Pharmacia). custom-made functionalized with 1,12-diaminododecane linker purchased from It was a macroporous styrene-divinylbenzene copolymer made. beads The Genecia assembler support of Pharmacia Pharmacia Monobeads ™ that are not derivatized with rinkers. Uge Rustad and Moke, 1980, Advanced Colloidal Interface Cy See Enns, 13: 101-140, which is incorporated herein by reference.   All protected amino acids are available from Backchem Biosciences Inc. Obtained from Ted. PCR and sequencing primers are based on Applied Bios The stem model 394 was synthesized using an oligonucleotide synthesizer. An authentic sample of a peptide is the Fmoc protected amino acid, HBTU / HOB t in situ activation chemistry, and with 40: 1: 1 TFA / water / ethanedithiol Applied Biosystems Model 431A Peptide With Deprotection Synthesized with a synthesizer. These peptides are water / acetonite as eluent Rainin C with Lil / 0.1% TFA₁₈Purified by HPLC on reverse phase column (> 95% purity), structure was verified by mass spectroscopy.   B. Parallel combination of 69-base oligonucleotide and opioid peptide dinorphin B Success   C of opioid peptide dynorphin B Terminal 7 amino acid fragment H-Arg-Gln-Phe-Lys-Val-Val-Thr-NH₂(RQFKVVT) ( SEQ ID NO: 2) is a 69-mer oligodeoxynucleoside on 10 μm diameter beads. Cide (ST08) was used to synthesize in parallel. The sequence of ST08 is Five'-ATC CAA TCT CTC CAC (ATC TCT ATA CTA TCA) TCA CC [TA TC CT AT TT TT  AC] CTC ACT CAC TTC CAT TCC AC-It was 3 '(sequence identification number: 20). This arrangement The underlined part of the row corresponds to the PCR priming site and the region in parentheses is It is homologous to the primers used to sequence this template. In parentheses The closed 14 base sequence represents the coding sequence of the template.   The beads were initially succinimidyl 4-O-DMT-oxybutyrate (Molecular Prob es), and N-Fmoc-2,4-dimethoxy-4 '-(carboxymethyloxy) -benzhi Drillamine (ie acid cleavable Knorr carboxamide linker) ) Or N-Fmoc-Thr (t-Bu) -OH (for non-cleavable experiments). Treated with a mixture of Nzotriazole. DMT protected hydroxy on beads The ratio of Fmoc-protected amino groups to residue is spectroscopically about 20: 1. It has been determined. The beads consist of the 3'-O-methyl-N, N-diisopropylate of the following nucleotides: 20 cycles on an automated synthesizer using a phosphoramidite Subjected to gonucleotide synthesis: N⁶-Bz-5′-O-DMT- (7-deaza) -2′-deoxy Adenosine (Very and Associates, Michigan, Ann Arbor), N^Four-B z-5'-O-DMT-2'-deoxycytidine, and 5'-O-DMT-thymidine (Glen Research ).   The beads are then removed from the device to remove the Fmoc protecting group. For this, it was treated with 10% piperidine in DMF for 5 minutes. The first amino acid residue ( After coupling N-Fmoc-Thr (t-Bu) -OH), the beads are loaded with possible unreacted amine. Treated with acetic anhydride and 1-methylimidazole in DMF to cap . All peptide conjugation reactions were performed for 20 minutes and 0.11M Fmoc- in DMF. Amino acids, 0.1M HBTU, 0.1M HOBt and 0.3M DIEA were included. Next The beads were loaded with 2 cycles of nucleotide addition on the synthesizer (TCA Detritylation; Tetrazole-catalyzed phosphitylation; Acetic anhydride In water; oxidation with iodine in acetonitrile / water). The successive steps of amino acid binding and dinucleotide addition are performed using the peptide sequence RQFK. Synthesis of VVT (SEQ ID NO: 2) and oligonucleotide coding region It was repeated until the build was complete. Perform an additional 35 cycles of oligonucleotide synthesis After that, the beads were treated with piperidine / DMF (1: 9, 8 minutes), thiophenol. / Triethylamine / dioxane (1: 2: 2, 4 hours), ethylenediamine / Ethanol (1: 1, 5 hours at 55 ° C), and TFA / water (20: 1, 1 hour) More sequentially processed to fully deprotect both peptide and oligonucleotide chains did. In experiments with acid-cleavable linkers, the TFA deprotection reaction The supernatant was concentrated under reduced pressure and the isolated crude peptide was then analyzed by HPLC. Was.   C. Build the encoded library   The parallel synthesis described above was used to construct the library. Site of peptide synthesis N-Fmoc-Thr (t-Bu) OBt and succinimidyl 4 on all beads as described above. By coupling a mixture of -O-DMT-oxybutyrate, D Different from the NA synthesis site. The placement of oligonucleotide tags in the library The columns differed from ST08 only within the coding region. Oligonucleotide The 3'-conserved region of ST08 was initially loaded with a total bead amount of 35 mg (approximately 1.75 x 10⁸Bi Above). The Fmoc protecting group was removed, and the above beads amounted to 7 Divided into equal parts. 7 different α-N-Fmoc protected amino acids (side Chain protecting groups are shown in parentheses): Arg (N^G-Pmc), Gln (Trt), Phe, Lys (^tBoc), V al, D-Val and Thr (^tOne of Bu) has been combined. Next, each part is Two rounds of reotide synthesis were submitted. Uniquely identifies different amino acid residues The respective sequences of the attached dinucleotides are TA, TC, CT, AT, T T, CA and AC. The beads are then pooled, mixed thoroughly and the whole bead Amount of Fmoc was deprotected.   Bead splitting, peptide binding, oligonucleotide dimer synthesis, bead replation This cycle of sollation and Fmoc removal was repeated a total of 7 times. The last Fmoc protecting group Was not removed. Instead, use 35 cycles of oligonucle Subjected to reotide synthesis. The library was then completely deprotected as above.   D. Library coloring and FACS analysis   The library section (typically 0.5-2 mg of beads) is loaded with blocking buffer Suspend in (PBS, 1% BSA, 0.05% Tween-20) and incubate at room temperature for 1 hour. I started. The beads were pelleted by centrifugation and a solution of mAb D32.39 (block Resuspended in 10 mg / ml in rocking buffer). Suspend the suspension on ice for 30 minutes. Incubate, pellet by centrifugation, and wash with blocking buffer. Was. The beads were then loaded with phycoerythrin-conjugated goat anti-mouse antibody ( Suspended in a solution of Molecular Probes) on ice for 20 minutes. Blocking beads Wash in liquid and Fluorescence Activated Cell Sorting (FACS) device (Becton Dickinson FACStar)  Plus) for dilution into PBS. Bee combined with mAb D32.39 Were identified by their acquired fluorescence. The most strongly colored 0 in the library. PCR each beads from 17% and from the category with the lowest fluorescence (about 98%). Sorted into small centrifuge bottles. The specific binding of D32.39 to beads was 10 μM. To the soluble peptide Ac-RQFKVVT-OH (SEQ ID NO: 2) at the final concentration Was blocked by preincubation of the mAb.   E. FIG. PCR of beads-bound template   Buffer system supplied by the manufacturer (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1%  Triton X-100, 2 mM MgCl₂) In 0.2 mM dATP, dCTP, and dGTP, 0.8 mM dUTP , 2 mM each plastic Immers, 3 unitsTaq  Polymerase (Promega) and 1 unit of uracil DNA Glycosylase (Gibco BRL) (total volume 70 L) is used to perform PCR amplification Was. Primer sequence, 5'-ATC CAA TCT CTC CAC-3 '(SP13) (SEQ ID NO: 2 1) and 5 '-(bithione) -GTG GAA TGG AAG TGA-3' (SP14) (SEQ ID NO: 22) ) Were homologous and complementary to template ST08, respectively. PCR reaction is 95 ℃ , 30 seconds denaturation, 50 ° C, 1 minute primer annealing, and 72 ° C, 1 minute It consisted of 45 cycles of extension. The reaction is 20% acrylamide or 2% low melting point aga It was analyzed by electrophoresis in a rose gel.   F. Sequencing of PCR products   Biotinylated PCR products from individual reactions were co-transfected with streptavidin. Separation was performed using magnetic beads (Dynal) loaded. Unbiotinylated chain Each bead sample was treated with sequencing cocktail after alkaline elution and washing of . The dideoxy sequencing was performed using the primer 5'-ATC TCT ATA CTA TCA-3 '(SP15) Row identification number: 23) andBstUsing polymerase (Bio-Rad), the manufacturer's instructions As indicated, except that deoxy-to dideoxynucleotide triphosphate (Ph armacia) at a ratio of 1: 100.   G. FIG. Determination of peptide binding affinity   The binding affinities of various peptides for the monoclonal antibody D32.39 are competitive. It was measured in a combined experiment. cAM D32.3 fused to a consensus substrate sequence for P-dependent protein kinase Tracer peptide containing a known epitope for 9 (LRRASLGGGR RQFKVVT (SEQ ID NO: 24); 50 pM) is [g-³³P] ATP (Re et al., 1989,Proc. Natl. Acad. Sci. USA  86: See 558-562. In the present specification by reference Incorporated) to give a highly specific activity radiolabeled and the peptide of interest Of various concentrations (10 μM to 1 pM). This peptide mixture was added to D32.3 9 (0.1 Gmg / ml) coated polystyrene wells. Sample 2 at 4 ° C Incubate for a period of time, count the radioactivity associated with each well, and Used to make the tube. IC under assay conditions₅₀Is the dissociation constant of the peptide ( K_d) Must be close to.   Example 2: Synthesis and stability studies of thiazolidinones   The following examples relate to the synthesis of thiazolidinones using the method of the invention. this Synthesis is based on US patent application no. 08 / 265,090 (June 23, 1994) Are incorporated herein by reference.   A. Preparation of double-labeled thiazolidinone   H₂N-S-TentaGel (500 mg) (commercially available polystyrene-based resin, doy Tukuni, Chubingen, Rapp Polymere, 1 g, 0.30 mmol / g load), α- Fmoc-Gly-OH (2- labeled on carbon¹³C, 99%, Massachusetts Cambridge Isotope Laboratori, Andover, County es). The resin is Ac₂Cap with O, deprotect with piperazine, and The Fmoc-photolinker was attached as its OBt activated ester. Press the resin again. Capped, deprotected and unlabeled as anhydrous with Fmoc-Glycine-OH. I responded. One additional round of capping and deprotection was to remove free amine resin. occured. PhCHO labeled at carbonyl (carbonyl-¹³C) 99%, 0.75M from Cambridge Isotope Laboratories, Inc., Andover, Sa. 70 ° C in ACN containing 3Å molecular sieves with 2.0M mercaptoacetic acid Reaction for 2 hours at 37 ° C. yielded a double labeled thiazolidinone resin. Thorough resin Washed (3X5 ml CH₂Cl₂ 3X5 ml DMF, 3X5 ml CH₂Cl₂, 3X5 ml MeOH, 3X5 ml  CH₂Cl₂， 3X5 ml Et₂O), and vacuum dried.   B. TFA stability study   Part of resin (20 mg) 95% TFA / 5% H₂Treat with O for 1 hour, then CH₂Cl₂, MeO H and Et₂Washed with O. Resin gel-¹³C NMR analysis shows two labeled charcoals. Shows no loss of thiazolidinone, as evidenced by the relative integrals of the primes Was. See panel B in FIG. Some of the photolinker or thiazolidinone The breakdown is believed to result in a decrease in the integral value of benzyl carbon. This experiment Both Azolidinone and Photolinker are stable to TFA treatment Indicated.   C. DNA synthesis stability studies   Put a portion of the resin (20mg) into a standard DNA synthesis cartridge and fill in A, C and T 40 of DNA synthesis using Leotide (used as their phosphoramide) Cycling was followed by iodine oxidation after each cycle. Mock dimethoxy trime Chill (DMT) was removed by adding 2% TFA / CH to the resin at the beginning of each cycle.₂Cl₂At It was done by reason. Remove resin from cartridge, wash with DMF, gel -¹³It was analyzed by C NMR spectral analysis. See panel A in FIG. Profit The spectra obtained show almost no destruction of the photolinker or thiazolicinone molecules. Or none at all. Photolysis of part of the resin (2 mg) in PBS buffer pH 7.4 for 3 hours The released thiazolidinone was analyzed by HPLC. Panels of Figures 31 and 32 See Le A. The data show that thiazolidinone was released in high purity and photo Both linker and thiazolidinone are significant by treatment with standard DNA synthesis reagents It was not possible to change to.   Example 3: Combinatorial synthesis   The combinatorial synthesis of YGGFL was performed using the above synthesizer. Synthesis is a reaction Performed in Vessels 1-6, Reaction Vessels 7-9 were unconnected. Added to beads The other six amino acids were L, E, G, Y, A and F. YGGFL is the other Identified with peptide. Beads were added to the parent container (29.4 mg) and placed in DMF. Suspended. Each synthesis cycle consists of redistribution, peptide bond, capping, amine Deprotection, a collection of deprotections for FMOC, at DMF The steps involved rinsing and recombining in the parent container.   Following synthesis, labeled Hertz antibody was introduced to the mixed beads. Hertz antibodies mostly bind to YGGFL. FACS analysis shows the existence of YGGFL Was identified. This is because this particular combination was synthesized by a synthesizer Prove that. The experiments featured a diverse collection of peptides, including YGGFL. And can be synthesized by a synthesizer.   The invention has been described in some detail by way of illustration and example for purposes of clarity and understanding. Although described in detail, certain changes and modifications may be made within the scope of the appended claims. Is obvious.   Example 4: Investigation of bead distribution using the device of the present invention   Investigate whether the mixed beads from the parent vessel are evenly distributed to the reaction vessel In order to do so, some beads were biotinylated. One biotinylated bead I put it in my reaction container. Eight other reaction vessels with non-biotinylated beads Got it inside. The synthesizer parented all beads from 9 reaction vessels. Copy in a container. Take a sample from the parent container and set the fluorescent streptavidin Allowed to bind with biotin on the mouse. Fluorescence activated cell sorter (FACS) Analysis shows that about 9.1% of the beads in the parent container were biotinylated. Then bee Rearranged into 9 reaction vessels and paired with biotinylated beads in each vessel. The percentage of total beads was determined by FACS analyzer. Table 5 shows certain reaction vessels The mixed beads in have a ratio of biotylated beads to total beads that is similar to the parent container. To indicate   It should be understood that the above description is illustrative and not restrictive. above Many embodiments will be apparent to those of skill in the art upon reviewing the description. Therefore, the scope of the present invention is , Which is not determined by the above description, but by the appended claims and Etc. should be decided according to the whole range.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI C12Q 1/68 9309-2G G01J 1/42 N G01J 1/42 0273-2J G01N 21/27 Z G01N 21/27 9282-4B C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ), AM, AT, AU, BB, BG , BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, JP, KE, KG, KP, KR, KZ, LK, L R, LT, LU, LV, MD, MG, MN, MW, N L, NO, NZ, PL, PT, RO, RU, SD, SE, SI, SK, TJ, TT, UA, US, UZ, VN (72) Inventor Kedder, Heim, USA 94306, Palo Alto, College Avenue 433 (72) Inventor Dower, William, Jay. United States, California 94025, Menlo Park, Brunner Avenue 2307 (72) Inventor Barrett, Ronald, W. USA, California 94087, Sunnyvale, Carlisle Way 562 (72) Inventor Gallop, Mark, A. USA, California 94022, Los Altos, Orange Avenue 511 (72) Inventor Niedels , Michael, C. United States, California 94024, Red Head City, Temesuka Le Way 727

Claims (1)

[Claims] 1. In a method of synthesizing various molecules on a plurality of substrates, a step of distributing the substrates into a plurality of reaction vessels, using different reagents in each of the reaction vessels, Coupling a first portion of various molecules, transferring the substrate into a mixing vessel through a flow tube, and mixing the substrate, passing the substrate from the mixing vessel through the flow tube into the reaction vessel Redistributing to a container, and coupling a second portion of the diverse molecule to the first portion of the diverse molecule to form the diverse molecule on the substrate. 2. The method of claim 1, wherein the redistributing step comprises redistributing at least a portion of the substrate through a common manifold. 3. An apparatus for parallel coupling reactions on a solid support, comprising: a parent vessel, at least one reaction vessel storage location connected to the parent vessel, wherein the reaction vessel storage location is the coupling reaction. A plurality of reaction vessels for performing in parallel), and a plurality of flow tubes between the parent reaction vessel and the reaction vessel (wherein the flow tube is a flow path between the reaction vessel and the parent vessel). A delivery system for delivering reagents to the reaction vessel storage location, and a programmable system for controlling movement of the solid support between the parent vessel and the at least one reaction vessel storage location. A device that includes a computer. 4. 4. The apparatus of claim 3 including a common manifold, the common manifold being connected to at least one of the plurality of flow tubes. 5. The delivery system further comprises means for delivering the reagent from the first part of the device to the second part of the device, and the first part has a higher pressure than the second part. An apparatus according to claim 3. 6. The apparatus of claim 3 wherein said reaction vessel storage location further comprises means for simultaneously agitating the contents of said plurality of reaction vessels. 7. The reaction vessel reservoir is further for delivering the reagent in a first mode in parallel to a selected one of the plurality of reaction vessels, and in a second mode, for reacting the reagent to the plurality of reactions. 4. The device of claim 3 including valve means responsive to command signals from the programmable computer for sequential delivery to selected ones of the containers. 8. An apparatus according to claim 3, wherein the parent container, the reaction container storage location and the delivery system are isolated from the atmosphere during the parallel coupling synthesis reaction. 9. A valve for transferring the solid support between the parent vessel and the plurality of reaction vessels, wherein the valve is while moving between the parent vessel and the plurality of reaction vessels, The device of claim 4 wherein the solid support is the only valve passing therethrough. 10. The reaction vessel reservoir is further for delivering the reagent in a first mode in parallel to a selected one of the plurality of reaction vessels, and in a second mode, for reacting the reagent to the plurality of reactions. 10. The apparatus of claim 9 including valve means responsive to command signals from the programmable computer for sequential delivery to selected ones of the containers. 11. In a method of performing a coupling reaction on beads in parallel, a step of transferring the beads in suspension from a parent container to a plurality of reaction containers, in parallel, on the beads In the coupling reaction, transferring the beads from the plurality of reaction vessels to the parent vessel, and mixing the beads. 12. The method according to claim 11, wherein the step is repeated a predetermined number of times. 13. 12. The method of claim 11, wherein the transferring step further comprises transferring at least a portion of the beads through a common manifold. 14． An optical array block for use with an optical detector for detecting the presence of liquid in a substantially translucent tube, wherein a beam of light from a transmitter of the optical detector is passed through the center of the tube to the optical detector. Means for directing onto a condenser and means for inhibiting light (other than the above beam of light) emitted from the transmitter of the optical detector from reaching a portion of the condenser of the detector. A block containing ,. 15. The optical array block according to claim 14, wherein the directing means includes a pinhole hole passing through the optical array block. 16. The optical detector comprises two forks, the transmitter is arranged on the first fork, the condenser is arranged on the second fork, and the optical array block is further arranged between the two forks. The means for frictionally engaging the optical array block and the tube are arranged so that the longitudinal axis of the pinhole hole intersects the radial axis of the tube at an angle of 90 degrees. 16. An optical array block according to claim 15 including means for: 17． 15. An optical array block according to claim 14 wherein said directing means further comprises means for placing said tube between the transmitter and the light collecting portion of said optical detector. 18. One two-way valve having a first port and a second port, one three-way valve having a first passage and a third port, the third port as the first passage A programmable computer responsive means for selectively contacting; a programmable computer responsive means for selectively contacting said first port with said second port; and said first Means for sealingly connecting the port of said to said first passage, thereby forming a manifold, for delivering a reagent. 19. The third port is connected to a first tube carrying a reagent, and the other of the first port and the second port is connected to a second tube carrying argon, where the 15. The system of claim 14 wherein a first tube carries the reagent either towards or away from the manifold. 20. At least one reaction vessel reservoir (wherein the reaction vessel reservoir includes a plurality of reaction vessels and a plurality of monomer reservoirs, each of the reservoirs being coupled to one of the reaction vessels); and A reagent reservoir, the common reagent reservoir being connected to the at least one reaction vessel reservoir for delivering a common reagent to the plurality of reaction vessels through a common manifold. Combined synthesis device for carrying out coupling reaction. 21. Each different molecule in the library is covalently bound to a solid support and tagged with one or more different tags, where each of the one or more different tags is A method for synthesizing a tagged molecular library comprising various hydrocarbon regions and molecular hooks, the tag also being covalently bound to the solid support, comprising: (a) Partitioning the supports among a plurality of reaction vessels by a stochastic method, (b) exposing the supports in each reaction vessel to the first chemical building block, (c) pooling the supports, (d) ) Distributing the support among a plurality of reaction vessels by a stochastic method, (e) exposing the support in each reaction vessel to a chemical building block, and (f) steps (a)-(e). Repeat at least 1 to 20 times No way. 22. 22. The method of claim 21, wherein the solid support is a linker. 23. 22. The method of claim 21, wherein the solid support is Monobead ™. 24. 22. The method of claim 21, wherein the molecule is attached to the solid support by a linker. 25. 25. The method of claim 24, wherein the linker is cleavable. 26. Each of the one or more different tags has a cleavable linker that attaches each of the one or more different tags to the solid support, a molecular hook, and various lengths that connect the linker to the molecular hook. 22. The method of claim 21, comprising a hydrocarbon chain of. 27. Each of the one or more various tags is an expression 22. The method of claim 21, wherein n is 1-10, X is a cleavable linker, and R is a molecular hook. 28. 27. The method of claim 26, wherein X is a photocleavable linker. 29. 22. The method of claim 21, wherein said molecular hook is selected from the group consisting of biotin, activatable groups and highly associated peptides. 30. 26. A tagged molecule library according to claim 25, wherein the molecule is cleaved from the solid support and then incubated with the receptor under conditions which lead to binding of the ligand to the receptor. Screening method. 31. The molecule is a peptide, the solid support is beads about 50-500 μm in diameter, the cleavable linker is a mixture of cleavable linkers, and only a portion of the molecule on the beads 31. The method of claim 30, wherein is cut prior to the incubation step. 32. A method of synthesizing a synthetic peptide library comprising a plurality of different members, each member comprising a peptide consisting of a sequence of amino acid monomers linked to beads, the beads also comprising the sequence of monomers in the peptide. Is linked to one or more oligonucleotide identification tags, wherein the amino acid monomer is Fmoc protected and piperidine is used to remove the Fmoc protecting group by 5-15%. A method comprising removing Fmoc by treating with piperidine for 5 to 60 minutes or with 15 to 30% of piperidine for 1 to 30 minutes. 33. 33. The improvement according to claim 32, wherein the beads are about 10 [mu] m in diameter and consist of macroporous styrene-divinylbenzene copolymers derived with dodecylamine linker. 34. 33. The improvement according to claim 32, wherein the beads are Monobead ™. 35. The amino acid monomer has a tBu side chain protecting group, TFA is used to remove the tBu side chain protecting group, and the oligonucleotide tag comprises 7-deaza 2'-deoxyadenosine. Improvement according to section 32. 36. A method according to claim 26 for detecting the presence of one or more different tags, the step of cleaving the one or more different tags from the solid support, the one or more different tags. Immobilizing said tag, linking an amplifiable detectable group to said molecular hook on said one or more different tags, amplifying said amplifiable detectable group, and said amplifying Detecting the presence of possible and detectable groups, wherein the presence of the amplifiable and detectable groups is indicative of the presence of the one or more different tags. 37. 36. The method of claim 35, wherein the amplifiable, detectable group comprises an oligonucleotide sequence capable of binding to the molecular hook. 38. A method for determining the synthetic sequence of molecules bound to a solid support in a tagged molecule library according to claim 21, wherein the one or more bound to the solid support is A method comprising individually detecting the presence of different tags, wherein the presence or absence of said one or more different tags is indicative of the occurrence of a particular synthetic step in the synthetic sequence of said molecule. 39. The individual detection of the one or more different tags physically separates the one or more different tags according to their structure, thereby obtaining a separation pattern for the one or more different tags, Immobilizing the one or more different tags to preserve the segregation pattern, treating the tags with an oligonucleotide sequence, thereby selectively binding the oligonucleotide sequence to the one or more different tags A step of amplifying the oligonucleotide sequence, a step of detecting the presence of the oligonucleotide sequence, wherein the presence or absence of the oligonucleotide sequence indicates the presence or absence of the one or more different tags. Identifying the one or more different tags present by the relative position of the one or more different tags in the separation pattern, which is indicative; The occurrence of a specific synthetic steps in the synthesis order of the child, said comprising one or more different presence or step of correlating with the absence of a tag, the method according to the paragraph 38 claims.