CN1134156A

CN1134156A - Synthesizing and screening molecular diversity

Info

Publication number: CN1134156A
Application number: CN 94193984
Authority: CN
Inventors: 杰弗瑞·H·苏格曼; 理查德·P·拉瓦; 海姆·凯答耳; 威廉·J·都瓦尔; 罗纳德·W·贝瑞特; 马克·A·盖罗普; 米歇尔·C·尼德尔斯
Original assignee: Affymax Technologies NV
Current assignee: Affymax Technologies NV
Priority date: 1993-11-02
Filing date: 1994-11-02
Publication date: 1996-10-23
Also published as: WO1995012608A1; BR9407947A; AU1128095A; GB9609254D0; AU703472B2; EP0726906A1; NZ276860A; CA2175587A1; JPH09508353A; GB2298863A; GB2298863B; EP0726906A4

Abstract

A device and method for efficiently synthesizing diverse molecular products on substrates. A parent vessel (200) contains a suspension of substrates. The suspension is pressurized with argon and transferred to a plurality of reaction vessels (201-209) in one or more reaction vessel banks where monomer addition reactions take place. Optionally, the substrates may be tagged with a tag monomer. A vortexing motor (300) vortexes the contents of reaction vessels (201-209) during monomer addition reactions to enhance synthesis. After the desired monomer and/or tag monomer addition reaction, the suspension is pressurized with argon and transferred back to parent vessel (200) for mixing. Thereafter, the suspension may be pressurized with argon and reallocated among reaction vessels (201-209) for further synthesis.

Description

Synthetic and screen multiple molecule

The application is that the United States Patent (USP) series number of application on November 2nd, 1993 is 08/146,886 and 08/149,675 part continuation application, and each piece of writing is all incorporated by reference by this paper.

The disclosed part of this patent file comprises content protected by copyright.The copyright owner does not oppose the facsimile copy of your patent document or patent disclosure, because it appears in the patent document or record of patent and trademark office, in any case but protect all copyright rights whatsoever in others.

The present invention relates generally to synthetic a large amount of differing moleculars and therefrom identify and separate method and apparatus with usefulness and required active compound.The invention still further relates to identification marking is mixed in these compounds so that have the identification of the compound of desired characteristic.

The part of macromole acceptor can be identified through a large amount of different peptide of molecular biology or synthesising chemical technology preparation by screening.The recombinant peptide storehouse is by being established in the group that degenerate oligonucleotide is inserted into coding filobactivirus glutelin and the conjugated protein LacI. of DNA-.See Cwirla et al., 1990, Proc.Natl.Acad.Sci.USA.87:6378-6382; Scott ﹠amp; Smith, 1990, Science 249:386-390; Devlin et al.1990, Science 249:404-406; Cull et al., 1992, Proc.Natl.Acad.Sci.USA 89:1865-1869; With PCT publication number WO 91/17271, WO 91/19818, and WO 93/08278, and each piece of writing is all incorporated by reference by this paper.These can comprise greater than 10 with hangar ⁹Different peptides, wherein each peptide is fused in the big protein sequence that is connected with its hereditary physical property of encoding.The interaction of these storehouses and acceptor, by affinity purification several times, the selectivity exposure or be presented at E.Coli and the dna single clone in be amplified to disclose and be responsible for being screened effectively with the carrier of the identity of the peptide of receptors bind with sequencing.Be also shown in PCT publication number WO 91/05058 and WO 92/02536.

The chemical process of setting up peptide or other library of molecules is not limited to use only synthesizing of 20 kinds of group amino acids coding.Make it comprise alpha-non-natural amino acid and other molecular structure unit by amplification structural unit group, increased the difference of accessible sequence and structure widely.In some describe as the method that produces the synthetic molecules storehouse, these reaction product are separated three-dimensionally, and each library member's identity is determined clearly by synthetic character, these synthetic Geysen et al. that see, 1984, Proc.Natl.Acad.Sci.USA 81:3998-4002; Geysen et al., 1986, in Synthetic Peptides as Antigens; Ciba Foundation Sympo-sium 119, eds.Porer, R. ﹠amp; Wheelan, J. (Wiley, New York) PP.134-146; Fodor et al., 1991, Science 251:767-773; U.S. Patent number 5,143,854 and PCT patent publication No. WO84/03564; 86/00991; 86/06478; 90/15070; With 92/10092, each piece of writing is all incorporated by reference by this paper.

By " tea bag " (tea-bag) polypeptide synthesis prepared the storehouse that has greater than 3,000 ten thousand dissolubility peptides.See Houghten, 1985, Proc.Natl.Acad.Sci.USA 82:5131-5135 and United States Patent (USP) 4,631,211, each piece of writing is incorporated by reference by this paper.Each storehouse is synthesized and screens and is the degeneracy peptide mixt, and wherein each amino acid in this sequence is clearly defined.Iterative method screening (as in the competition binding analysis) and resynthesis are used in these mixtures of fractionation and the definite storehouse the active peptide of tool.See Houghten et al., 1991, Nature 354:84-86; Pinilla et al., 1992, Peptide Research 5:351-358; Blake, J.﹠amp; Litzi-Davis, 1992, Bioconjugate Chem.3:510-513; With PCT patent publication No. WO92/09300, each piece of writing is all incorporated by reference by this paper.

The synthetic agreement of cracking of utilization FurKa etc. sees the 14 international biological chemistry conference (1988) summary, Prague, Czech.5:47 (also see FurKaet al., 1991, peptide protein matter research international magazine 37:487-493; With Sebestyen et al., 1993, Bioorg.Med.Chem.Lett.3:413-4187, Lam and his colleagues have prepared and have comprised 10 ⁶The storehouse of the peptide that resin beads individual and 100-200 μ m diameter links to each other.See Lam et al., 1991, Nature 354:82-84; Lam et al., 1993, Bioorg.Med.Chem.Lett.3:419-424; In PCT patent publication No. WO92/00091, each piece of writing is all incorporated by reference by this paper.These beads are by screened with the acceptor incubation of a mark: be determined by visual inspection and be selected under the help of micromanipulator with the bead of receptors bind.Each bead comprises 50-200Pmol can be by edman degradation or the directly definite single peptide sequence of mass spectroscopy.In principle, can be in this way produce and have greatly different storehouse by the size that reduces these pearls.The sensitivity of peptide sequence determination technology is limited to 1Pmole, yet the scope of direct peptide sequencing analysis is had tangible restriction.Yet,, do not have analytical procedure to provide directly and the obvious sequence analysis when the structural unit group in storehouse is expanded when comprising D-or other alpha-non-natural amino acid or other chemical structure unit.

The high-level efficiency screening of a collection of chemosynthesis molecule and natural product (as microbial fermentation liquid substratum) plays a part crucial in the research of the lead-in wire compound of new pharmacological reagent traditionally.To making and the interest of the combinatorial chemistry of estimation molecular difference and correlation technique remarkable surging means important milestone in the development of drug discovery paradigm.See the Pavia etal. that is quoted by this paper, 1993 Bioorg.Med.Chem.Lett.3:387-396.Today, chemistry of peptides has become the main tool of exploitation combined method utilization in part identification.See the Jung ﹠amp that is quoted by this paper; Beck-Sickinger, 1992, Angew.ChemInt.Ed.Engl.31:367-383.This ascribes the validity of the different amino acid monomer of a large amount of structures to, relatively the high yield solid phase coupling of kind chemistry and with the synergy of the biological method of making the recombinant peptide storehouse.And the potential and special biologic activity of many low-molecular-weight peptides makes these molecules become the attractive starting point that medicine is found.See Hirschmann, 1991, Angew.Chem Int.Ed.Engl.30:1278-1301 and Wiley ﹠amp; Rich, 1993, Med.Res.Rev.13:327-384, each piece of writing is all incorporated by reference by this paper.Disadvantageous pharmacokinetic properties as the difference the oral bioavailability rate and removed rapidly in vivo, restricted the widely exploitation of peptide compounds as medicine.Nearest this understanding has evoked the researchist combination organic synthesis conceptual expansion outside the chemistry of peptides (has been seen the Bunin ﹠amp that this paper quotes to making known medicine such as benzodiazepine ; Ellman, 1992, J.Amer.Chem.Soc.114:10997-10998) and polymerizable molecular such as few the N-glycine (" peptide ") and the few carbamate that replace.See Simon et al., 1992, Proc.Natl.Acad.Sci.USA89:9367-9371; ZucKermann et al., 1992, J.Amer.Chem.Soc.114:10646--10647: and Cho et al..1993.Science261.1303-1305, each piece of writing is all incorporated by reference by this paper.

Though the large-scale storehouse of molecule can have big value in the performance of useful compound of identification or raising lead-in wire compound, the difficulty of screening these storehouses, particularly large-scale storehouse has restricted the effect that these storehouses should be obtained in reducing as drug discovery and cost of developing.As a result, the exploitation of making and screening the method for library of molecules is become the object of making earnest efforts studying, wherein each molecule in the storehouse comes mark so that the evaluation of compound with a special identifier.(see the PCT patent publication No. WO93/06121 that this paper quotes; Also see U.S. Patent Application Serial 946,239, proposed on September 16th, 1992 and proposition on September 18th, 762,522,1991, supra).In this method, chemical synthesis process typically be on the resin beads combination synthetic product by with synthetic in each coupling or other product produce that reactions steps is consistent to be connected the identifier mark and clearly to be determined with these beads.Each mark stipulated to take place in the interested reactions steps, for example in a certain specific step of which amino acid monomer in the peptide synthetic method by coupling.The structure and the evaluation of a compound on any pearl, for example the sequence of peptide can be released by the mark group of reading on the bead.Ideally, these marks have high information content, are suitable for very high sensitivity and detect and decipher and the stable reagent to adopting in synthetic.The notion of the chemosynthesis of oligonucleotide coding was also proposed by Brenner and Lerner.See that this paper quotes 1992, Proc.Natl.Acad.USA 89:5181-5183.

Adopt coding method to show at first to spread out from the diamines of quadrature differentiation to connect thing, parallel combination synthesis method can be used to produce a storehouse that comprises the solubility chimeric peptide of " adherent " line and " coding " line.See this paper Kerr et al. incorporated by reference, 1993, J.Amer.Chem.Soc.115:2529-2531.By the natural or natural amino monomers of amino acid encoded recording of four L-amino acid compositions on foundation " coding " line and the coupling of cement line.From equimolar peptide mixt, select compound by the acceptor affinity purification, and differentiate by HPLC.Determine the sequence of the coding line of each purifying molecule by the structure of Edelman degraded announcement cement line.Similarly the peptide coding method also is reported among the peptide research 6:161-170 in 1993 by NiKolaiev etc. recently.

The scope of this respect of coding method that the qualification of the sensitivity of Edelman method and efficient is had final restriction analysis the storehouse of limit difference.The utilization of oligonucleotide provides bigger hope, but still needs improving one's methods of synthetic oligonucleotide tagged molecule storehouse.And, still need the replacement methods that are labeled library of molecules synthetic and that screening is very a large amount of.

Be desirably in the synthetic a collection of different branch period of the day from 11 p.m. to 1 a.m on a large amount of solid supports such as the bead, other problem can occur.The example that use has a bead of the different molecular product of synthetic thereon be disclosed in by this paper incorporated by reference for example below application in, they are that the U. S. application series number that proposed on April 29th, 1992 is 07/876,792; The application serial no that on September 18th, 1991 proposed is that the application serial no that proposed on September 16th, 07/762,522 and 1992 is in 07/946,239.

Though obtained considerable success, above-mentioned technology has still run into certain restriction.For example, the synthesizing when beginning on bead of different products, it is essential that many Artificial Control of these beads are become.For example on April 29th, 1992 propose by this paper U. S. application series number 07/876 incorporated by reference, in 792, must hang a collection of bead in carrier separates bead, the monomer of finishing on the bead of separate groups adds reaction, sometimes separately mix the bead of these reorganization again, and repeat this process with selective reorganization synthetic bead like this.When comprising that a large amount of monomers and reaction comprise that many monomers add step, it is very dull that manual operation becomes.Remove this, the many products of " metering " synthetic become one makes the timid task of people.

From as seen last, the library of molecules of the synthetic and mark that screening is very a large amount of improve one's methods and equipment is expected for people.The present invention has satisfied these and other needs.

The invention provides improving one's methods of preparation and screening library of molecules, wherein each molecule is with specific in the storehouse, and the demarcation of decoding accords with mark easily.A kind of fast and effeciently equipment and the method for synthetic differing molecular product also are provided.

In one embodiment, the invention provides the product mark of combinational chemistry method and the reagent to the synthetic chemistry storehouse that structure is encoded.In an important embodiment, the invention provides a kind of by alternately on trickle bead, finishing the synthetic method of peptide and oligonucleotide with compatible synthetic method.Synthesis peptide library by a large amount of oligonucleotide codings of this combination synthetic method preparation is made up of many beads, comprises artificially encode significantly many copies of single stranded DNA mark of structure of related peptide of simple peptide (tool is determined in proper order) and sequence in each storehouse.Interactional problem between the fluorescently-labeled biological acceptor that can propose relevant storehouse effectively and obtain by flow cytometry and each bead of choosing by the energy that utilizes the single bead of FACS device class.Dna marker on the bead of classification amplifies by round pcr and the structure of the peptide part that is encoded is determined in order-checking.These storehouses can be used to, and high-affinity (nM) part of for example seeking acceptor is as anti--peptide mono-clonal.

Synthetic molecules of the present invention storehouse can prepare by synthetic compound on each of a large amount of solid carriers, and this compound is because different solid carriers and difference.Prepare compound with the method that comprises the following steps: (a) in multiple reaction vessel with the equal fractional bearer of random fashion; (b) carrier in each reaction vessel is exposed in the first chemical structure unit; (c) carrier is compiled; (d) in many reaction vessels with the equal fractional bearer of random fashion; (e) carrier in each container is exposed in the chemical structure unit; (f) repeating step (a) to (e) 1 to 20 time at least.Typically, the solid carrier of equivalent will be divided equally to each reaction vessel substantially.In an embodiment of present method, the chemical structure unit is selected from the amino acid group, and the compound that obtains is the peptide oligopolymer.

More specifically, the present invention relates to coupling chemistry some improvement relevant with these methods.The improvement of one of them relates to and is used for removing the chemistry of Fmoc protecting group in the alpha-amino group of joint or growthing peptide chain from these synthetic beads.Preferably, this removal effect is by with 5% one 15%, is preferably 10% piperidines and handles 5 to 60 minutes, be preferably 5 to 10 minutes and become effectively, though can adopt other condition as 15% to 30% piperidines processing 5 to 30 minutes.Other improvement relates to the activity chemistry of peptide coupled reaction, because when some automatic equipment is used to finish peptide storehouse synthetic of oligonucleotide mark, the invention provides a simple mixtures that reduces the HoBt/HBTU of agent delivery bottle.

In others, the present invention relates to the method for the library of molecules of complex sign, wherein each molecule in the storehouse and solid carrier is covalently bound and with one or more different chemical unreactive hydrocarbons marker marks.Wherein said marker comprises variable hydrocarbon zone and molecular hook.Preferably, this marker comprises the joint of the cleavable that this marker is linked to each other with solid carrier, the hydrocarbon chain of molecular hook and the tool variable-length that molecular hook is linked to each other with the cleavable joint.More preferably wherein said marker comprises those examples of following formula:

Wherein n is 1 to 10 or bigger, and x is the joint of cleavable, and R is a molecular hook.These molecular hooks preferably are selected from vitamin H, and complement that high association peptide is right and protected activable group if can be by the groups of photoactivation.The joint of preferred cleavable can be by the joint of photodestruciton.

The method that these unreactiveness hydrocarbon markers exist that detects also is provided.This method comprises that cracking goes out one or more different markers from solid carrier, then marker is fixed on second solid carrier.Then oligonucleotide sequence is amplified, detect its existence, wherein whether the appearance of oligonucleotide sequence is the sign whether marker occurs.

Another embodiment of the present invention provides the method for order of the synthesis step of the synthetic molecules that a kind of solid carrier of having determined to be labeled in the library of molecules links to each other, wherein the molecule in the storehouse with the unreactive hydrocarbons label by the chemical process mark.This method comprises the existence that detects the one or more not isolabelings on the described solid carrier respectively and as the existence of described each mark of the sign of the special synthesis step of appearance in the described molecule synthesis or there is not situation.

On the other hand, the present invention relates on the bead synthetic too little can not be on conventional flow cytometry equipment the method and apparatus in the isolating synthetic chemistry storehouse that is encoded.The size that these beads make the storehouse that obtains never bead the storehouse 10 ⁹Individually increase to 10 ¹³, for the Ku Keda 10 that bead is arranged ¹⁸Size.The invention still further relates to the method in these storehouses of screening.

The invention still further relates to the synthetic storehouse that is encoded of screening to define method with compound.An important aspect the invention provides and prepare, mark, and screening natural product storehouse has with the relevant important improvement in natural product screening field of the method for active compound with identifying with difference.

On the other hand, the present invention relates to a kind ofly identify fast effectively improving one's methods of compound the storehouse from library of molecules of the present invention.In the method, will show desired characteristic (as with receptors bind) a chain of and clone is so that the sequencing of a large amount of marks in the simple sequence assaying reaction from the oligonucleotide mark that is labeled compound library.If the compound that is labeled is a peptide, adopted the code pattern that is based upon on the genetic code basis, each the mark subclone from concatermer can have been selected and plasmid and the phage system of expression system as partly describing in above-mentioned background to other for peptide is analyzed further so.

In another embodiment, the invention provides the apparatus and method of effectively synthetic differing molecular product fast.According to special aspects of the present invention, synthetic different polymkeric substance in as the substrate of bead.Optionally, these beads with the building-up reactions of molecule marker in be " marked " simultaneously, only by embodiment, the synthetic molecules on bead can comprise peptide, and molecule marker can comprise oligonucleotide.Certainly, basic " structural unit " no matter when molecule has relevant molecule unanimity with other, the same available method described herein of other molecular product is synthetic.Embodiment comprises 2,3-benzodiazine, prostaglandin(PG) and β-corner mimetic crystal.

According to one embodiment of the invention, with transparent vessel mixing bead suspensoid.By common branch manifold the blended bead is allocated in a plurality of independently reaction vessels, in these reaction vessels, bead is exposed among the different selecteed monomers, these monomers are in reaction on the bead and preferably be coupled on it by covalent linkage.These beads optionally are exposed among the chemistry " marker " of same covalency or logical additive method and these bead couplings.Then reconfigure to transparent vessel these beads and mixing by branch manifold.Then in a plurality of reaction vessels, will mix the bead suspensoid and separate again, continue add monomer, process such as mix bead and redistribute.This method has caused a collection of the have bead of different components that forms on its surface or the formation of other substrate.

According to an aspect of the present invention, the present invention includes a kind of equipment and method of on substrate, synthesizing differing molecular.Substrate is dispensed in the reaction vessel of selection from a transparent vessel.Then reagent is added in the reaction vessel with synthetic a part of molecule.Then substrate is moved in the transparent vessel and mix.For further synthetic, substrate is reassigned in the reaction vessel.Continue this operating process up to having synthesized required one group molecule.Between synthesis phase, whole synthesizer and external environment sealing.

Broadly, the invention provides the equipment and the method for preparation and screening library of molecules, wherein each molecule in the storehouse is with specific, easily the identification tag of deciphering.

Further understanding to essence of the present invention and advantage can be with reference to following description and figure.

Fig. 1 represents the synoptic diagram of synthesizer of the present invention;

Fig. 2 represents the synoptic diagram of reagent reservoir;

Fig. 3 represents to be used for the 3-porthole valve of synthesizer;

Fig. 4 represents to be used for the 2-porthole valve of synthesizer;

Fig. 6 A represents to have the replaceable equipment of working face of reaction vessel that supports the rotatable circular disc travelling belt of many group reagents reservoir;

Fig. 7 represents low branch manifold valve, introduction valve, interconnecting between reagent reservoir and the reaction vessel;

Fig. 8 represents with round to the greatest extent agitator;

Fig. 9 represents the top of reaction vessel working face;

Figure 10 A and 10B represent-lower reaction vessel retaining clip;

Figure 11 A-11D represents that the optical sensor that utilizes according to an aspect of the present invention detects haply light straightening device for the existence of the liquid in the translucent pipe;

Figure 12 represents reaction vessel according to an aspect of the present invention;

Figure 12 A is illustrated in the reaction vessel temperature control cover on every side of Figure 12;

The cover among Figure 12 B presentation graphs 12A and the cross-sectional view of container;

Figure 12 C represents to have alternative embodiment that temperature is controlled the reaction vessel of cover;

The cover among Figure 12 D presentation graphs 12C and the cross-sectional view of container;

Figure 13 represents transparent vessel;

Figure 14 is the sketch of the electronics of control synthesizer;

Figure 15 represents the sketch of pilot circuit;

Figure 16 represents that the Be Controlled computer adopts the step of discharging all liquid in the reactor;

Figure 17 represents that the Be Controlled computer adopts the speciality step of cleaning the bottom manifold;

Figure 18 represents that the Be Controlled computer adopts the step that stirs the content in the transparent vessel;

Figure 19 A to 19D represents that the Be Controlled computer adopts the step that the bead suspensoid is redistributed to reaction vessel with transparent vessel;

Figure 20 represents pack into the step of reaction vessel of reagent that the Be Controlled computer adopts self-pressurization in the future to transport system;

Pack into the step of reaction vessel of the reagent that Figure 21 A-21D illustrative Be Controlled computer adopts self-pressurization in the future to transport system;

Figure 22 represents that the Be Controlled computer adopts amino acid monomer is added to step in the reaction vessel;

Figure 23 A-23C illustrative Be Controlled computer adopts amino acid monomer is added to step in the reaction vessel;

Figure 24 represents that the employing of Be Controlled computer is transferred to blended step the transparent vessel with the bead suspensoid from reaction vessel;

The data movement of the primary clustering of Figure 25 illustrative control software;

Figure 26 illustrative command decoder structure;

Figure 27 illustrative is used to obtain the synoptic diagram of valve data; With

Figure 28 illustrative is used to obtain the synoptic diagram of sensing data.

Figure 29 is illustrated in the equipment of synthetic combinatorial chemical library on the trickle bead.This equipment is made up of a vacuum manifold or the magnetic sheet that links to each other with solid carrier, and wherein solid carrier has the synthetic surface of the reaction site of a series of synthetic compounds of a tool.Division plate is made up of the corresponding reacting hole of a series of and described reaction site, and it is used to divide the member in storehouse after mixing step each time.This equipment also can be used to the synthetic of aid mark chemical libraries.

Figure 30 illustrates monitoring thiazolidine stability ¹³The use of CNMR.Panel C represents carrier-bound thiazolidine ¹³CNMR spectrum, wherein thiazolidine is at 2 of ring be that quilt is gone up in α position (position that is labeled with " * " expression) for the joint carbon back ¹³The C double-tagging.Panel B is illustrated in 95%TFA and handles carrier-bound double-tagging thiazolidine after 1 hour ¹³CNMR spectrum.Panel A be illustrated in the DNA in 40 cycles synthetic after carrier-bound double-tagging thiazolidine ¹³The CN-MR spectrum.

Figure 31 further specifies monitoring thiazolidine stability ¹³The use of CNMR.Panel C is illustrated in the α position of ring and is that (mark position with " * " expression) used on the α position for the joint carbonyl ¹³Atom carries out the carrier-bound thiazolidine of double-tagging ¹³The CNMR spectrum.Panel B is illustrated in the thiazolidine of the carrier-bound double-tagging of photodissociation after 90 minutes in the PBS damping fluid ¹³The CNMR spectrum.Panel A is illustrated in the thiazolidine of the carrier-bound double-tagging of photodissociation after 3 hours in the PBS damping fluid ¹³The CNMR spectrum.

Figure 32 represents to follow the trail of by the HPLC with carrier-bound thiazolidine reaction mixture synthetic and that 3 hours photodissociation prepares in the PBS damping fluid through the DNA in 40 cycles.

Figure 33 explanation is provided as the illustrated operator's interface (" GUI ") on the control computer.As shown in the figure, GUI comprises the rectangular window that has a working space 1303.At the top of window is the menu area 1305 that has user's command selection 1306-1313.Each command selection comprises the auxiliary next stage menu of control synthesizer operation.The user can be by selecting suitable command selection to operate with mouse.

Figure 34 has described GUI, shows that the next stage menu in the big menu is selected.

Figure 35 has described the dialogue case that moves big menu.

Figure 36 has described GUI, and the next stage menu that is presented in the group menu is selected.

Figure 37 has described GUI, and the next stage menu that is presented in the variable menu is selected.

Figure 38 has described GUI, and the next stage menu that is presented in the diagnostic menu is selected.

Figure 39 has described the valve diagnostic screen.

Figure 40 has described the sensor diagnostic screen.

Figure 41 has described GUI, and the next stage menu in the display file menu is selected.

Figure 42-44 expression starts synthetic dialogue case, it allows the user to select to be used for synthetic reaction vessel (Figure 42), select the beginning in each reaction vessel, circulation and end macros (Figure 43) and input amino acid symbol and oligonucleotide password (Figure 44).

Figure 45 has described GUI, and the next stage menu that is presented in the synthetic menu is selected.

Figure 46 has described situation about occurring the term of execution of synthetic or macros gas.

Figure 47 has described user's fault dialogue case.

Figure 48 has described GUI, and the next stage menu that is presented in the edit menu is selected.

Data traffic in the primary clustering of the control software of Figure 49 illustrative under the Window environment.

The present invention relates generally to prepare and the improving one's methods of selection markers chemical libraries.The invention still further relates to a kind of in a collection of differing molecular such as above-mentioned marking storehouse synthetic useful device.

In order to understand the meaning of improving one's methods, it must be understood that the basic technology of the signature library that not only prepares and use, and also various synthetic and the screening step how to cooperatively interact and how selective reagents all influences the result who is obtained.The mark chemical libraries is synthetic on solid carrier usually, and the selection of carrier and joint is successful key.Joint can be used to carrier is linked to each other with marker, carrier is linked to each other with the storehouse molecule or is not having under the situation of solid carrier, and marker is linked to each other with the storehouse molecule.With the chemical structure unit, the marker selection relevant with synthetic method can be same key, but also is subjected to the influence of the characteristic of available solid carrier and joint.Analysis and application to these signature library influence these selections equally, and available equipment and reagent.

Though equipment of the present invention and method mainly describe according to synthesizing of oligonucleotide and peptide, the invention is not restricted to this.The present invention will seek as polyose, phosphatide, polyurethane, 2,3-benzodiazine, the application in prostaglandin(PG) and β-corner mimetic crystal and other material synthetic.Disclosed as (Holmes) in this paper U.S. Patent number 5,242,974 incorporated by reference, can form annular material.

As using and synthetic being disclosed in further detail in the following application of examining of the different substances of oligonucleotide and peptide, they are all incorporated by reference by this paper: the U. S. application series number 07/876 that on April 29th, 1992 proposed, 792, the U. S. application series number 07/762 that on September 1st 8,1991 proposed, the U. S. application series number 07/946,239 that on September 16th, 522 and 1992 proposed.

By explanation of the present invention is provided shown in the following table.

The synthetic general introduction of I, mark chemical libraries

II, solid carrier

A. type

B. joint

C. molecular vehicle

III, chemical structure unit

A. oligomer and monomer

B. other structural unit

IV, marker

V, synthetic method

A. oligonucleotide mark peptide storehouse

B. the peptide storehouse of synthetic oligonucleotide-mark improves one's methods

C. micromolecular synthetic

D. the method for preparing the solubility storehouse

VI, analytical procedure

A. the screening analysis in bead storehouse

B. screen shla molecule

C. screen the natural product storehouse

VII, equipment and reagent

VIII, the general introduction of parallel coupling building-up reactions apparatus embodiments finish

Except top notion, provide following term so that the description of this invention, some abbreviations and term are prescribed to have common definition and is used to describe the present invention by this paper.

Abbreviation: HBTV, O-(benzotriazole-1-yl)-1,1,3,3-tetramethyl-alditol phosphofluoric acid ester; HOBt, I-hydroxybenzotriazole; HATU, [O-(7-azepine benzo triazol-1-yl)-1,1,3,3-tetramethyl-alditol phosphofluoric acid ester; TFA, trifluoroacetic acid; TCA, trichoroacetic acid(TCA); DIEA, diisopropylethylamine; DMF, dimethyl formamide; Fmoc, the 9-fluorenylmethyloxycarbonyl; DMT, two pairs of methoxy trityls; Trt, trityl; Bz, benzoyl; Pmc, 2,2,5,7,8-pentamethyl-benzo dihydropyrane-6-sulphonyl; ^tBoC, tertiary butyl oxygen carbonyl; PBS, phosphate buffered saline buffer; BSA, bovine serum albumin(BSA); MAb, monoclonal antibody.

Complementary or complementary in essence: these terms refer to a compound and another bonded ability, and for example part and its complementary acceptor combines.Typically, these terms are used in the description of the base pairing between the Nucleotide of nucleic acid, for example, and between the primer binding site between two chains of double chain DNA molecule on the Oligonucleolide primers and the single-chain nucleic acid that will check order or increase.Complementary nucleotide is generally A-T (or A-U), C-G, and also there is a variation widely in the synthetic or modified nucleotide of knowing for those of ordinary skills that closes characteristic of signing an undertaking.When " in essence complementary " is present in a RNA or DNA chain and hybridizes to complementary nucleic acid under hybridization conditions.Typically, hybridization occurs in a chain that has 14 to 25 Nucleotide at least and exists at least about 55% when complementary, but the hybridization of highly selective will occur in when complementarity and increase to 65%, 75%, and 90% and 100% o'clock.See this paper Nucl.Acids.Res.12:203 incorporated by reference, 1984.The highly selective hybridization conditions is known as " tight hybridization conditions ", is defined as follows.

Epi-position: this term is used for describing the part by the antigen molecule of describing with the interactional zone of acceptor subclass that is known as antibody.

Identification tag: in general significance, this term is used for representing a kind of physical property, and it provides a kind of method of discerning chemical reaction, for example the oligomer on solid carrier synthetic in the monomer addition reaction of an independent solid carrier experience.Identification tag is used for writing down the step of the synthetic series reaction that is used for a chemical libraries.This identification tag can have any discernible characteristic, for example comprises: can debate other shape, size, quality, color, optical density(OD) etc. with microscope or alternate manner; Differential absorbancy or light emission; Chemical reaction velocity; Magnetic or electrology characteristic; Or any other information needed of encoding, the distinctive mark that can translate out at (or a some) molecular level.The preferred of identification tag is oligonucleotide, because the nucleotide sequence of oligonucleotide is the firm form of the information of being encoded.Whether use solid carrier in no matter synthesizing, " identification tag " can directly be coupled on the synthetic oligomer.In the embodiment of back, identification tag can be thought the effect of playing oligomer synthetic " carrier " equally from conceptive.

Part: this term is used for representing typically by combining the molecule of discerning with a special receptor.Be designated as " part " with the medium of receptors bind or reaction, it has only its meaning is just arranged with regard to the acceptor of correspondence.As long as the material studied can in conjunction with or by alternate manner and receptor response, term " part " does not comprise any specific molecular size or other structure or composition characteristic.And, part " can be used as the native ligand with receptors bind, or as the functional analogue of stimulant or antagonist.Include, but are not limited to this by the triable part of the present invention, the stimulant of cell-membrane receptor, antagonist, toxin and venom, virus epitopes, hormone, sugar, cofactor, peptide, enzyme substrates, cofactor, medicine (as narcotic, steroide etc.) and protein.

Monomer: this term is used for representing being joined together to form any member of component of other molecule or group of molecules, for example one group of oligomer or polymkeric substance.Useful set of monomers among the present invention, but be not limited thereto, for example synthetic for peptide comprises L-amino acid, D-amino acid or synthesizing amino acid.Monomer used herein refers to any member of the synthetic basic group of an oligomer.For example, the amino acid whose dimer of L-is formed a basic group that is used for polypeptide synthetic 400 " monomer ".Different monomers is organized substantially and be can be used in the consecutive steps of polymkeric substance in synthetic.Person of skill in the art will appreciate that " monomer " only is a class " chemical structure unit ", the chemical structure unit of any kind can be used among the present invention, no matter whether synthesizing oligomer or little organic molecule or some other molecules.

Oligomer or polymkeric substance: these terms are used for representing the molecule that the method by chemistry that relates to the monomer subunit or enzyme addition forms.Comprise for example nucleic acid as oligomer, the linearity of polysaccharide and phosphatide, ring-type and branched polymers and have a α, the peptide of β or omega-amino acid, heteropolymer, polyurethane, polyester, polycarbonate, polyureas, polymeric amide, polymine, polyaryl alkene thioether, polysiloxane, polyimide, poly-acetic ester or other polymkeric substance, open according to the present invention, they are conspicuous for those skilled in the art.

Peptide: this term is used for representing that monomer whose is the oligomer of the a-amino acid that links together by amido linkage." peptide " also can be used to refer to " polypeptide ".Within the scope of the invention, be interpreted as amino acid and can be L-optically active isomer or D-optically active isomer, though may usually be called as " polypeptide " greater than 20 amino acid whose peptides, peptide is usually greater than 2 amino acid monomer length, be more often greater than 5 to 10 amino acid monomer length, even can adopt a general letter abbreviations to represent amino acid greater than 20 amino acid.Adopt a general letter abbreviations to represent amino acid (representing proline(Pro)) as P.These abbreviations are included in the Stryer incorporated by reference by this paper, and Biochemistry is among the Third Ed. (1988).

Oligonucleotide: be used for representing general single stranded DNA or RNA molecule by the synthetic method preparation.Though for suitable, the oligonucleotide that the present invention adopts is generally 50 to 150 length of nucleotides to the oligonucleotide of different lengths, is preferably 80 to 120 Nucleotide under some conditions.For example, oligonucleotide tags can be set up with the Nucleotide consistent with monomer that is used for synthetic oligomer and monomer addition step and the addition step of Nucleotide.In addition, the very short oligonucleotide that 2 to 10 Nucleotide are promptly arranged can be used to expand existing oligonucleotide tags to determine the monomer coupling step.Can by by this paper incorporated by reference by Beaucage and Carruthers, 1981, the phosphoramidate method of describing among the Tetr.Lett.22.1859-1862 or according to Matteucci et al., 1981, three ester methods among the J.Am.Chem.Soc.103:3185, or by preparing suitable oligonucleotide as other method of using the oligonucleotide automatic DNA synthesizer DNA of purchasing.

Connect: this term refers to the functional sibship between a nucleic acid fragment and another nucleic acid fragment feasiblely.For example, if promotor causes or influenced transcribing of encoding sequence effectively by alternate manner, promotor then is connected on the encoding sequence feasiblely.Usually, connect feasiblely mean connected nucleic acid fragment or sequence be right after and must be connected with two protein coding regions that are right after in reading frame.

Parallel coupling: this phrase refers to that two structural unit compounds are coupled on the independent special site of substrate synchronously.This substrate can be the solid carrier with the specific groups that links to each other with these structural units, or form have two independently with other compound of the special basic usefulness of each structural unit, coupling synchronously referred to before joining new structural unit compound at first by the link coupled structural unit coupling of two compounds and these special sites.Therefore, term used herein " synchronously " is not limited to these two structural units accurately simultaneously by coupling.

Acceptor: this term refer to for have specific collection and property to part molecule.Acceptor can be nature and produces or the synthetic molecule.Acceptor can their unaltered natural or separate stage or is used with other material bonded state.Acceptor can covalently or non-covalently be connected with other material.Can adopt the acceptor in the method in the present invention to include, but are not limited to this, antibody, cell-membrane receptor, monoclonal antibody, with the antiserum(antisera) (as virus, cell or other material) of antigenic determinant reaction, polynucleotide, nucleic acid, Sugar receptors, polysaccharide, cell, cytolemma and organoid.Acceptor is also known as " anti--part ".When two molecules be generally macromole by molecular recognition when forming complex body, then formed " ligand-receptor to ".Other acceptor includes, but are not limited to this, is essential special transport protein matter or enzyme for the antibiotic microbial survival of needs; The binding site of enzyme, the ligand-binding site point in the antibody molecule; Nucleic acid; As by this paper incorporated by reference at Lerner et al., 1991, the catalytic polypeptide of describing among the Science 254:659; And hormone receptor such as Regular Insulin and growth hormone receptor.

Substrate or solid carrier: the material on the surface of tool rigidity or semihard represented in these terms.Though can adopt other form, these materials preferably adopt globule, ball, and dish or other be form easily.In certain embodiments, at least one surface of substrate can be flat basically.Preferred coarse spherical surface.

Tight hybridization conditions: this phrase refers to the hybridization conditions of highly selective, wherein have only when correlated series for fully or height (promptly greater than 80%) when complementary, nucleic acid is stablized with the just maintenance that combine of other nucleic acid (or other section of identical nucleic acid molecule).These conditions generally comprise less than salt concn 1M, as less than 500mM, and generally include salt concn less than 200mM.The hybridization temperature of oligomer will be generally greater than 22 ℃, for example greater than about 30 ℃, and usually above about 37 ℃.May demanding hybridization temperature for the segment that special hybridization is long.(these factors comprise based composition because other factors may greatly influence the stringency of hybridization, the length of complementary strand, the situation of organic solvent, the degree of base mispairing), so carrying out absolute mensuration to any one factor only, the binding ratio of these factors wants much important.

Synthetics: when by external chemistry or enzymic synthesis and when preparing, compound then is a synthetics.Can with synthetic storehouse of the present invention with, for example can be on bacterium, virus of breeding among the host that yeast or other are lived or in the plasmid vector those compare.

I. the synthetic general introduction of mark chemical libraries:

The present invention relates generally to the method for synthetic and selection markers chemical libraries.In essence, chemical libraries of the present invention each this " book " by interested pharmaceutical chemicals or molecule, identifies that the marker of interested pharmaceutical chemicals or molecule or their some material circumstances and the connector between pharmaceutical chemicals interested or molecule and the marker form.In an important embodiment, interested pharmaceutical chemicals or molecule are oligomer, as peptide, marker is an oligomer, as nucleic acid, connector is solid carrier or particle, therefrom few collective and mark and optionally split, as for the ease of the storehouse of detecting or provide solubility, can screen these storehouses and separate with receptors bind or have each oligomer of some other desired characteristic.The general method of preparation mark chemical libraries is described by the preparation of the different oligomer of large quantities of height, wherein each library member is at other library member, for having the oligomer (though this storehouse generally comprises identical " book ") of specific monomer order.This storehouse or collection for example can comprise, the X of all combination results that are assembled to X different monomers in the oligomer that length is n in one group of monomer ⁿIndividual different compound.This collection only for example also can comprise one or a small amount of position and has the different monomers unit, and the oligomer that all other positions have identical sequence.

The general method of synthetic these oligomer generally includes-makes up at random the chemistry and/or the enzyme assembling of (at random) process and monomeric unit.A kind of method comprises step: (a) a large amount of solid carriers are divided equally to a large amount of reaction vessels; (b) use first monomer in each differential responses container and the various combination of marker, first monomer and first marker are coupled on the carrier in first reaction vessel; (c) carrier is compiled; (d) carrier is divided equally to a large amount of reaction vessels; (e) use second monomer in each differential responses container and the various combination of second marker, second monomer is coupled on first monomer, second marker is coupled on the solid carrier or first marker; Optionally repeat coupling and all step by step 1 to 20 time or more with different markers and different monomers.Usually, the solid carrier of equivalent will be divided equally to each reaction vessel basically.Those skilled in the art see can adopt identical chemical structure unit in different coupling steps, identical chemical structural unit can adopt in the linked reaction of more than one simple coupling step of tool (reaction vessel).

In order to be easier to detect the present invention, can consider at first that from three kinds of different monomers A the length of the set of monomers of B and C assembling is unmarked storehouse synthetic of all oligomer of three residues.The bead of trisection is divided equally in three reaction vessels, monomer A is coupled in first reaction vessel, monomers B is coupled in second reaction vessel, monomer C is coupled in the 3rd reaction vessel.Then compile bead from all reaction vessels.This compiling comprises roughly three kinds of dissimilar beads of equivalent, and each bead is a feature with the monomer that is coupled on the bead.Compile mixing with this, and reassign to and comprise A respectively, B or C are as in will next step monomeric independently monomer reaction container of link coupled.

After this linked reaction, each reaction vessel comprises the bead of all three kinds of different monomers that have on the position 1 and is included in monomer on the position 2 in each second specific reaction vessel now.All beads are compiled again, have prepared the mixture of a bead like this, and wherein each bead provides a kind of in nine kinds of possible dimers.Again compiling of this bead is distributed in three reaction vessels, and is coupled on these three kinds of different monomers, prepared three kinds of monomeric all trimerical complete groups (3 like this ³=27).The use that is readily appreciated that enough a large amount of synthetic beads help to guarantee this group fully representative arbitrarily, the monomeric various combinations of adopting in Zu He the synthetic method at random.

Modification to this completely random method also is possible.For example, set of monomers can go on foot another step and is expanded or dwindles from one; If the coupling chemistry can reach.For next step can change set of monomers (as the amino acid in the step, the nucleosides in another step, the carbohydrate in another step) fully.Peptide synthetic monomeric unit for example can comprise single amino acids or bigger peptide unit or both.A kind of is in some combinations that are formed on the various orders that some step of synthetic distributed on the solid carrier in the different monomers group.By this method, also can set up the oligomer of the different lengths that has relevant or irrelevant order, and can be when changing other residue some monomer residues be fixed on and set up the oligomer skeleton on some positions, wherein change some residue or zone to form difference.

Synthetic these combination synthesis steps that comprises usually of mark chemical libraries.Because identification marking can the easily decoded significance of reporting each oligomer, yet the mark chemical libraries can be bigger and complicated than unmarked storehouse widely.In fact, the method in the synthetic storehouse of the coding of synthetic compound of the present invention make screening large quantities of by multistep suddenly the compound that can not check order of synthetic preparation become possibility.

Particularly, the use of oligonucleotide mark and oligonucleotide coding provides strong mechanism for the structure individual character that writes down by each member in the huge storehouse of making up the qualification compound, particularly peptide that synthesize preparation.As long as but parallel synthetic method keeps quadrature and compatibility, this method is widely used in encoding in the combination assembling of other non-peptide structure.Only provide the order of the reaction of mono-product species, obviously defined combination synthetic net production for producing very high throughput.This situation and standard peptide and DNA synthetic chemistry are approximate, and the product structure that obtains gives clear and definite regulation by the structural unit of use in synthetic and/or the order of linked reaction.

Yet the synthetic organic reactions of great majority are for speciality relatively, the productive rate that changes and usually be multiple product (as regional and stereoisomerism structure).Utilize this chemistry to come the combinatorial libraries on the synthesis of solid carrier in the storehouse, to produce a mixture of products that is positioned on each bead.The most generally speaking, synthetic coding can not determine uniquely the to be correlated with chemical structure of body.Otherwise this coding method can be taken as coding-construction library member's correct synthesis condition (for example reagent, reaction conditions etc.) more accurately.Screening this storehouse discerns and then can produce on preparative-scale again and be divided into several parts of " effectively prescription " that separate the bioactive composition of tool.The application that the code database technology is used at the discovery medicine for the scope that enlarges combinatorial chemistry and it and the exploitation of a large amount of useful compounds have sizable potentiality with separating.According to synthesizing of tagged molecule storehouse, the importance that the present invention may be better understood, for example the solid carrier in the storehouse is synthetic using and selecting.

II, solid carrier

A. type

Usually, mark chemical libraries of the present invention is made up of a collection of solid carrier, for example bead or particle.These solid carriers can be Any shape, although be preferably coarse shape.These carriers needn't be same size, shape or composition; Though these carriers usually and be preferably homogeneous.In some embodiments, carrier can be preferred especially very uniformly for size.Yet in other embodiments, two groups or how visibly different solid carrier can be used for certain purpose, and promptly solid carrier can be by individual particle or more linked granulometric composition.

Solid carrier can be made up of many materials, and any one ability to many chemical active radicals of mainly being derived is with chemical property or the matching of other molecule synthesis and the restriction that mark is connected of oligomer.Suitable carrier substance comprises glass, latices, highly crosslinked polystyrene or similar polymkeric substance, gold or other glue state metal particle and other material well known by persons skilled in the art.Referred except other, the chemical active radical of these solid carriers that can be used to derive is to be used in each molecule or oligomer solid-state synthetic those usually, so they will be known those skilled in the art.Term used herein " solid carrier " comprises that to have oligomer synthetic and mark in some embodiments connects and/or the particle in the suitable site of synthetic.The solid carrier that has many present invention of being useful on synthetic oligomer storehouse preparation.Solid carrier is generally used for for example above-named peptide, in the solid phase synthesis of nucleic acid and other oligomer, therefore is well known to those skilled in the art.Solid carrier of the present invention does not comprise viable cell, virus or as the cloning vector of phage vector or plasmid.Monobeads ^TM(from Pharmacia Fine ChemicalsAB, Uppsala Sweden buys) or their equivalent are all particularly useful as the solid carrier of all respects of the present invention.Monobeads ^TMGood big or small homogeneity and the small size of 10 μ m are provided.Further, these Monobeads ^TMNot aggegation in the organic or inorganic solvent, and provide suitable carrier for oligonucleotide and peptide synthetic.At last, Monobeads ^TMFor initial amino acid (100nmole/mg) provides very high load.

Be selected to finish a size that importance is a carrier of specific solid carrier of the present invention.If desired, with enough solid carriers and effectively coupling, can prepare the close set of some oligomer.Usually, the solid carrier size is in the scope of 1nm to 100 μ m, but the available sometimes solid carrier that reaches the big quality of 1mm size.The suitable size of solid carrier depends on the number and the identification marking connection site in the synthetic site of (1) required oligomer; (2) will synthetic the number (with the number of the solid carrier that has each oligomer that need screen) of different compounds; (3) size of solid carrier to the influence (for example by the cell sorter of fluorescent activation) of the particular screen strategy that will use (FACS).

As special example, diameter is that the solid carrier of 1 μ m can be used for the present invention.If comprise the solid carrier of nearly 0.2ml in each reaction vessel, and oligomer will need 100ml solid carrier or nearly 10 altogether so by 50 monomers synthetic (50 parallel reactors) ¹³Solid carrier.If want to prepare sexamer, exist more than 1.5 * 10 so with these 50 monomers ¹⁰Individual possible order, and that particular order will appear at about 10 ³Solid carrier on.Divide the capacity of resin according to normally used peptide, the capacity that estimates of each bead is about 0.1pg peptide/bead.By this estimation, each solid carrier is with the about 100amol of tool or 10 so ⁸The oligomer chain.

In order to improve detersive efficiency, can adopt than synthetic atresia bead or other solid carrier that lacks the hole of general peptide; Yet for some application of the present invention, porous small ball or resin work are good and be generally preferred.Non-porous support will have the low density of growing chain, although the capacity of several magnitude reduces, for effective screening, can prepare enough big oligomer density.The few hole of utilization carrier, more most oligomer in screening process can with receptors bind.And the mark that few hole carrier will reduce from a reaction to next reaction shifts, and has therefore improved the accuracy of the reading of main (standard) mark.

Mention as top institute, another embodiment comprises the use of two solid carriers such as bead, they are physically linked together, and one of them is connected with the synthetic site (or joint) of molecule or oligomer, and one is connected with the connection site (or joint) of identification marking.This structure allows molecule or oligomer separately, and identification marking enters discontinuous " zone " and permission is used the greatly different chemical reaction bases and the chemical constitution of material in order to connect.Solid carrier can be derived respectively, then is connected under all or nearly all synthesis of solid carrier will have the condition of the solid carrier that the mark that drawing connects.Solid carrier can be different sizes, for example the synthetic bead of a big bead that is connected with the connection of difference (perhaps many) tick marks.In one embodiment, first solid carrier will have that at least one links to each other with amino acid, and second solid carrier will have at least one bead that links to each other with Nucleotide.

The method that connects two beads is subjected to the restriction of oligomer synthetic chemistry.The most conspicuous method that connects bead be to use with each solid carrier on assorted two functional crosslinkers of main chemical reactions radical reaction (example of this reagent is seen Pierce Immuno Technology Catalog and Handbook PP.E10-E18 (1991).These linking agents can reach the various purposes of partly describing as hereinafter.

B. joint

In the time of on being connected to a solid carrier, the common general one or more molecule joints of the mark that oligomer is relevant with it are connected on the carrier.This linkers had a suitable functional group at each end before connecting, a group is suitable for linking to each other with carrier, and another group is suitable for linking to each other with oligomer formula inspection note thing.In some embodiments, adopt the joint of cleavable so that analyze or detect step.

A large amount of available different reagent that connect are provided, identification marking can be connected on oligomer or other the interested storehouse molecule be connected to solid carrier or mark that is pre-existing on.For example, identification marking can be connected on the monomer that is attached in the oligomer, or be connected on the structural unit that is attached in the non-oligomeric compounds.For the peptide oligomer, the side chain of cysteine residues provides a site easily for the connection of mark.In other cases, as long as the clean synthetic reduction of required oligomer can easily be allowed to, mark even can be connected and cover a small amount of oligomer chain.Mark can be directly connected to oligomer (or other compound of interest) is bonded on the joint of solid carrier.In this embodiment, before connecting, the joint tool is suitable for the 3rd functional group of the connection of identification marking.

According to using and required effect, certainly in conjunction with a large amount of joints.For example, can select to transmit hydrophobicity, wetting ability or three-dimensional part are to obtain the joint in the required effect aspect the characteristic of for example coupling or joint efficiency.In one aspect of the invention, the branch joint promptly for example the joint that has huge side chain of joint Fmoc-Thr (tBu) be used to provide stability be used for controlling the interval of the molecule on the solid carrier in the storehouse or the storehouse in interval between molecule and the mark.

As mentioned above, can adopt the effective effect of acquisition of the joint of cleavable.The preferred light degradable joint of the present invention comprises that 6-nitro veratryl oxygen carbonyl (NVOC) linker compounds relevant with other NVOC (see PCr patent publication No. WO90/15070 and WO92/10092 incorporated by reference by this paper, also see the U.S. Patent Application Serial NO.971 that on November 2nd, 1992 proposed, 181).In another embodiment, joint is the nucleic acid that has one or more restriction sites, part among the library member (mark, oligomer or other compound of interest or both, or solid carrier) can be by suitable optionally cracking from another of Restriction Enzyme like this.Nucleic acid that this is new and joint have illustrated the joint that can be used in a large number obtaining for the beneficial effect of the object of the invention.

C. molecular vehicle

As mentioned above, the present invention can also a kind of method finish, and does not wherein have solid carrier, directly mark (generally by a joint) is connected to just on synthetic oligomer or other molecule.Use another kind of method, can on solid carrier, synthesize oligomer or other molecule with and mark of correlation, and before screening or other use, split or remove from solid carrier by other method.This method below will more fully be described.No matter whether solid carrier exists, and the size in storehouse and composition will be by coupling and mixing steps, and the number of used monomer or other structural unit decides in synthesizing.

III, chemical structure unit

A. oligomer and monomer

Broad applicability of the present invention may be the most easily understood in the synthetic and screening in the big storehouse by considering different oligomers and polymkeric substance.The polymkeric substance that oligomer is made up of monomer; For biological polymer, the monomer sequence in the oligomer has been determined important biological characteristics usually.Interested preferred oligomer comprises peptide, oligonucleotide, glycine and polycarbonate that few N-replaces.As mentioned above, for the purposes of the present invention, monomer is can be joined together to form-any one member in component of oligomer or polymkeric substance, i.e. amino acid, carbonic ether, sulfone, sulfoxide, Nucleotide, carbohydrate, urea, phosphoric acid, lipoid, ester, their mixture and analogue.Therefore, monomer can be can be carried out chemical coupling by suitable activation maybe can be accepted and be used for carrying out enzymatic link coupled any kind.

Require the suitable coupling chemical property of given monomeric unit group of utilization or structural unit from the method for the monomer of many types assembling oligomer.Can be connected to structural unit on another structural unit in type progressively any group can be used as set of monomers.Can be by chemistry, enzyme, or the arbitrary combination of other method or these methods is transmitted this connection.The oligomer that obtains can be linearity, ring-type, branch or take those to those skilled in the art for conspicuous various other structures.

B. other structural unit

Invention described herein mainly is the preparation about the molecule that comprises aminoacid sequence, but the present invention can easily be applied to oligomer preparation and can be in the compound of step by any group of synthetic in the type of step, these all can be understood by those skilled in the art.For example, as benzodiazepine , glycolylurea and peptide phosphonic acids can (be seen the U.S. Patent Application Serial 08/119 that on September 9th, 1993 proposed by using this paper to prepare, 700, it is that the at present resigned series number for U.S. Patent number 5,339,115 part continuation applications of proposition on June 21st, 1993 is 081,577 part continuation application, each piece of writing is all incorporated by reference by this paper here.

In one embodiment, the present invention can be used for setting up the branched polymers storehouse.Yet in many examples, the linear polymer storehouse for example has greater than the peptide of 3-4 residue very usefully, and the shape of these linear molecules becomes long and narrow.Perhaps part is not because the high degree of flexibility most drug of molecule has the shape of this expansion.The branch trunk polymer can cause shape of molecule and known drug similar.Therefore, in one embodiment, the present invention relates to monomer and at least three kinds of other monomers and can be connected to combining of functional group on it.

If only use this monomer, branch is synthetic so fully will cause usually by the last monomeric height ratio of link coupled (tying other used monomer in synthetic mutually).Certainly the different monomeric mixture of branch of fusion changes this ratio, have any problem most but then on the structure of identification compound of interest, have, be that the monomeric mixture of branch is complicated more, the available information about the synthetic specific molecular of mark is just few more.In one of the present invention is improved one's methods, in each monomer coupling step, mix two kinds of monomeric mixtures, a kind of can branch, a kind of can not, produced one like this and comprised a large amount of difform storehouse that has high information flag.In this case, mark will be determined the monomer in each coupling step appearance, but whether uncertain monomer can branch.Yet, only use those the monomeric simple resynthesis be included in from the compound of the selection group in first storehouse will discern the structure of these compounds easily.

VI, mark

Identification marking has an identifiable feature, promptly for example in shape, and size, quality, electric charge or color aspect can be distinguished by microscope or other method.These identifiable features can come from the optics of mark, chemistry, and electricity or magnetic properties, or come from the combination of these characteristics.In fact, mark is as tagged molecule on the level of (or a plurality of) molecule or solid carrier and the identifiable information of coding.By utilizing identification marking to follow the tracks of the path that each member taked of chemical libraries, can push away the structure (being the monomeric order of any oligomer) of declaring any chemical substance in the storehouse by reading identification marking.

Can have identifiable different size with being built into, shape or color or with the bead of bar code mark, these marks can be the luminous or radio-labeling of " machine can be read " with the discernible mark of microscope.Identification marking also can be the molecular structure of codified, and these information can be coded in size (polymkeric substance length) or the molecular composition.Perhaps the best example of the mark of latter type is a nucleotide sequence, promptly assembles and next DNA or DNA from natural or modified base.

For confirm to be marked at synthetic and the screening chemical libraries in role, consider for example to use in oligomer is synthetic, be connected on each bead in the identifiable alphanumeric indicia of microscopically.Mark " A " refers to participate in step 1 bead of A-monomer reaction; " C2 " refers to participate in step 2 bead of C-monomer reaction, and " B3 " refers to the B-monomer that is added in step 3, by that analogy.When the 3rd goes on foot end of synthesis, a bead will have three kinds of connected marks; A for example ₁, C ₂And B ₃, this shows that the sequence of the peptide on the bead is ACB.This method requires many different identification markings to equal the product of different monomers number and synthesis step number (in this embodiment 9) at the most, if mark is with such sequence of steps A, A-C, A-C-B links to each other with another, the number of identification marking then is reduced, have only in this case with the as many identification marking of monomer to be used, to add and which kind of monomer, the mode of the record unanimity that the step adds where is assembled identification marking.

In another embodiment, the molecule that mark can be sought by a series of light, for example fluorescigenic, phosphorescent compound, their spectral response curve can be changed (for example photobleaching) and therefore be used for storage information, and these information are used to each bead or other solid carrier in the signature library.In a such method, bead in conjunction with multiple all can be by photobleaching optionally, becoming thus to fluoresce maybe can not weaken fluorescigenic fluorophore.In each coupling or chemical reaction step, irradiation bead (or not having) makes the fluorophore photobleaching (or not having) of one or more particular types, has therefore write down the monomer individual character in the oligomer that is synthesized.See by this paper Science 255:123 incorporated by reference (on March 6th, 1992).

Therefore identification marking is discerned other reactions steps of each monomer coupling or each library member or solid carrier experience, and other chemical reaction that has write down the step that each monomer is added in the synthesizing series or finished.

For convenience and with the identification marking type, mode of connection and oligomer chemistry character or other molecule synthesis adapt, can be before monomer addition or other reaction, among or immediately mark is connected afterwards.As mentioned above, by variety of way, or directly by a link molecule, or by the solid carrier that oligomer is synthesized thereon, identification marking can be linked to each other with oligomer.In the method for back, also mark can be connected on another solid carrier that is connected to conversely in the solid carrier that oligomer is synthesized thereon.The solid carrier that has experienced specific monomer addition or other chemical reaction step by physical property link together, and can be labeled as a group time, promptly before next step compilation steps, identification marking is added.

One in the case, when only the monomeric unit of a small amount of oligomer was changed certainly, as when think only to change this amino acid of one in the peptide, may need definite only was those monomers that change in oligomer.For example, may want to change only 3 to 6 amino acid in the peptide of 6 to 12 amino acid lengths, maybe may want to change in the polypeptide that reaches 50 amino acid lengths some as 5 amino acid.By provide one to determine only reformed amino acid whose identification marking in each sequence for each solid carrier, can discern the sequence of each peptide uniquely, these will easily be understood by those skilled in the art.In this case, all solids carrier can be retained in in the same reaction container of the addition of common monomeric unit and divide equally in the differential responses container for the unitary addition of particular monomers.

The synthetic oligodeoxynucleotide especially is preferably the identification marking that has information.Oligonucleotide is natural, the high density information medium for storing.The individual character of the monomer type relevant with chemical synthesis process, addition step or any out of Memory can easily be coded in the oligonucleotide sequence of a weak point.Oligonucleotide also at an easy rate with many solid carriers, oligomer, joint links to each other with other molecule.For example, oligonucleotide can easily be connected on the synthetic bead of peptide.

A distinctive remarkable benefit is by polymerase chain reaction (PCK in the coding method of use oligonucleotide, see PCK Protocols:A Guide to Meth-ods and Applications (Innis, M, Gelfand, D, Sninsky, J.andWhite, T, Academic Press, San Diego 1990); Also see U.S. Patent number 4,683,202 and 4,965,188, each piece of writing is all incorporated by reference by this paper) and other nucleic acid replication and amplification technique obtain the ability that the target of enormous amount increases.Though being most commonly used to external DNA cloning method is PCR, suitable selectable amplification method comprises for example amplification of nucleic acid sequences (Compton, 1991, Nature 350:91-92) and the amplification sense-rna (see by Van Gelder etal. for referencial use on this paper, 1988, Proc.Nat.Acad.Sci.USA 85:7652-7656) and certainly continue formula sequence replicating system and (see 3SR incorporated by reference by this paper, see Guatelli etal., 1990, Pro.Natl.Acad.Sci.USA 8): 1874-1878).PCR only needs on a small quantity (use highly selective and effective ways, even a single copy being just enough) DNA masterplate, can make people use the solid carrier and the bigger storehouse of acquisition of microscopic size like this.

Use nucleic acid marking to be convenient to considerably beyond the foundation and the screening in the synthetic storehouse of the otherness that is limited storehouse method acquisition by other.And the bead raw material of amount easy to control is adopted in these storehouses, and the biological reagent of the practical amount that therefore may be utilized distributes receptors bind.A conditioning step in the processing that relates to the ESL storehouse with oligonucleotide mark of improving one's methods of the present invention---amplification, chain separates and from the sequencing of the mark of each bead.This method has improved at least one sets of numbers of efficient of sequencing, and relating to the mark chain of rings (connection) step, wherein many differences of generally coming from library members' amplification of selecting are banded in together before being marked at the clone of oligonucleotide mark or sequencing.

In an embodiment of the inventive method, the mark of amplification is connected, and then the clone is the mark of linearly aligned 10 to 20 (or more) in the conventional sequencing carrier.Preferably, suitable restriction site is installed in adjacent with " coding region " (sequence with information content) of oligonucleotide mark, and after the mark on the one group of bead that increased, restriction site is cut, and segment is connected to form concatermer.Then this concatermer clone is reduced in the carrier of suitable sequencing, as long as find to have more than at least 10 marks on each template, each template can be used for altogether for example two-way sequencing of 500 to 800 bases so.This method also will provide the scheme of choosing of avoiding separating with FACS each bead.Bead or tagged compound can be classified to the storehouse, are used for being amplified of sequencing, in the storehouse of connection and clone's mark.In addition, because broken away from the requirement of operating each bead, can use less than the bead of 1 μ m (generally this size is too little for conventional FACS divides plate) and set up and screen the storehouse.This selection can be passed through affinity purification (pan, magnetic ball), is done easily, and then the bead of enrichment is increased as described above and clones.

The oligonucleotide identification marking can be before corresponding monomer coupling (synthetic for oligomer) or other chemical reaction step, among or afterwards, by the then base ground assembling of a base.Follow in the base of oligonucleotide mark under a kind of situation of synthetic of base, per step be labeled as a mononucleotide, or mostly be very small amount of Nucleotide (i.e. the piece of 2 to 5 Nucleotide) most.Follow in the method for piece at piece, tool 2 to 5 to 10 or more the coding nucleotide of polybase base (codon) add as the protected piece that is activated.Each piece carries monomer type or out of Memory, and the order of monomer addition or other reaction is represented in a tag block and Next addition in proper order.In addition, piece codified oligomer is synthetic or the information of other reactions steps number and monomer type or other structural unit.This method has kept the sequence of steps in the linear array with the oligonucleotide chain of the parallel growth of oligomer.For the chemical compatibility of keeping parallelism synthesis step (for example oligonucleotide and peptide), can revise the character of the synthetic chemistry composition of standard, it is a following importance of the present invention that will further go through.

Also can connect in each step and contain the amplimer site, monomer customizing messages and reaction sequence information, length are the oligonucleotide of the protection (or not protection) of 50 to 150 bases.When a series of n oligomer synthetic (monomer coupling) or other chemosynthesis step finish, will have the n group different be encoded with the storehouse in each oligomer sequence or the relevant oligonucleotide identification marking of other compound.After identification had the oligomer of ligand activity, relevant oligomer can increase and is carried out the situation that sequencing is deciphered oligomer or other compound by PCR.

As below will more fully discussing, the selection of the base of using in the oligomer identification marking is to decide by other chemical reaction condition that oligomer synthetic chemistry or mark standard are exposed to wherein.Therefore, when adopting the chemical process need utilize strong acid and chemical phenomenon, the use of the oligonucleotide of being made up of only pyrimidine C and T and double density sign indicating number can show valuable.Use similar approach, purine nucleotides can overcome by using purine nucleoside analogs the instability of strong acid, for example the 7-denitrification assorted-Desoxyadenosine and 7-denitrification be assorted-pancreatic desoxyribonuclease (sees the Barr et al. that this paper is incorporated by reference, 1986, Bio Techniques 4:428-432 and Scheit, Nucleotide Analogs:Synthesis and Biological Function PP.64-65 (John Wiley and Sons, New York).Use these or other analogue will allow to use quaternary or other coding method opposite with binary.Therefore, in preferred embodiments, identification marking will be the oligonucleotide of 50 to 150 length of nucleotides of having an appointment, and it is formed by pyrimidine or pyrimidine and purine analogue or at the nucleosides that is used to assemble Undec any kind under the coupling condition in oligomer storehouse.The oligonucleotide identification marking can comprise one 5 ' and one 3 ' expansion site and optionally comprise for each step of oligomer synthetic may be the primer sites of a specific determined dna sequence.

The method that is combined into that coding has an oligonucleotide provides a method for the ambiguity that runs in the direct organization analysis that is positioned at a small amount of part of separating from big storehouse and the major limitation of sensitivity.The heavy body of DNA information storage can be developed the fine detail of returning a library structure of retaining.In the following embodiments, adopted one to comprise three base (C ⁷DA, dC, " password system " structure of 2 adjacent nucleotides T), it can be encoded and combine 3 ²=9 amino acid structures unitary synthetic (only seven structural units are used for the synthetic of this storehouse).As c ⁷DG also is included in the coding templet, adopts being combined into of 1000 different monomers can pass through to use only 5 Nucleotide (4 so ⁵=1024) " password system " size is supplied with.

Information can be coded in the length rather than or except in the nucleotide sequence or be coded in any other polymerization oligomerization mark of this respect.As long as length is used to represent the addition of each specific monomer and oligomer, deciphers mark that the situation of oligomer can be by aforesaid amplification oligonucleotide so and come identification marking to be carried out by any of the many technology that connect size separation by what comprise poly amic acid gel or capillary gel electrophoresis.Each different monomers that adds in the given step of oligomer synthetic or each different chemical reactions steps is represented by the oligonucleotide mark of unique length.The oligonucleotide mark comprises the amplification site, PCR primer sequence for example, and it is feature that its sequence is designed to the given step number in oligomer or other chemosynthesis.The oligomer of any given position is formed in the sequence so determines to comprise that the step of utilization in synthetic is that the PCR primer sequence of feature carries out amplification label and utilizes technology well known in the art such as gel or capillary electrophoresis (utilization just at the oligonucleotide of mark as standard) that amplified production is separated by size.When one of the hope preparation compound library relevant with homing sequence, this embodiment is particularly useful.During the step in a synthetic site that is simulateding, only need a mark.

Except length, the oligomer sequence information also codified in the base sequence that comprises the oligonucleotide mark.This type password not only connects in each coupling step in the embodiment of a different oligonucleotide mark, and all has value in the embodiment of the expansion one oligonucleotide mark that is pre-existing in each coupling step.For example, can adopt the oligonucleotide that reaches about 100 bases (or longer), as described below, wherein each has 7 (or more) districts.

District 1 is 3 '-primer sites (20-25 base).This site is used to be connected in the original amplification by PCR with another PCR site (at 5 ' end of oligonucleotide).Also can use another amplification method.

District 2 is " step is distinctive " determined dna sequence primer sites (15-20 base).This site is distinctive for the distinctive step to numbering in the synthesizing series.The whole oligonucleotide that joins in all beads in particular step will have this sequence jointly.Each will have a special primer sites of height of representing that step for the step of numbering.

District 3 is a transcribed spacer (20-30 bases).But the transcribed spacer section of measured length, but it is long to be preferably 20 to 30 bases, and the coding site is positioned over from the sequencing primer sites enough far so that the good reading to monomer coding or cog region to be provided.

District 4 is monomer cog region (8 bases).In this illustrative embodiment, in binary coded unit of each base representative of 8 bit strings, wherein for example T=0 and C=1.Each group step distinctive identification marking is by 8 based compositions that have 1 (C) or an O (T) on each of 8 positions.Each monomer type is encoded by 1 to 8 the mixing of these " switches ".

District 5 is that step number is determined district's (difference 4 bases on each limit that are 3 zones add 2 bases).Four unit encoding step number in this short scope.This is unnecessary for the sequencing primer, but it is decoded to can be used to confirm that the primer that suits is used with correct step.

The district 6 is repetitions of monomer identification marking (8 bases), and this zone has as distinguishing 4 identical information, and is used to confirm the monomer situation.This second monomer coding region is installed has also increased the possibility of acquisition good sequence mensuration " reading ".

Zone 7 is 5 '-PCR primer sites (20 to 25 bases).This site is as the very big site that is used for the 2nd PCR primer of sequence amplification.Have whole seven these characteristics, some of them are the length of oligonucleotide optionally, will be generally between 75 and 125 bases.

256 kinds of different monomers types of one eight binary digit form codifieds.The step number that can be encoded is by the oligonucleotide decision of distinctive group of existing step (8 every group).Use 10 groups (80 Nucleotide), codified reaches 256 different monomers that are assembled on the oligomer that arrives 10 unit lengths (therefore provides coding to reach 256 ¹⁰=1.2 * 10 ²⁴The ability of individual oligomer sequence).Can adopt the identification marking of coding, so the designated specific bit of each monomer (routine Ala=00000001, Gly=00000110 etc.).It is correct binary coded that the suitable oligonucleotide of combination provides.

For the ease of the identification of oligonucleotide mark, many selections are arranged.For example, can be by the direct read flag from the ball of sequencing formula hybridization.Also can increase the oligonucleotide mark so that mark identification can be passed through vivo clone or the amplification of external for example PCR method by the oligonucleotide identification marking that a single solid carrier or oligomer carry.If the order that is restricted to 100 molecules that detects needs each oligonucleotide mark 100 or more copy on the ball to the major general so.Any by several different methods, wherein some will be described below, preparation single stranded oligonucleotide, double-strandednucleic acid, or the marked copies of the form of mixtures of single and double-strandednucleic acid and the material that increases carried out sequencing.In one embodiment of the invention, wherein adding one in each monomer addition step separates and special oligonucleotide mark (opposite with the existing mark of each step expansion), the institute that can increase immediately is underlined, and material and the primer that adopts different sequencings for the mark of each type that then will amplification) have and the as many independently sequencing of oligomer synthesis step reacts.In this embodiment, by the suitable selection of primer sequence, but also mark can be amplified each mark from other mark respectively.Finish the sequencing reaction, measure on the gel at standard sequence and carry out electrophoresis, the oligomer sequence is released by the password that the sequence information that obtains discloses.

Yes-no decision is to use common PCR primer and common sequencing primer (sequencing primer even can be overlapping with the PCR primer sites whole or in part), by hybridize to the few nucleic acid of pearl in the distinctive sequence complementary of each step oligonucleotide probe on discern this step.The sequencing of single group be reflected at all amplifications from carrying out on the oligonucleotide of single pearl, reaction product is electrophoresis on the Dan Zudao of gel, then reaction product is transferred on the Hybond membrane that suits, hybridize to the distinctive probe of one step and (see that this paper is incorporated by reference, Maniatis et al., Cold Spring Harbor Laboratory, ColdSpring Harboo, NY (1982)).Behind the signal that detection obtains, probe to be washed off from film, the distinctive probe of another step is hybridized.Also can use be described in this paper EPO publication number incorporated by reference be 237,362 and the PCT publication number be 89/11548.

Parallel hybridization provides a kind of selection for succession hybridization.The sequencing reaction is divided into the many aliquots containigs that equate with peptide synthesis step number, is carrying out electrophoresis on the independently group road at each on the sequencing gel.After reaction product being transferred to a suitable film, this film is cut separately respectively to be organized, and then each road group hybridization is extremely in the distinctive oligonucleotide probe of many steps.(seeing " Uniplex DNA Sequencing " and " MultiplexDNA Sequencing, " the in Plex Luminescent kits Protuct Catalog that this paper is incorporated by reference, Bed-ford, MA, 1990).

As mentioned above, single synthesis of solid carrier (or a connection pearl that has a mark, or in the solution in " well ") can only comprise a hundreds of copy of each oligonucleotide mark.Can be by PCR or other method well known to those skilled in the art with these mark amplifications so that the DNA that will be carried out sequencing exactly of capacity to be provided.The ability of decoding oligomer depends on the number of obtainable oligonucleotide identification marking, amplification amount that can reach from obtainable mark and the accuracy of the DNA of that amplification being carried out sequencing.

If adopt the pcr amplification of oligonucleotide identification marking, may run into " PCR product pollution ", it is caused that by the product of PCR reaction pollution is used for increasing, and other has the PCR reaction mixture afterwards of the mark of identical PCR primer binding site.By unstable being introduced the product sequence, the possible pollution that processing reaction afterwards so that elimination are got off from the front reaction zone can prevent this problem.A special embodiment of this method introduces product with dUMP, and a complete set of wherein buying is sold by PECI and Life Techndogies.Handle the DNA that each new PCR reacts any dUM of containing of the appearance of having degraded with uridylic N-Glycosylase, prevented the amplification of polluting like this.The template DNA that does not comprise dU (only containing dT) is unaffected, certainly, before the amplification beginning Glycosylase is removed or inactivation.

More above-described peptide complex sign tools only have the unique features that comprises pyrimidine.This means UNG method (PerkinElmer Cetus Instruments (PECI) Catalog that this paper is incorporated by reference, Alameda (1991)) will these chains that produce only in half be those contain T ' S (or U ' S) in work.DUMP can not be incorporated into complementary only has in the chain of purine; Yet the damage of splitting that the purine chain is caused by the alkali of sour depurination and main chain very easily.The combination of these processing can reduce the problem that product pollution is brought widely.The another kind of method that prevents the pollution of carrying is incorporated into a restriction site (EarI can be used as many pyrimidines mark) in the oligonucleotide mark before being included in and suspecting the reaction amplification that is labeled pollution, and digests with corresponding Restriction Enzyme.This method only just works when the mark that will increase is not split by enzyme, and it is the situation of single stranded oligonucleotide mark normally.

For the DNA of the amplification of sequencing, expectation produces single-stranded template usually.Can produce by any method.The method of one of them is an asymmetric PCR, wherein uses excessive a kind of primer to increase a chain to 10 to 100 times the amount that is higher than other (seeing that this paper for example US patent No. incorporated by reference is 5,006,584).The another kind of method that one single-stranded template is provided is by a kind of primer of biotin labeling, by being adsorbed to the immobilization chain affine purifying usually of poison or removing gained chain (the Pierce Immunotechnology Catalog andHandbook that this paper is incorporated by reference, 1991).Yet another kind of method comprises the rna transcription thing (representative is a chain only) that produces from rna polymerase promoter and with ThermoScript II sequencing transcript (the Sommer et al. that this paper is incorporated by reference, Chapter 25, InPCR Protocols:A Guide to Method and Applications, Supra).If mark only is made up of pyrimidine nucleotide, all purine can be handled by cancellation by acid/base so, stay the pyrimidine chain of sequencing.

The sequencing reaction easily of the independence of the distinctive primer of each step of service requirements of the independently sequencing primer of the distinctive oligonucleotide of each step.By the use of the primer of distinctiveness mark allow identification marking from single solid carrier in single reaction by sequencing, and in the single track group on the gel (when only for many pyrimidines is 2 roads; If use 4 different bases then be 4 roads) in electrophoresis.The primer of buying (by this paper ABI Catalog incorporated by reference) that has the recognizable fluorophor mark of the usefulness that is suitable for this purpose at present also can use a series of chemiluminescent labelings that can buy at present, and (the Bron-stein et al. that this paper is incorporated by reference, the product that BioTechniques 8:310-314 (1990) is amplified can easily be carried out sequencing or be identified the situation of the peptide deciphered on the ball or other molecule or be connected to by other method on the mark of oligonucleotide by other method.For this purpose, can use a kind of in the multiple sequencing method, comprising sequencing by the distinctive probe hybridization of sequence.Adoptable determined dna sequence enzyme comprises the Taq archaeal dna polymerase among the present invention, E.Coli archaeal dna polymerase (or Klenow segment), T7 polysaccharase, Sequenase ^TMSequenase and Sequenase II ^TMSequenase (adorned T7DNA polysaccharase), BSt archaeal dna polymerase and ThermoScript II are (from this paper AMV incorporated by reference, MMLV, RSV, etc, see USB Enzymes for DNA Sequencing, VS BiochemicalCorp, 1991, Clevaland OH).The sequence of oligonucleotide mark also can be discerned by high reliability DNA hybridization technique, and for this reason, having the immobilization polysaccharase of oligonucleotide in a large number synthetic may be (the seeing the PCT patent publication No. 92/10587 and 92/10588 that this paper is incorporated by reference) of benefiting.

No matter mark is oligonucleotide or some other molecular structures, and the selection of mark depends on the method for attribute and synthetic these molecules of the molecule of forming the storehouse, and they are discussed below.

When the synthetic chemistry process comprises use with inconsistent reagent of above-mentioned oligonucleotide mark and reaction conditions, be expected to use the alternate marking method.Therefore, the method in synthetic tagged molecule of the present invention storehouse also expects to utilize the unreactiveness hydrocarbon tagged molecule that can individually resolve by serial of methods such as chromatographic process.

The use of these inert hydrocarbon marks in the library of molecules has obtained description.See all by this paper Michael HJ Ohlmeyer incorporated by reference, et al., Proc.Nat ' 1A-cad.Sci.90:10922-26 (in December, 1993) and disclosed PCT application number are WO94/08951.

The mark utilization of describing resembles binary coding method as herein described, binary codedly is used to stipulate each chemical structure unit, the i.e. amino acid that will add in synthetic.The length of password can be dependent on the overall number of the structural unit of adding.For example, when seven structural units that will add are only arranged altogether, available triad password.This allows seven passwords that independence is different, and from 001 to 111, each is used for stipulating of seven structural units, for example Methionin=001.The number of structural unit is big more, and spendable password is big more, for example, and an eight-digit binary number password as described herein.

The utilization chromatographic process prepares many marks, and wherein each mark has resolution different with other mark or separated graphics.If a special marking, it will show one " 1 " on each position of the final mark password of given synthetic molecules.Therefore, have four structural units at a molecule, during with the tri-bit encoding system coding, have 12 possible password figures, each structural unit has three figure places.For each step in synthetic, the solid carrier that tagged molecule will be synthesized thereon, so that not only show the structural unit that adds, and show the step that adds fashionable place.For example, mark be 1 or the appearance of " T1 " show that on first of the binary marks password of the structural unit that the first step adds one " 1 " is arranged.Similarly, the appearance of T7 shows on the 7th of all passwords one " 1 ", in the password of three numerals, it will be equally corresponding to first position of the structural unit of the 3rd adding.Therefore be defined as the structural unit of password 111, if 1 adding in the position, will be by mark T occurring ₁, T ₂And T ₃Encode.In other words, if add in step 2, identical structural unit will be by mark T occurring ₄, T ₅And T ₆Encode.

The special hydrocarbon mark of being described by Ohlmeyer has following structure:

Wherein n is 1 to 10.Length and reformed halo group that hydrocarbon chain changes make it can pass through chromatographic process, are in particular and capture physical sepn or the resolution that the chlorine phase chromatography carries out mark.

Yet, the restriction of the examined method sensitivity of the detection of these hydrocarbon marks.Therefore, in order to ensure the detection of mark, necessary with a large amount of marks.This requires a large amount of fractionated microballoons, reduced like this and can be synthesized, and be increased the reaction times assurance mark maximum be coupled to the size in all storehouses on the solid carrier.At last, in the more macromolecular place of screening, more hydrocarbon mark is injected towards in the solid carrier, mixing of a large amount of hydrocarbon on solid carrier, for example, big synthetic compound with a large amount of marks, because the space, hydrophobic or ionic interaction might have a negative impact to the screening of the compound on the synthetic and/or solid carrier continuously.

The detectability relevant with these hydrocarbon marks, mark time and screening or synthetic interferential problem can use method of the present invention to prevent.Especially, a kind of marking method of employing the hydrocarbon mark is provided in one embodiment of the invention, wherein this mark has one " molecular hook " and replaces the detected electrophoresis as descriptions such as Ohlmeyer, this " molecular hook " is defined as allowing to connect and can increasing at this paper, but therefore the functional group on the mark of detection moiety makes the detection of the mark on the solid carrier become possibility.This hook generally comprises a stable functional group or a molecule, this molecule with can expand detectable group and form covalent linkage or have the affinity of height with the part of that group.This hook comprises the biological example element, has connected affine mould being incorporated on it of strepto-of the detectable group that can increase, or the complement of high binding peptide.This high binding peptide generally comprises the complementary pair to the peptide of another tool high-affinity.Therefore, complement refers to a peptide of such centering.It is in 08/321,933 that high binding peptide briefly is described in the U. S. application series number that proposed on October 12nd, 1994, and it is that the U. S. application that proposed on May 24th, 1993 number is 08/067,387 part continuation application, and each piece of writing is all incorporated by reference by this paper.On the other hand, but hook can comprise protected activating group, but it can be activated the covalency ground detection moiety that can increase and is connected on the mark.Particularly useful in fortune kind of application by the activating group that light is protected.Activating group is generally known in the art, comprises these groups, as carboxyl, and hydroxyl, amino, thiol and analogue.Employ blocking group that light is not wanted for example those of the description in the disclosed PCT application number WO93/22680 incorporated by reference by this paper for all purposes can protect these groups.The group that obtains is can be photoactivated.

Remove the above, molecular hook can comprise multifunctional group.For example, molecular hook can comprise that two or more can be coupled to the different functional groups on two different bodies.Can be this situation, for example wish mark is coupled on another solid carrier, for example the responsing well in the microtiter plates again for testing goal.First functional group can be used to optionally in conjunction with the complementation group on the solid carrier.In case by coupling, but second functional group can be used for increasing in the selectivity coupling of detection moiety.This hook generally comprises functional group described herein or can optionally be attached to the combination of other group on other this group.Embodiment for example, this hook is connected to the vitamin H and the digoxin of hydrocarbon mark with can comprising orthogonality.In case separated, these marks can with solid carrier for example the microtitration well contact, this solid carrier be capped a kind of can with a functional group on the mark group of the antibodies of anti-digoxin for example, and be allowed to combine with them.After the repeated washing step, solid carrier is contacted with oligonucleotide, oligonucleotide is coupled on the group that can be incorporated on the oligonucleotide that second functional group Streptavidin as previously described connects.Then detect the bonded oligonucleotide as previously mentioned.The synthetic of hydrocarbon mark of the present invention can be undertaken by method well known in the art.Referring to for example March, Advanced Organic Chemistry (John Wiley﹠amp; Sons, 3rd Ed., 1985), Larock, Comprehensive Organic Trans-formations CVCH Pubishers, 1989).

Because unreactive hydrocarbons mark of the present invention provides the more detection of sensitive hydrocarbon mark, the amount of the special marking on the solid carrier can be lowered under the situation of the detectability that does not influence it.Further, by reducing the amount of the mark on the solid carrier.Mark is coupled to the time required on the carrier to be shortened.

Mark useful among the present invention will usually comprise available hydrocarbon district and molecular hook.More preferably, this mark will comprise the joint of the cleavable that mark is linked to each other with solid carrier, the hydrocarbon chain of described molecular hook and the variation length that molecular hook is linked to each other with joint.Isolabeling will not have the hydrocarbon chain of different lengths or different molecular hooks so that their physical sepn and detection.

It is preferred to have being labeled as of following formula:

Wherein n is 1 to 10, and X is the joint of cleavable, and R is a molecular hook.Preferred molecular hook comprises the biological example element, but high binding peptide and activating group, for example can photoactivated group or their combination.

But the joint of useful cleavable for example comprises the joint of the photodestruciton of describing in the U. S. application that proposed 23 days June in 1994 incorporated by reference by this paper for all purposes number 08/265090 among the present invention.But the joint of this photodestruciton comprises those materials with following array structure:

After synthetic and mark, mark is removed from solid carrier by the photodissociation of for example joint.Then with the method for keeping the mark separated graphics, for example to the HPLC of part collection, or other chromatographic process of example gel or capillary electrophoresis is separated mark separately.Because mark is usually in a usual manner as non-detectable measuring now such as absorbancys, they must be to allow subsequent detection, and the mode relevant with their separated graphics is separated.As an embodiment, cracked is marked on the HPLC post separatedly from the solid carrier, and is collected in the Fraction Collector.When mark is carried out last detection, will describe in further detail below, mark/synthetic institute is underlined for being used in, and the part that expressive notation exists is relevant with known wash-out profile or separated graphics.

Then isolating mark is fixed according to their separated graphics.Thisly fixing can take the point sample each several part, for gel be basic separation point sample, or in the i.e. fixed form in microtiter plates of responsing well.

In case be fixed, mark can be attached to hook to increase to be increased on the detectable group, but the detection moiety that can increase generally comprises the compound or the structure that can be amplified or produce the signal that can be amplified.The compound of the exponential amplification of energy is preferred.But the useful especially detection moiety that increases is an oligonucleotide sequence.Can increase and linking of detectable group can take various ways with hook.For example, when hook comprises a vitamin H group, with oligonucleotide be coupled to tightly with vitamin H bonded streptavidin on.On the other hand, can then add in the back in the intermediate steps of biotin labeled oligonucleotide and add Streptavidin.Select in the embodiment at one, mark can comprise the complement of high binding peptide.In this case, oligonucleotide combines with other complement of peptide, and oligonucleotide can combine with mark tightly like this.Yet in another embodiment; mark comprise by as be the activating group of the photolabile blocking group protection described among the WO93/22680 in the past by disclosed PCF application number incorporated by reference; this group can be activated by the photodissociation to the unstable group of light, and making then, oligonucleotide is coupled on the mark by method well known in the art.In this case, if but use the photolabile blocking group that to expect to select to have the photodissociation characteristic different with the joint of photodestruciton.This will allow in optionally cracking from the solid carrier of mark under the situation of activated molecule hook not.

In case be connected on the mark by hook, use the round pcr described herein oligonucleotide sequence that can increase, when fixing when adopting the trace form, must increase with the diffusion of the oligonucleotide that keeps partial concn and avoid increasing, it can be detected and corresponding with separation method.For example, these amplified reactions in can be by the gel that carries out trace overlapping are finished.

The oligonucleotide that is hooked and the detection of mark thus can this sequence be that the known oligonucleotide sequence that is amplified is finished in the oligonucleotide that be amplified or by surveying wherein by a mark is incorporated into.The detection of mark is corresponding with known mark separation method.The mark that to so discern is incorporated in the underlined password of institute that is synthesized molecule again.Only be the embodiment purpose,, connect with hook if separate according to HPLC, amplification and detection, digital #17 expressive notation is corresponding.For example, if this is mark #15, " 1 " can be used to represent all the binary coded slot #s 5 that are synthesized molecule on the special carrier so.This coding method described herein is only for the purpose of embodiment, it should be appreciated by those skilled in the art that to multiple coding method can be used in the method for the present invention.

Those skilled in the art also will recognize does not need the password of sequence of synthetic molecules that will report to be included in the single polymerization sequence of each mark.On the contrary, password can by on the solid carrier each not the existence of isolabeling do not exist and embody.Though these marks may be by couplings mutually sequentially, the technician with understanding make each not isolabeling be connected to benefit on the solid carrier individually.Especially, any He all markers step are only needed single coupling chemical process.Further, can avoid having the protection/de-protected complicated agreement of the mark of differential responses group.

The V synthetic method

Method of the present invention can be used in any group the synthetic chemistry reaction of carrying out in the program of the different compounds of preparation.Though the present invention generally uses the chemical structure unit to illustrate, more generally uses the monomer structure unit, should understand general essence of the present invention.The carrying out of most of synthetic chemistry reactions has a great difference with general monomer linked reaction; General organic chemical reactions provides variable productive rate and produces multiple product, for example zone and stereoisomerism structure.The present invention can be used to discern the useful products of such series of chemical, because can utilize this method to make the information of label coding synthetic product replace the clear reaction product structure of determining.

Yet in order to simplify discussion, the present invention almost is counted as a series of monomer coupling steps.Because various coupling step of the present invention can carry out in single other reaction vessel at disengaging time, even for example unitary structural unit with greatly different coupling chemical processes also can be used for the assembling of compound of interest in the storehouse.Though the present invention can be by being exposed to solid carrier in structural unit and the identification marking simultaneously, or sequentially (be exposed to structural unit earlier, be exposed in the mark again or be exposed in the mark earlier and be exposed in the structural unit again), this has the method for succession to allow an additional snappiness according to the coupling chemistry.Under any circumstance, carrying out that linked reaction preferably arranges is to carry out different linked reactions abreast.

After the coupling step of each parallel series is finished, in reassigning to each container of next coupling step before, other compound in oligomer and storehouse synthetic solid carrier is thereon compiled and is mixed.This blending means has prepared a big storehouse of the compound of each different members that has the storehouse on the different solid carriers.If each synthesis step all has high coupling efficiency, the compound on all single solid carriers has same structure so basically, if or compound be oligomer, then have identical sequence monomer.By synthesis path decision structure or sequence at any given solid carrier that synthesizes latter stage.The maximum length of oligomer is generally less than about 20, be generally 3 to 15 monomer length, but the length of 8 to 12 monomers (residue) is preferred in some cases.

Given the mark and the structural unit of the different numbers that are suitable for purposes of the present invention, many chemical processes that prepare chemical libraries of the present invention are arranged.Yet, must guarantee that no matter to be each coupling step of mark or oligomer can not produce the unwanted reaction of underproof amount or damage mark or the oligomer that has occurred on carrier.In one embodiment; must guarantee to have the solid carrier of the chemical reaction group that mark is connected with oligomer by use; desired reaction only takes place, and wherein being connected of mark and oligomer passes through to use the blocking group of two differences or " quadrature " type protected.Solid carrier is exposed on first and goes in protective material or the activator, has removed first type blocking group from the chemical reaction group of example as the synthetic site of oligomer.With first monomer reaction after, arbitrary optionally protect step after, then solid carrier is exposed in second activator of the protecting group of removing second type, for example expose chemical reaction group as the identification marking connection site.With the mark coupling, repeat these steps again, be generally 1 to about 20 times.

A. the peptide storehouse of oligonucleotide mark

In an important embodiment, the present invention relates to large-scale storehouse synthetic of different peptides.Though many other compounds and oligomer can (be seen the Gait Oligonucleotide Synthesis:A Practical Ap-proach incorporated by reference by this paper by method, IRL Press, Oxford (1984), Friesen and Danishefsky, 1989, J.Amer.Chem.Soc.111.6656; And Paulsen, 1986, Angew.Chem.Int.Ed.Engl.25:212) prepare, but the solid phase synthesis process of peptide is particularly important and (sees the Mer-rffield incorporated by reference by this paper as everyone knows, 1963, J.Am.Chem.Soc.85:2149-2154) and the peptide storehouse all extremely useful for various purposes.In the Merrifield method, amino acid covalently is attached on the carrier of being made by insoluble polymer.Another has the amino acid of alpha-amino group blocking group and is formed a dipeptides by the amino acid reaction with covalent bonds.Remove blocking group, the 3rd amino acid that will have the α blocking group adds 1 in dipeptides.Continue this step has obtained expectation until peptide length and sequence.Available blocking group well known by persons skilled in the art prevents that false coupling from (seeing the The Peptide of this paper incorporated by reference, Vols.1﹠amp; 3 (eds.Gross, E., and J Meinhofer, Academic Press, Orlando (1979 ﹠amp; 1981)) or allow one to control coupling.For various purposes of the present invention to photo-labile, unstable and unsettled blocking group of acid and their combination can all be to use to alkali.

In addition, L type and D type amino acid all may be employed in the peptide synthetic method described herein.As what describe in the U. S. application series number 08/309,45/ that 21 days September in 1994 incorporated by reference is proposed by this paper by all purposes, adopt the D-amino acid can be useful in " peptide backward-oppositely " (retro-inverso peptide) synthetic.These backward-oppositely peptide will comprise identical aminoacid sequence, but have and use the opposite stereochemistry of L-amino acid synthetic peptide.

When the present invention is used for preparation and screening peptide storehouse, selection be labeled as nucleic acid.Synthetic and circulation of oligonucleotide identification marking is followed a round-robin and is connected and has many compatible chemical property and process for peptide.Yet,, may need to use the various combination of blocking group and/or synthesizing ribonucleotide to avoid the degraded of synthetic mark or oligomer in order to keep the integrity of peptide oligonucleotide mark between synthesis phase.Usually, many pyrimidines oligonucleotide is marked under the general peptide synthesis condition relatively stable, and this is opposite with the oligonucleotide mark that comprises natural purine nucleotides, but but the amplification of how many refractory PCR of many pyrimidine nucleotides mark.May need purine radicals or analogue such as 7-impurity elimination nitrogen-Desoxyadenosine and 7-impurity elimination nitrogen-pancreatic desoxyribonuclease, their tested abilities that stands the peptide coupling condition are incorporated into the expectation efficient that the acquisition in the mark is increased.For the purposes of the present invention, mark optionally comprises 10% to 90%, preferably is 35% to 50%, more preferably is purine or the purine analogue Nucleotide of 33%-35%.The oligonucleotide mark optionally mixes vitamin H or other reporter group so that purifying, hybridization, amplification or detect (seeing the Pierce ImmunoTechnology Catalogand Handbook incorporated by reference by this paper, 1991).

Therefore, in chemical process and the character of selecting to be used for to prepare the peptide storehouse of oligonucleotide mark of the present invention, must (1) select to have the solid carrier of functional group of suiting; (2) select amino acid coupling chemistry; (3) select oligonucleotide mark coupling chemistry; (4) select various marks, the blocking group of monomer and oligomer and (5) are selected to go to protect and cracking chemistry (for mark or peptide) in certain embodiments.Those skilled in the art recognize that not all in each case top selection all needs to make, because some use the identical problem that can not propose with other.For example, can not all require one or more blocking groups for all application.In the ordinary course of things, one during these are selected is important.

Consider and the coupling chemistry; the relevant factor of selection of the synthetic blocking group in the peptide storehouse of oligonucleotide mark need to be considered the amino acid of the Fmoc protection of buying with the Merrifield chemical coupling of standard and synthesizing with the phosphoramidate chemical coupling oligonucleotide of standard.This method can think to have following steps: (1) removes aminoterminal Fmoc blocking group from the joint or the peptide that are connected in bead; (2) amino acid with Fmoc protection (side chain also can be protected) is coupled on the free amine group that produces in the step (1); (3) any unreacted free amine group being carried out selectivity covers; (4) the DMT blocking group from being connected hydroxyl on thereon the mark, bead or Nucleotide mark is removed; (5) accidentally have 5 '-blocking group on DMT blocking group and the phosphoric acid ester and the ring of base be for the Nucleotide phosphoramidate of amine; (6) optionally cover any unreacted free hydroxyl group; (7) the phosphorous oxidation of oligonucleotide mark; (8) peptide and oligonucleotide mark go the protection.Each step in these steps is discussed below.

(1) before connecting next amino acid monomer, must be from the joint that links to each other with bead or peptide the aminoterminal Fmoc blocking group of removal.Usually, in DMF, handle to finish in about 1 hour and thisly go protection (also see step 8), but one aspect of the present invention to relate to synthetic for the peptide storehouse of oligonucleotide mark, use the guard time that goes of the piperidines that reduces concentration or shortening with 30% piperidines.Piperidines can cause methyl three esters protections the oligonucleotide mark go protection, the ester protection group of O-methyl phosphorus has bigger base stability because of the β-cyanoethyl group than known standard to piperidines cracking sensitivity.It is 5% to 15% that the preferred Fmoc of the present invention removes protective condition, is preferably 10% piperidines and handles 5 to 60 minutes, is preferably 10 to 20 minutes, and 15% to 30% piperidines is 15 to 30 minutes.Another of known effect Fmoc removal is treated to DBU handles (1,8-diazabicylo (5.4.0) 11-7-alkene), and for example 5%DUB handled 5 minutes.Yet by this paper Palom et al. incorporated by reference, the report of Tetr.Lett.34:2195-2198 thinks that this processing can cause the methylation of the N-3 of thymidine.Though the removal of the Fmoc of DBU-mediation some should in can be effective, should admit the possibility of base modification.

(2) (see The Peptides, Supra can be coupled to the amino acid of Fmoc protection on the free amine group group of bead or peptide with conventional BOP coupling chemistry.Usually, adopt the amino acid (110mM) of the Fmoc protection in the solution of forming by 1: 1 DMF/DCM, HBTV (100mM), the amino acid whose coupling of the mixture of HOBt (100mM) and DIEA (300mM).Other activating chemical process also can be used in this situation, for example substitutes HBTV/HOBT with HATV.Yet in one embodiment of the invention, reaction mixture is by 3: the amino acid of the 55mM Fmoc protection in the solution that 1DMF/DCM forms, and 50mMHBTV and 150mMDIEA form; This embodiment is preferably used and can be omitted the equipment that reagent transports bottle, and side chain also can be protected; Because the commercial availability of structural unit is multiple purpose, preferred Fmoc/ ^tThe Bu protection.The useful amino acid structure unit of other band side chain protected comprises Arg (Pmc), Gln (Trt), HB (Trt), Asn (Trt), Asp (O ^tBu), Glu (O ^tBu) and Lys ( ^tBOC) and the amino acid of the band side chain protected that provides by photolabile blocking group.

(3) by handling with diacetyl oxide and 1-Methylimidazole or other methods known in the art can obtain that unreacted free amine group group is carried out selectivity and cover.

(4) by being CH with trichoroacetic acid(TCA) (TCA) ₂Cl ₂Middle 10%TCA handles and the DMT blocking group will can be removed coupled bead or the hydroxyl on the mark from the Nucleotide mark.If the ring to sour unstable protection group and Nucleotide in the use phosphoric acid ester (is a Deoxyribose cytidine for amine; 7-impurity elimination nitrogen-Desoxyadenosine; with 7-impurity elimination nitrogen-pancreatic desoxyribonuclease), so these groups will enough firmly resist 5 '-TCA (being generally 1-3%) that uses in the O-detritylation.

(5) coupling have 5 '-the Nucleotide phosphoramidate of DMT blocking group can obtain by using conventional phosphoramidate chemistry, though need must to consider on the phosphoric acid ester oxygen ring with the base of oligonucleotide mark for the protecting group on the amine.For the photolabile blocking group of nucleic acid, see PCT patent disclosure WO92/10092 and Baldwin et al. incorporated by reference by this paper, 1990, Tetr.Lett.46:6879-6884.As mentioned above; suitable phosphoric acid ester blocking group comprises O-methyl and β-cyanoethyl; but O-allyl group and/or the IV-allyloxycarbonyl (3-that promptly quotes (allyl group N; N '-di-isopropyl) phosphoramidate) also can be used to protect the ring of phosphoric acid ester oxygen and nucleoside base for amine; (see HgyaKawa et al. incorporated by reference respectively by this paper; 1990, J.Amer, Chem.Soc.112:1691-1696.Contain three (dibenzyl subunit acetone), two palladium chloroform mixtures by use, triphenylphosphine and n-Butyl Amine 99/formic acid then carry out the THF washing, moisture N, and N-diethyl curing carboxylamine sodium and water washing can be removed allyl-based protection group.The phosphoramidate coupling is passed through as the 1H-tetrazolium; 4-oil of mirbane tetrazolium; Pyridine hydrochloride/imidazoles.Recent phosphoramidate activator with the pseudoreaction on the nitrogen on peptide or the oligonucleotide low-level be cost caused optionally 5 '-the O-phosphitylation (sees the Gryaznov andLetsinger incorporated by reference by this paper, 1992, Nuceic Acids Research 20:1879-1882).

(6) by handling with diacetyl oxide and 1-methyl tetrazolium or covering by handle the selectivity that can reach with diacetyl oxide/lutidine/DMAP to any unreacted free hydroxyl group.

(7) by handling or use I with iodine and pyridine ₂, collidine, the MeCN in the water handles the phosphorous oxidation that can reach in the oligonucleotide mark.On the other hand, by adopting the phosphorous mild oxidation agent of oxidation ^tBuOOH can reduce amino acid first propylhomoserin, and the oxidation of tryptophane and Histidine (see Hayakawa et al. incorporated by reference by this paper, 1990, Tetr.lett.27:4191-4194).

(8) by sequentially using in the methylene dichloride 1% TCA, use thiophenol/NEt again ₃/ diox (1: 2: 2); then with 1; 2-quadrol/EtOH (1: 1) handles down at 55 ℃ and remove blocking group from mark; then use trifluoroacetic acid (95: 5TFA/ water, cation removal agent) remove to the unsettled blocking group of acid can realize peptide and oligonucleoside mark go the protection.Purine nucleotides to the unstable of strong acid (as TFA) can by utilize purine nucleoside analogs 7-denitrification assorted-2 '-Desoxyadenosine and 7-denitrification be assorted-2 '-phosphoramidate of pancreatic desoxyribonuclease avoids (seeing the Barr et al..1986.BioTechniques4:428-432 incorporated by reference by this paper, and Scheit, Nucleotide Analogs:Synthesis and Bio-logical Function PP.64-65 (John Wiley and Sons, NewYork).

A preferred embodiment in the peptide storehouse of following part explanation synthetic oligonucleotide mark.

Improving one's methods of the peptide library of B. synthetic few nuclear person acidity scale note

The method of setting up a kind of bead base oligonucleotide encoded peptide library of practicality need satisfy several gordian technique standards.These standards comprise the exploitation of the mutual coupling chemical process of (i) parallel secretory piece and oligonucleotide; The bead that (ii) has suitable physical character, the selection of material; (iii) separate miniature bead wherein part and the target receptors bind be loaded with part easily; (iv) successfully read the code labeling thing, promptly by pcr amplification with from single bead ordering template marker DNA from single spherolite.The invention provides improving one's methods of a kind of synthetic this class library, describe as this part and embodiment 1, it shows how to use strand oligonucleotide marker to encode synthesizing of component peptide on 10 μ m diameter polystyrene spheres.

In this was improved one's methods, peptide assembled with parallel alternately synthesis mode with nucleic acid, so that each bead all has a strand peptide sequence and unique oligonucleotide identification tag.These oligonucleotide share common 5 '-and 3 '-the PCR priming site; Therefore these beads can be used as pcr template.For this method is described, synthetic and screen about 8.2 * 10 ⁵The coding synthetic library of seven peptides with anti--dynorphin B monoclonal antibody D32.39 (Cull etc., 1992, Proc.Natl.Acad.Sci.USA 89:1865-1869, draw herein and be reference) combination, use fluorescence laser active cell sorting (FACS) instrument to select and the indivedual beads of the strong bonded of antibody.After selecting to carry out the oligonucleotide tags pcr amplification on the bead, DNA is sorted to determine the characteristic of peptide part, these are described in greater detail in hereinafter.

Important method of this method is described in the additional detail part of following embodiment 1, and it is to be peptide and the synthetic solid phase carrier of selecting of marker.Preferred solid phase carrier, promptly 10 μ m diameter beads are made by macropore vinylbenzene-divinyl multipolymer and are derived with the n-Laurylamine joint.By with the complete acidylate of Fmoc-glycine, piperidines is removed the Fmoc base and is reached piperidines dibenzo fulvene (piperidine-dibenzofulvene) affixture (e that the spectrophotometric standard measure discharges admittedly subsequently ₃₀₂=7,800 1mol ^-1Cm ^-1, estimate that the amino on these beads is～100 μ mol/g.5 * 10 ⁹Bead/g, its corresponding maximum peptide carrying capacity is～the 20fmole/ ball.Use the amino acid of due care and the mixture of Ω alcohol acid to carry out amino and the hydroxyl that the bead acidylate obtains the quadrature differentiation, can develop peptide and nucleotide chain respectively thus.Change is coupled to the amino acid of initial bead amount (vide infra) and the ratio of alcohol acid is controlled the average stoichiometry of every bead peptide to oligonucleotide by putting.The test of use standard Fmoc chemical process is having trifluoroacetic acid cleavable Knorr joint (Bernatowicz etc., 1989, Fetr.Lett.30:4645-4648, here draw and be reference) bead on synthetic (5 peptide to 12 peptides of peptide, find that it has the fidelity of height, with synthetic the having no difference of on conventional peptide synthetic resins, carrying out, analyze the result who is measured as rough cleavage of peptide acid amides HPLC.

Parallel synthesis strategy requires to use a cover blocking group on mutually orthogonal amino acid and nucleotide structure unit, and each bar polymer chain is stable for employed reagent in employed in synthetic and the second chain deprotection.Though can use various protections/deprotection scheme (as mentioned above) in principle, preferably on the peptide structural unit, use Fmoc/ ^tThe Bu protection obtains because the natural and alpha-non-natural amino acid that this mode is protected can extensively be bought.Yet, ^tThe peptide side chain protected group of Bu-base requires to use strong acid (being generally trifluoroacetic acid) to handle to slough; condition be can cause oligonucleotide comprise 2 '-Desoxyadenosine (dA) or 2 '-the quick depurination of pancreatic desoxyribonuclease (dG) (sees Capon; 1969; Chem.Rev.69:407-498, draw here be reference).Around this problem be exactly in the template oligonucleotide tags with 7-denitrogenation-2 '-Desoxyadenosine (c ⁷DA) replace dA.The antiacid catalytic hydrolytic action of the glycosidic linkage of deazapurine nucleosides is (referring to Scheit, 1980, Nucleotide Analogs:Synthesis and Biolgical Func-tion (John Wiley and Sons, New York) PP.64-65, here draw and be reference), duplicate fully by the thermostability polysaccharase that uses among the PCR and to be mixed with these monomeric oligonucleotide (referring to Mc Conlogue etc., 1988, Nucl.AcidsRes.16:9869, with Barr etc., 1986, Bio Techniques 4:428-432, wherein each piece of writing is at this all as a reference), antiacid guanosine also can be comprised in the template DNA.All parallelly all use 5 in synthetic '-O-dimethoxytrityl 2 '-deoxynucleoside 3 '-(O-methyl-N, N-di-isopropyl) phosphoramidate.In the DNA synthetic schemes, be used for Nucleotide phosphorous acid ester intermediate is converted into the reagent (I of phosphotriester ₂/ collidine/H ₂The O/ acetonitrile), do not find that its commute oxidation residue Trp and Met or other protected amino acid have disadvantageous effect.Use in the methylene dichloride 1% trifluoroacetic acid (TCA) in～40 seconds with 5 '-the O-DMT group is removed on the few nucleic acid chains that is increasing fully, and all routines are used for Fmoc/ ^tAcid labile Side chain protective group in the Bu chemical process removes tyrosine-derived ^tOutside the Bu ether, processing all was inertia in 1 hour to 1%TCA.Fmoc-Tyr (OBz) prove the peptide that contains tyrosine synthetic in suitable substituting, this O-benzoyl fat is used to remove α-N-Fmoc protecting group in synthetic to peptide TCA and piperidines all are stable.The quantitative deprotection of alpha-amino group residue requires to handle 5-10 minute with piperidines/DMF (10%v/v), and also can cause the part demethylation (t of protected polynucleotide phosphotriester _1/2～45min).Control experiment shows; the any unusual phosphitylation effect meeting of product phosphodiester class is changed and goes back by final oligonucleotide deprotection steps (referring to Lehmann etc. in nucleotide chain propagation process subsequently; 1989, Nucl.Acid Res.17:2379-2390, draw here be reference).During parallel synthetic finishing, use thiophenol ester (thiophenolue) (phosphoric acid ester O-demethylation) to use ethanol quadrol (protected cytidine and 7-denitrogenation-VITAMIN B4 residue take off benzoylation) to make the complete deprotection of DNA then.These gentle anhydrous ammonias separate condition not to protected peptide sequence have a negative impact (referring to Juby etc., 1991, Tetr.Lett 3:879-882, draw here be reference).Under standard conditions, use TFA can make protected peptide sequence deprotection.

The C-terminal district that had before shown opioid peptides dynorphin B (YGGFLRRQFKVVT) (SEQ ID NO:1) has represented the epi-position of anti--dynorphin B mAbD32.39 (referring to Cull etc., above-mentioned): solubility seven peptide RQFKVVT (SEQ ID NO:2) and D 32.39 with high affinity (Kd～1nM) combine.On the bead of the quadrature differentiation of carboxamide (Knorr) joint of the Fmoc protection that has a sour cleavable, carry out the parallel synthetic of this peptide and the few oxygen Nucleotide of 69 bases.After adding 20 initial Nucleotide, with piperidines/DMF and first peptide residue that is coupled to unhindered amina (Fmoc-Thr ( ^tBu)-OH) handle bead.Bead is in two circulations of phosphoramidate chemical reaction process and the next amino acid of coupling (Fmoc-Val-OH) then.Repeat this process, be synthesized fully until seven peptide sequences and nucleotide coding district, and then with 35 following Nucleotide extended DNAs so that 5 of a transcribed spacer and PCR '-priming site to be provided.At last bead is exposed in whole oligonucleotide and the peptide deprotection condition subsequently, the TFA supernatant liquor that contains the peptide under the cracking is with the reversed-phase HPLC analysis.HPLC result shows, the rough peptide of parallel synthetic is formed (with real RQFKVVT (SEQ ID NO:2) co-elute) by a kind of single principal constituent, and this thick product is compared not obviously difference with the synthetic peptide that is produced of the control peptide that the oligonucleotide chemical reaction does not take place.

Contain T, dC and c ⁷The stability of the parallel synthetic chemistry of the template DNA of dA is compared with the similar target thing that contains standard purine nucleotides dA.Utilize the single spherolite clone ability of the positive cell counter of fluorescence-activated cell sorter (FAC Star Plus cytometer); be sorted in miniature centrifugal (microfuge) pipe from the indivedual deprotection beads of two synthetic; and bound widow is nucleic acid-templated to increase by PCR45 circulation.Only make " clean " amplification product of desired size and nucleotide sequence from the template that contains deazapurine.Therefore in parallel peptide synthetic process, kept the integrity of this oligonucleotide, proved that the template from single bead can easily be increased and sort.

Use 7 amino acid structure unit Arg, Gln, Phe, Lys, Val D-Val and Thr make up encoded libraries by the combination synthetic method, design this library and contain 823,543 (7 ⁷) individual different seven peptides that are connected on the 10 μ m beads.α-N-Fmoc-Thr (tertiary butyl-oxybenzene and triazole (protected Threonine) and 4-O-DMT-oxidation butyric acid succinimide ester residue at first with all bead couplings, obtain being used for the amino and the hydroxyl of the differentiation of synthetic quadrature.Usually, every bead has the strand peptide sequence of 20 molecules of per molecule dna marker thing.By setting up the amino acid of each adding of distinctive dinucleotides cell encoding, after the 7th circulation of peptide link coupled, merge bead, DNA is synthetic to be finished.Initial used total ball amount is 35mg (1.75 * 10 ⁸Individual bead), guaranteed that each peptide sequence is present in the library～200 different beads on.Conclusive evidence is analyzed in the micro-ordering of the peptide in one equal portions library, and seven amino acid is distributed in every kind of situation of seven peptide mixts (noticing that L-Xie Ansuan and D-Xie Ansuan are undistinguishables in the Edman degradation process) of degraded randomly.

By wandering cells analysis of accounts mAb D32.39 and contrast bead and with the combining of bead library.Have the forward control sequence RQFKVVT (SEQ IDNO:2) and the bead of 69 no oligonucleotide tags and dye strongly, and blank bead is not dyeed with antibody.By contrast, have only a fraction of encoded libraries to be combined with D32.39.10 ⁵Inferior analysis revealed～2%.Library dyeing is higher than background level.Obviously, this is special at binding site with combining of D32.39, because it can be by mAb and solubility RQFKVVT (EDY ID NO:2) preincubation and is blocked fully.Indivedual beads from the library have and can contrast the fluorescence intensity that bead is compared with forward, its be sorted into serve as a mark in miniature centrifugal (microfuge) pipe thing by pcr amplification (collect the ball that fluorescence is arranged at the top, account for total amount 0.17%).Amplified reaction contains duTP and the impurity of ura DNA glycosidase to prevent to leave over, have soluble products from the front amplification procedure (referring to Longo etc., 1990, Gene 93:125-128, draw here be reference).Obtain nucleotide sequence from the bead of 12 sortings, the peptide sequence of inferring is listed in table 1.The fluorescence that the representative peptide sequence that obtains from single ball has is not obviously to be better than background, and it also is listed as being form as a comparison.

Table 1

High fluorescence intensity bead

Sequence Kd, nM (SEQ ID NO:3) TFRQFKVT 0.29 (SEQ ID NO:4) TTRRFRVT 4.3 (SEQ ID NO:5) TVRQFKTT 8.8 (SEQ ID NO:6) QvRQFKTT 16 (SEQ ID NO:7) RQFRTVQT 76 (SEQ ID NO:8) KQFKVTKT 340 (SEQ ID NO:9) QQFKVVQT 370 (SEQ ID NO:10) KQFKVTQT 410 (SEQ ID NO:11) TQFKVTKT 560 (SEQ ID NO:12) TFRvFRVT 1400 (SEQ ID NO:13) FRRQFRVT not tested (SEQ ID NO:14) RQFKQVQT not tested

Low fluorescence intensity bead

Sequence

Kd、mM(SEQ?ID?NO：15)?????????QTvTvKKT??????????＞1(SEQ?ID?NO：16)?????????QQVQRQTT??????????＞0.4(SEQ?ID?NO：17)?????????KTQvVQFT??????????not?tested(SEQ?ID?NO：18)?????????QvTQvRVT??????????not?tested(SEQ?ID?NO：19)?????????FVVTVRVT??????????not?tested

Data in the table 1 are consistent with early stage research, proved that the preferred recognition sequence of D32.39 is positioned at six amino acid segments of RQFKVV of dynorphin B (referring to Cull etc., above).Preferred positive charge residue arginine and Methionin are in first and the 4th of this epi-position, phenylalanine show as this motif exclusive the 3rd residue.In this library preferred second for glutamine residue significantly the amino acid Xie Ansuan of preferred aliphat b-side chain (only L-isomer) and Threonine as the 5th and the 6th residue.The D-Xie Ansuan shows as outside the common motif the most received residue on the position.According to the experiment of designed binding analysis, (Kd～0.3-1400nm) is the predictable detection that has another antibody divalence primary antibody that is labeled to selected peptide affinity scope.The operation of valency (for example, using the monovalence acceptor of direct mark) can improve the ability of only separating high-affinity part with the severity of wash conditions.C. small molecules is synthetic

Though at first synthetic or other big polymkeric substance library angle has been described all respects of the present invention from peptide, as preceding described, each side of the present invention can be applicable to other chemosynthesis with being equal to.Especially, apparatus and method of the present invention can be used to finish various chemosynthesis steps in many synthetic schemess, comprise as small molecules synthetic.These devices can be used to add reagent with selectivity in the chemosynthesis scheme, and are consistent with the synthetic schemes that is used for specific molecular.

Further, can carry out different synthetic schemess abreast,, wherein in different step, add different reagent from obtaining multiple small molecules library on the solid carrier as carrying out with the above-mentioned peptide that carries out is synthetic.These multiple libraries can be with method screening described here, to have required character.In addition, when this multiple library of small molecules of needs, can carry out mark so that synthesis step is encoded to this library, these synthesis steps are included in each segregant synthetic of molecular library.

This small molecules synthetic example on solid carrier comprises, as thiazolidone (thiazolidinones), metathiazole alkane ketone and derivative thereof, described as the embodiment 2 that comprises here, and U.S. patent application No.08/265, application on June 23rd, 090,1994, draw at this and to be reference, be used for all purposes.D. the method in production solubility library

Be some application, people can imagine a kind of " no ball " or " soluble " molecular library.Shla molecule, mark with unlabelled, can be used for various purposes, comprise the activity (referring to following VI.B part) and the amplification label thing of analysis of compounds.Have the whole bag of tricks to produce the shla molecule library, mark and be not labeled, and dissolving synthesizes the compound on solid carrier, mark and be not labeled.Usually, in this method, adopt following of cleavable.

For example, shown in above-mentioned part II.B, the joint of cleavable can be used for cracking molecule that be labeled or that be not labeled on bead or other solid carrier, makes institute's molecule (s) of interest dissolving thus.For producing the soluble molecule that is labeled, the cleavable joint will be connected on bead or other solid carrier and have at least two functional groups: one is to be used for synthetic molecule (s) of interest, and another is used for the complex sign thing.Be connected on the same joint molecule and marker are synthetic thus, this joint combines with solid carrier conversely.In case synthetic this molecule and marker, just cracking is fallen joint and is obtained a soluble molecule that is labeled.

Utilize the great scale immobilization to aggregate into (VLSIPS ^TM) technology, and before screening from the carrier the cleavable synthetics.Referring to U.S. patent No.5,143,854 and PCT patent application No.92/10092, it all draws at this and is reference.In one embodiment, at VLSIPS ^TMSynthetic one group of oligonucleotide on the sheet, and each oligonucleotide is connected joint such as disulphide (ginseng U.S. patent application No874 applies for 849,1992 April 24, draws to be reference) here with sheet by a cleavable joint.Oligonucleotide tags has a free functional group, as amine, is used to be labeled the connection of molecule, and this molecule is generally oligomer, is preferably peptide.This marker optionally only contains pyrimidine or contains pyrimidine and the similar base of purine.This marker also can contain the binding site that is used to increase, i.e. PCR primer sites, and the ordering primer sites that can select, and contain sequence monomer partly short that simple coding is labeled oligomer.Then, synthetic oligomer, joint promptly amino from the free terminal of marker or that be connected in marker synthesizes, so that every oligomer is connected with marker.That collects is labeled oligomer and can be released from sheet by the cracking joint, produces the oligomer library that is labeled of a solubility.

Can recognize other advantage by the solubility library that produces molecule.In any one ball Ji Wenku, the size that the size of bead (quantity) will the assembling library of physical constraints.For example, can need a few gram beads to contain 10 to assemble one ⁹Individual difference is labeled the library of molecule.People the invention provides and a kind of synthesizing be labeled improving one's methods of molecular library, so that can obtain much practical, much bigger library.This improving one's methods provides a kind of means, and wherein compound discharges from solid carrier before mixing step, but is linked on the solid carrier again before each coupling step.

In the method, be labeled molecule and be fixed on the solid carrier, in each mixing step of this method, allow to discharge the molecule that is labeled from carrier with a kind of reversible manner.In one embodiment, by a kind of ultra-filtration membrane (suitable film, referring to as " Commercial Compatibilty Chart " in the Millipore catalogue, it has shown all kinds of SOLVENTS that is used for synthetic method and the stable film of chemical) this reversible combination is provided.The film of about 2,000 to 10,000 daltons of molecular weight cut-off (as Amicon YM5 film) is suitable for most of libraries, and in coupling step, the molecule tunicle in library keeps, and coupling agent and other reagent can pass film by vacuum take-off.In mixing step, eliminate vacuum, so that molecular mixing.

In another embodiment of this method, in coupling step poly-in the covalently bound molecule that is used to be labeled of reversible be connected with carrier.Suitable reversible chemical connect example comprise (1) by, be labeled molecule and thioester bond that the N-hydroxyl-the succinimide base carrier provides as mercaptanization (thiolated), this ligation can be by NH ₂The control of OH concentration; (2) disulfide linkage that is provided by molecule that is labeled as mercaptanization (thiolated) and 2-pyridyl disulfide carrier (as the sulfo-agarose, from Sigma), this connection can be controlled by DTT (dithiothreitol (DTT)) concentration.VI. analytical procedure

Utilize big combinatorial library to find part, depend on the effectiveness of the Methods Biochem Anal of stable affine sensitivity strongly.The invention provides the new analytical procedure of using with the synthetic molecules library of having encoded in a large number, this molecular library comes also to have widely anyway to be used.For example, this library can be used in the assay determination to differentiate the part of bind receptor, as with the peptide and the nucleic acid of protein bound, medicine with the target receptors bind for the treatment of, by the epi-position of antibody recognition (natural and synthetic), and the identification all cpds, have pharmacy, agricultural and treatment diagnostic use.Corresponding to these multiple application of being given, there is a large amount of analytical procedures to be relevant to the present invention.Two kinds of important kind analyzing though there is some overlapping, comprise analysis of bead base and shla molecule analysis.

Yet this two alanysis generally comprises the following step usually.By analyzing the library is screened, wherein to the ability of each different molecular assay in the library itself and receptors bind interested.This receptor contacts with the synthetic molecules library, can combine product with any intermolecular formation of receptors bind under the analysis condition in acceptor and library.Differentiate binding molecule by the marker inspection that is relevant to this molecule then, in one embodiment, the library is being a kind of acceptor mixture in conjunction with the acceptor that is exposed under the condition wherein, each acceptor is associated with the discriminating marker that is specific to this receptor type, so check two markers after the binding analysis.A. bead base library screening is analyzed

When in the acceptor screening, isolating special bead, can separate spherolite by variety of way and be absolute version, comprise infinite dilution, micrurgy or wandering cells counting preferably, the acceptor that is labeled with solubility in binding analysis is assessed bound ligand library most effectively, by adopting the solid carrier or the bead of cell size, can use the wandering cells counting to carry out sensitivity receptor binding assay and fire ball operation.

The wandering cells counting typically refers to fluorescence-activated cell sorting and becomes FACS, is considered to be equal to for reaching " fluorescence-activated cell sorting " or " sorting of fluorescence-activation bead " that the object of the invention is carried out.The technician who is familiar with the FACS method can implement analytical procedure of the present invention at an easy rate, and FACS is applied to the clone's of express cell surface antigen or acceptor mammalian cell here.Usually, these assay determinations comprise combining of the acceptor that is marked with fluorescent marker and bead mixture, and this bead mixture has shown various molecules in the molecular library.Wash away after the acceptor of unconjugated or non-specific combination, utilize the FACS instrument to come sorting bead and discriminating and physical sepn that indivedual beads of height fluorescence are arranged again.Referring to Methods in Cell Biol-ogy, Vol.33 (H.A compiles Aca-demic Press for Darzynkievic2,2.and Crissman); With Dangel and Herzenberg, 1928, J.ImmunolMeth.52:1-14 all draws at this and is reference.In case required bead is separated, people's example can confirm that marker determines the characteristic (or molecular structure) of institute's molecule (s) of interest on the bead with example, form, or synthesis condition).

It is～104 times/second that standard FACS instrument allows bead (cell) fluorometric analysis speed, and when operating with single ball clone mode, sorting speed can reduce 5-10 doubly.When analyzing very large library (as＞＞10 ⁷Individual spherolite), before the use cell sorter separates indivedual beads, the pre-sieve form of some of available selection affinity.For example, be surrounded by acceptor the sub-micron size super paramagnetic particle through be commonly used to from big population mixture by magnetic activate sorting affinity purification specific cell (referring to Miltenyi etc., 1990, Cytometry 11:231-238, draw here be reference).Survey littlely in conjunction with situation for high probability, every kind of different compound all should be present on many beads in library in the library.For size is the encoded libraries that makes up from 10 μ m particles, and supposing has 100 times redundance, is limited to about 10 on the reality ¹⁰～10 ¹¹On the bead synthetic 10 ⁸～10 ⁹Individual compound uses less bead can prepare even bigger library, but conventional cell counter be it seems can not survey or operate and is far smaller than～particle of 1 μ m diameter.Certainly, as each place note here, the invention provides the various uses of this bead in the synthetic and screening of molecular library.For example, the application of the invention oligonucleotide tags series process, people needn't use the FACS technology to come molecule in the sorting library.

However, people do not answer underestimation to be used for the effectiveness of the FACS instrument of the object of the invention yet.In an analytical procedure of the present invention, the molecular library that is labeled synthesizes on the fluorescence bead.This bead is littler than cell, and is made up of fluorescent material.This library is with the cell suspending liquid incubation, this cell be high level expression interested cell surface receptor, as linking the proteic acceptor of G-.Certainly, people can also do many control experiments, as use in steps and do not have the cell of high level expression cell surface protein, and utilize these contrasts to confirm wrong positive cell (positives).

In either case, the cell of expressed receptor can both combine with the arbitrary library molecule with receptors ligand.Use the FACS instrument according to scattering of light or another kind of fluorescent signal as discerning fluorescently-labeled cell at an easy rate from endonuclear signal, and it is never separated and separates from unlabelled cell in the library bead of combined with fluorescent.After the sorting, marker on detection and the bead that cell links to each other, to confirm to be specific to the part of acceptor, according to this purposes, people can by as only select the brightest cell sorting to go out highest level and express and requiredly selected the brightest cell, and can adjust in conjunction with condition so that the maximization of specific combination situation.Be specific to interested receptors ligand and be specific to other cell surface receptor part by differentiating, people can detect the marker relevant with bead, these beads be combined with high level expression the cell of interested acceptor and the cell of non-high level expression.

Method of the present invention also can make people can utilize the FACS instrument to be sorted on the molecular library that synthetic is labeled on the bead, this bead than the FACS instrument of current use can sorting minimum spherolite much smaller.In this method, the synthetic library that screening has been encoded is at the effective active of conversion of signals approach.Make up this synthetic library with several modifying method: (a) bead is 1 μ m or littler, but and in FACS, need not to be sorting, this is used for some examples with regard to allowing littler spherolite; (b) marker to environment in the born of the same parents have resistivity (particularly nuclease being had resistance) oligonucleotide, thiophosphatephosphorothioate (physphorothioates) is a preferred substance of realizing this purpose; (c) peptide (or other number of chemical entity) is connected with the bead carrier by a joint, and the environment cracking in born of the same parents of this joint is fallen.This class joint comprises that as phosphodiester bond or disulfide linkage, but under any situation, the joint of cleavable must be stable to parallel building-up process by the joint of the use cleavable of harmless external factor of cell such as light with to the joint of environment sensitive in the born of the same parents.

Preferably the library bead is imported the report cell by mechanical means such as brolish plan; in some cases; can utilize the approach of the biological chemistry mediation that causes internalization, but this approach can cause not meeting the mixing of cellular compartment (being that lysosome is located) of needs usually.In case these beads are in the cell, peptide or other compound of institute's sensitization interest will discharge.If having proved to have, the bead of 10 μ m holds 10 ¹⁰Individual peptide (or other) synthetic site, and capacity scale represents that with volume then the bead of the same material of 1 μ m will hold 10 ⁷The synthetic peptide of individual molecule.(volume is released in～0.5pl) single (spheric) cell, will draw so～concentration of the free peptide of 30 μ m if all synthetic peptides are at 10 μ m diameters.This concentration can be controlled by the integral density on the bead, and lower loading density can be stipulated more strict screening form (i.e. the active higher compound of screening).The preparation recipient cell produces fluorescent signal at interested pathway activation of institute or deactivation.Select to produce the individual cells of required effect by the FACS instrument, and increase and sort, to confirm activated synthetic compound still being connected in bead and being contained in intracellular marker.

In another embodiment, adopt huge ball and screen the cell mass of expressed receptor (being beta galactosidase enzyme) with it, this receptor can produce fluorescence or other detectable signal, promptly produces a kind of detectable compound by the cracking substrate.These beads mix with cell mass then, and cell mass can be connected with bead.If with the acceptor on the compound irritation cell surface on the bead, just can produce detectable compound so, thus provide sorting in the activating cells never to be connected in the basis of the activating cells on the bead.People can use suitable reagent (be unlabelled or be not labeled) to select the high-affinity part to greatest extent.

The Cytometric system of selection of many instead of flow is arranged certainly in order to screening and selection institute molecule (s) of interest library.In one embodiment, screen a kind of coding synthetic library, has bacterial activity, to find to suppress bacterium or the compound of other microorganism growth or killing bacteria or other microorganism arbitrarily, these microorganisms can be paved plate with two dimensional form, as the cell of virus infection, many eukaryotic cells comprise cancer cells and some protozoons.By peptide or the compound sustained release from its bead that is synthesized, can screen big library relevant or incoherent chemical structure, act on the cell in the agar culture.

This method step is as follows: (1) paves plate to the cell of feel interest on agar plate; (2) with another layer agar overlapping auxilliary layer on cell, wherein suspending with the extent of dilution that is enough to provide suspension in the second layer agar has the bead of synthetic peptide/medicine, and so for example use kapillary can be picked out the discrete bead from solid agar; (3) initial release peptide/medicine from the bead; (4). culture plate so that the agar that the diffusion of peptide/medicine is fixed from bead expand to around agar and below contain in the agar of indicator cells; (5) read out the influence degree of the test compounds that has spread of certainly indivedual beads to the growth/form/phenotype of indicator cells; (6) select indicator cells to indicate district's band of required reaction (as the death of bacterium lawn), and use kapillary or similar tool choosing to choose the agar district that contains original bead, wherein testing drug is from this bead diffusion; (7) read the code labeling thing, as by the pcr amplification of coding material on indivedual beads is read, with the structure of peptide/medicine of determining to provide required reaction; (8) the suitable medicine/peptide of chemosynthesis selectively, and confirm required effect.

There is multiple mode from bead, to discharge test compounds, for example, use TFA from bead top cleavage of peptide/medicine, and the peptide under the permission cracking is in the bead surface drying, and this form makes subsequently in water suspending again in (agar) allow the release of peptide/medicine and will be released compound and is positioned agar district around the specific bead.Use can be connected medicine/peptide the chemical means people of the specific change sensitivity of bead environment with bead, wherein this specific variation can by and indicator cells dull and stereotyped on the agar upper berth cause or paving plate and agar curing after be initiated, as photosensitive connection, the connection of mercaptan sensitivity, salt-sensitive connection of Periodic acid or the like, if necessary, these chemical reagent itself can be diffused in another thin agar overlapping layer.This release chemical process must with the integrity of test substances, the healthy state of the integrity of en-cryption and bottom indicator cells has consistency on the bead.Certainly the special release chemical process that adopts also can depend on the type of the chemical process that is used for synthetic library and the character of indicator cells.Particularly preferably be the method in screening beta-lactam antibiotics library, be used to differentiate new antibiotic, this new antibiotic may kill new development to existing beta-lactam that the bacterial isolates of resistance element is arranged, and the method for preferred screening peptide library, this peptide library is the library of the analogue of known antibacterial peptide such as melon toad antibacterial peptide.

Also can use other method screening bead base molecular library.Affine adsorption technology can be used with library of the present invention.For example, the mixture of bead can be exposed to surface that acceptor is immobilized (referring to PCT patent application No.91/07087, draw here be reference).The washing substrate is after removing unconjugated bead, just condition (as low pH, acid treatment or the alkaline purification) elution of bound that can use the avidity that can reduce oligomer/acceptor interaction is in the bead on surface.Work if desired can use eluted bead to carry out affine adsorption process again.These methods and relevant varient all can be implemented in many ways as the use that above-mentioned phosphorus is selected; For example solid carrier can be the resin of chromatographic column of packing into.

In the another kind of method of the present invention, with the source of bound library of compounds as structure diversity, the form that is is suitable for the affinity purifying of gang's associated molecule, as receptor family interesting on the pharmacology.Usually, this method relates to the molecular library that uses a kind of bound molecule library screening another kind that is labeled not to be labeled.Library that be labeled, bound molecule screens complicated mixture soluble proteins, oligonucleotide, sugar, antibody etc. as a kind of affinity purified reagent.After the affinity purification, differentiate and many bonded of combinatorial library member molecule by separation and authentication method that wash-out is suitable.Then, combinatorial library is divided into the small portion that makes up the synthetic compound, measures which kind of compound mediated cohesive process exactly with a few experiments number of times by essential recirculation process.

With similar mode, the acceptor that uses the combinatorial chemistry library to differentiate and clone.Many acceptors are to have sequence homology (having reflected the multiple evolutional form from ancestors' parent usually) but present the not protein families member of homospecificity/affinity, and wherein specificity/affinity is aimed at the part/isoreceptor of one group of structurally associated.Each member of receptor family (Rn) can represent at special pharmaceutically-active isolating target, and is position in the health by utilizing their different character thus, and specificity is carried out the discovery and the exploitation of medicine with affinity of part or the like.If identify an acceptor (R1) its be enough in conjunction with character interesting so that the discriminating of other acceptor of same family will be favourable, just can adopt following method to differentiate the character of the acceptor relevant so at its binding site with R1.People at first differentiate and R1 bonded part, produce the combination of compounds molecular library that is labeled very relevant with this part on the structure then.

Next step, from think can expressed receptor other member's of family cell preparation polysome preparation.This polysome comprises and mRNA bonded rrna, is having sagging acceptor with nascent peptide to this mRNA of each stage of this protein synthesis of protein that almost completely prepares.The synthetic receptor protein that is close to when finishing can be expressed and the special receptor property of one or more combinatorial library member bonded, use is bound by the combinatorial library of solid carrier, by and the arbitrary member of combinatorial library between the avidity affinity purification have the polysome of acceptor.This affinity purification can comprise column chromatography method, separates the immobilization composition in batches from liquid phase, or aqueous solution two-phase separation method; The relevant mRNA that has the solid phase that connects acceptor and the acceptor of encoding from non-adhesion polysome with separation.

Next step, it is synthetic to use standard technique (reversed transcriptive enzyme etc.) to carry out cDNA from coding homoreceptor group's mRNA, and cDNA group is cloned into is suitable in the carrier that rapid serial analyzes.According to the existing knowledge of possible receptor sequence and the sequence reserving degree of expectation, can use the cDNA of PCR or another kind of amplification method amplification with this method enrichment.By what proper number cDNA was cloned, can differentiate the cDNA that has enough sequence homologies with known receptor R1 sequence _s(no matter be whole length or be not whole length), other member of the same receptor family (Rn) that representative is generally acknowledged.By the standard cloning process, optionally prepare these new cD-NA _sFull length cDNA clone (or its relevant portion, be relevant to part bonded extracellular region area part) as coding, and express these cDNA (promptly in eukaryotic expression system, being expressed as suitable solubility or embrane-associated protein) by standard method.Use the standard form that detects receptor-ligand interaction, detect the combination that is combined into individual compound from the colony of combinatorial library mixing cpd.In this way can accurately identify that compound and the new receptors bind of differentiating from the library.

For example by limiting dilution assay or those are with the similar approach of cell with the acceptor incubation that is coupled to little super paramagnetic spherolite, but the indivedual beads of physical sepn, use superpower magnetic force to extract the cell of expressed receptor part then (referring to Mil-tenyi etc., 1990, Cytometry 1.1:231-238, draw here be reference).Shown in last, use FACS can further analyze and sorting magnetic selection cell.The radioactive nuleus thuja acid also can be used for labeled receptor, can be differentiated by the bead of radioactivity mark and separate bead by selecting like this.B. screen shla molecule

People also can adopt the molecular library that is labeled to be effective in the new analysis of the present invention, wherein part with interested receptors bind before dissolvedly be mark or unlabelled form.For the screening solubility is labeled (not having bead) the especially big library of molecule, the preferred affinity chromatography of using under weak affine condition.For example, with contain hundreds of μ g the sample of 10mL affinity column of acceptor interested can screen 10 ⁸The 30mg library of individual molecule.Oligonucleotide is the preferred marker in this library, its can be very easy by pcr amplification and to be cloned into this carrier of TA cloning vector (In-vitrogen, company) that commerce can purchase be a kind of form that makes things convenient for that is used for sorting indicia thing information before the dna sequence analysis.In addition, oligonucleotide tags can be together in series by aforesaid mode, is labeled library of molecules (pools) thereby allow people to collect solubility, clone selected storehouse by placed in-line marker, measure then than flag sequence and differentiate required compound.

Use the immobilization acceptor also can screen the molecule that solubility is labeled.The molecule that is labeled contacted with the immobilization acceptor and the molecule of the non-specific combination of flush away after, any method in ining all sorts of ways discharges the molecule that bonded is labeled from acceptor.Select the molecule of amplification label to discharge from acceptor.Select the amplification label thing, then it is examined and determine, decoding is to differentiate and receptor-specific bonded molecular structure.But use a kind ofly, for example use a kind of fluorescently-labeled part replication by the oligomer that is labeled in the acceptor assay determination solution that is immobilized with the bead joint face.Can will have the bead of immobilization acceptor restore, and use FACS sorting bead with differentiate positive bead (fluorescence that weakens because the library molecule and be labeled part strive unexpectedly cause).Then with relevant identification marking amplification, decoding.

But the shla molecule of synthetic library on bead; Before analyzing then the molecule cracking is got off.In one embodiment, the micro-bead of molecular library is placed in the very little compartment or hole, and these are " manufacturing of millimicro level " goes out on silicon or other surface that is fit to chamber or hole.Be enough to produce in the loading buffer liquid of the spherolite in every hole by bead is scattered in, bead is loaded in the aperture.In one embodiment, bead solution is placed in the pond on aperture top, allows the bead sedimentation to go in the aperture.Use chemistry or hot system can finish the cracking of oligomer from the bead, but the system that preferred light is split.Institute's molecule (s) of interest can get off from cracking on the bead, produces unmarked molecule (marker still is connected on the bead) or in solution or produce tagged molecule in solution.Under any situation, the cracking from the bead of institute's molecule (s) of interest, but still remain in the cell with bead and identification marking.

In one embodiment, the surface or the part surface of aperture are coated with acceptor, add binding buffer liquid and fluorescently-labeled known receptors ligand in aperture, and to carry out the solution phase competition analysis of part, part is specific to acceptor here.Can estimate combining of fluorescent mark part and acceptor in the example by the same burnt image of individual layer immobilization acceptor.Receptor surface is the hole of weakening fluorescence and shows that d/d part and tagged ligand compete, and presents the bead or the marker of competition in the inspection window, to disclose the characteristic of competitive part.

Take indivedual beads away by micromanipulator from the hole, optionally the identification marking bead to positive hole restores.Another kind of mode is included in the bead production process or uses the bead that is combined with fluorescence molecule in the labeling process, the bead that only in positive hole, uses the laser bleaching of suitable wavelength to retain, then all beads are all removed, and use the FACS sorting, to differentiate the positive bead of being bleached, the mark of correlation thing can be amplified afterwards, and decoding is to differentiate and receptor-specific bonded molecule.

In another embodiment of the present invention, adopt the relatively large bead that is labeled, wherein the molecule of institute's sensitization interest in a system response from the bead cleaved down.In the method, little spherical diameter is 50 to 500 μ m, and capacity is equivalent to the peptide of every ball 100 to 500pmol, if preferably make up peptide library, uses the 100 μ m beads as 200pmol of capacity.The typical sizes in this library is from 10 ⁶To 10 ⁸, preferred 10 ⁷Individual differing molecular.The library is divided into 100 storehouses, and every storehouse contains about 100,000 spherolites.For example under the situation of peptide library, institute's molecule (s) of interest of a certain per-cent of cracking about 25% from the storehouse produces the peptide of every 1mL volume 500nM.

Then first cracked storehouse is at war with and analyzes or the functional analysis test.Identify and have the most highly active storehouse, recover the molecule that retains in original then, and will retain molecule and be divided into 100 storehouses, 1000 spherolites in every storehouse, repeat this process, have a spherolite, from this spherolite mark-sense thing and identify institute's molecule (s) of interest until every storehouse.This method has avoided the resynthesis of Houghten method and framework to limit, and advantage is that the storehouse is at random rather than relevant compound.Because the cumulative function of many low-affinity associated molecules, the chance of activated mixture has reduced.C. screen natural product libraries

Owing to have the automatic high throughput analysis that can obtain, the restricted condition when the screening natural product at first is the ability that obtains and handle (disperseing dissolving, mark or the like) sample now; The secondth, the desired actually operating of the active ingredient of qualitative positive.The invention provides the method that produces and screen the library of natural product, this library provides a large amount of samples that is easy screened form and has differentiated sensitization composition in the sample.The basis of this method is the combination of biological chemistry and Chemical Diversity, has from " natural product " promptly from the metabolic diversity of nature, and simple example comprises to microorganisms cultures feed supplement peptide gleanings.Each microorganism strains can produce the peptide (metabolite library) of many modifications.Because every kind of culture all can contain (potentially) very complicated metabolite mixture, need a kind of effective screening method.

Can adopt several approach, and these approach can be categorized as by quadrature factorization or that be labeled and solubility or bound.Describe for clear, be considered as the soluble peptide library of raw material, typical bacterial strain incubation in every kind of microbial fermentation screening procedure such as increment such as grade with this library, and the substratum of screening canonical form, positive then culture is with the set incubation in library and screening again, continue factoring (factoring) process,, advance to separate the qualitative of active metabolite according to the knowledge of possibility precursor molecule then until the input of differentiating the peptide that produces maximum active metabolites.Therefore, the active bio body is differentiated in screening for the first time, and step is subsequently differentiated active precursor, finally differentiates active metabolite by the standard analysis means.

Yet in form of ownership, factoring is the most loaded down with trivial details process, by cut off synthetic that produce and from the resin cracking get off freely that the library produces soluble compound, these compounds are used for cellular uptake and complete organism metabolism.Yet, indivedual compound concentrations quite low (relevant with the diversity of cultivating gleanings on the contrary), the metabolite as a result that causes invalid enzymatic conversion and extremely low concentration, ferment with every kind of microorganism isolate separately by biology product library subclass and with each subclass, can improve compound concentrations.By fixing one or more positions and make the randomization of remaining position make up sublibrary, for example, 500 pentapeptide sublibraries are arranged, it has used 50 structural units to comprise all arrangements of 20 fixed positions.In these sublibraries each all comprises 125,000 compounds.The use in mark library is in that it has an enormous advantage aspect convenience degree and the sensitivity, but to the compound gleanings being exposed to the method for metabolic activity, requires to change to some extent.Combination raw materials needn't only be a peptide, but can be made up of any one combinatorial chemistry gleanings.

Oligomer and other molecular library can be implemented in anabolic process, and per step encodes with identification tag, and this can carry out by directly connecting with marker synthesize parallel with oligomer.If oligonucleotide is used as marker, complex body is can be relatively big so, but still little as to be enough to insert in the cell, is activity form, and this merges by liposome, and electroporation, solvent are changed thoroughly etc. and to be carried out.In case enter in the cell, this complex body will to the metabolic mechanism of cell, degrade by using the Nucleotide of modifying to be connected can to avoid oligonucleotide tags to predispose to damage with Nucleotide.By the active metabolite in the culture that reclaims dissolved cell, the screening sample and the marker of decoding are to disclose precursor compound.The active bio body that carries out together with active precursor enlarges the active metabolite that fermentation will produce q.s and is used for qualitative test.By on the bead the library of compounds of coded combination process preparation can be exposed to bacterium, fungi is in the solute of vegetable cell etc.By this way, avoid in intact cell inserting the needs that are labeled complex body, and in the many molecules on the spherolite only the molecule of relatively small amount need detect (as in the fluorescence-activation binding analysis is measured).

The another kind of process useful of the present invention comprises the feed supplement as another kind of culture of the product that uses a kind of microorganism culturing.Since describe from the collection body of 100 different microorganisms isolates of extensive culture (～1 liter).By the supernatant liquor of each culture of filtered and recycled, and with its be divided into 100 10mL etc. increment.Each equal portions is all with inoculation of the strain in 100 strains and incubation.From 100 parts of microorganism isolates, produce 10,000 samples (metabolite of metabolite) thus.The kind very big by difference can extend to continuous metabolism with the metabolic method of this combination: for example with the microbial fermentation product with exotic plant solute incubation, or with the extraction of plant tissue part with the fungal cultures incubation.These methods can act on: any one product of the Chemical Diversity method of generation can both be used for these continuous metabolism product exposing step.

In another aspect of this invention, screen the natural product diversity, only a kind of composition of the preferred packing natural product of each liposome library of compounds or a kind of simple mixture by the liposome mixture that produces composite marking.The cut that the present invention allows to analyze 1000-10,000 chemical compounds or natural product extract simultaneously and analyzes 100 chromatographic separation, these cuts are derived by the natural product extract that has positive signal.In this relation, the meaning of " simultaneously " is to analyze together at the cell of identical degree Guan Zhongyu read-out system.

The liposome mixture of composite marking is prepared as follows.For for each independent natural product extract or chemical products in the storehouse, prepare isolating liposome, material is surveyed in the examination of this liposomes enclose aqueous phase, and liposome marker specific when packing is mixed in the Liposomal formulation.Can be with lipidosome freeze-dried so that standing storage at low temperatures, this is very helpful for storing to gleanings and near collect the site natural product sample, and will lipidosome freeze-driedly be convenient to natural product extract and be a kind of form of suitable combination experiment subsequently and standing storage.Lipoid in the preferred liposome preparation is identical to all samples and selects liposome with the equal skim that produces required size and integrity according to type and composition.Reagent such as trehalose can be wrapped into when liposome forms, to allow freeze-drying and to rebuild complete liposome by adding water subsequently.When the mark liposome generation/regeneration of packing extract/chemical substance, can use pressure technique, this technology allows the water volume of packing greater than the volume that calculates by liposome, thereby can test the test material of more volume, can read the more large-signal of cell based thus.

Existing liposome technology is convenient to be combined with the production of the liposome of high per-cent (＞80%) water (relevant with the result of use of every kind of test substances).Available various " washing " method is removed unconjugated water.In addition, can produce liposome, it can not leak or change the water (be relevant to the specificity of mark and do not have the blended water that is wrapped) of packing, and can produce liposome, it can not change the composition (glycolipid/proteantigen that the thing that serves as a mark inserts can not be changed) that is inserted in its lipid monolayer.

This method can adopt various markers, for example, marker can be that (a) has the different fluorophores that excite with emission characteristic, these character allow film that every kind of fluorophore can detected-fluorophore when other kind fluorophore or its binding substances exist can selectedly be dispensed into the aqueous phase of packing or rebuild liposome mutually in, towards the outside; (b) metallic cation of different rare earth elements, its by atomic absorption spectrum can be identified-the rare metal atom will be designed to salt form and be allocated in the packing aqueous phase of rebuilding liposome; (c) different antigen, discern these antigen-by the specific reaction of itself and suitable monoclonal antibody and elementary/secondary fluorescence detection antibody/fluorophore as required at protein, antigen on glycoprotein and/or the glycolipid can selectedly be dispensed into rebuild liposome film mutually in, towards the outside; (d) antigen, the binding substances of fluorophore and/or metal ion, it can improve the quantity of the possible signal that is used to screen greatly, the number that the additional levels of mark different liposome increases can identify the difference " quantity " of every kind of composition in the signal mixtures thus from the fluorophore/metal ion/antigenic use of different levels.

Also can adopt a kind of general fluorescent mark, it is total for all liposomes, can select the cell that merges with liposome fast from those cells that do not merge with liposome.This marker is different with arbitrary composite marking that is used for indivedual Liposomal formulations, and itself and produce the lipoid of liposome, signal mark, and the aqueous sample of medicine/natural product is mixed when liposome produces.Also can adopt fluorescent mark, it is automatic quencher (in liposome membrane) when high-density, but presents fluorescence in the liposome fusion with at cytolemma.Amalgamation mode according to liposome and cell, for example also can be incorporated into the fusion rotein of a kind of viral starting point or glycolipid, it will mediate the tight adhesion (depend on lectin class adhesion process, by suitable receptor-mediated, acceptor is to import the clone that is used to read as required) of liposome and cell.This dvielement can not influence the interaction (avoid situation about taking place) of liposome-liposome, but can improve the efficient of liposome-cytogamy.

This method can be utilized the cell read-out system, use a kind of reporter gene (as luciferase) that contains in the promotor downstream, the clone of beta-galactosidase enzymes, here promotor is being replied exogenous hormone or part such as steroid, cytokine, prostaglandin(PG), antibody, antigen or the like are activated when being added in the cell.The activation part activates a cascade signal with combining of cell surface receptor or intracellular receptor, and it finally causes replying the activation of promotor and transcribing of signal gene.The expression of signal protein causes being activated from discrete and produces signal the cell, and these cells can be by detection by quantitative.When seeking compound, this compound is as any part of antagonist action in the intracellular signal transduction cascade, can be by adding outer source signal agonist (cytokine, hormones etc.) the whole colony of pair cell carries out pre-treatment, and signal output reduction on the cell base of measurement at individuality after liposome merges.When seeking compound, this compound acts on arbitrary part of intracellular signal transduction cascade as agonist, source signal outside needn't in cell, adding, and after the liposome fusion, the appearance that can measure the signal on the individual cells basis.

The liposome mixture of mark and a large amount of excessive cytomixis of reading, excessive cell number is vital, only produces following products liposome one cytogamy after: (i) not with the cell of liposome fusion; The (ii) cell (recipient cell) that merges with liposome.It is very necessary that this step effectively mixes, and can use the cell suspending liquid of continuously stirring or linear stream to reach effective mixing, wherein the liposome mixture is joined in the cell suspending liquid at a slow speed.Merge with the standard method initiation, as adding PEG or using high-voltage, if desired, can be by in cell-liposome membrane, being incorporated into the identification of fusogen or ligand-receptor to merging with enhancing.Fusion steps adds the aqueous portion of single liposome effectively in recipient cell.Thus, the natural product extract aqueous solution just can work at any point of intracellular signal transduction approach now from the test compounds of chemical libraries (inventory) or chromatographic separation level part of natural product extract.Fusion steps also can add the specific marker thing in indivedual recipient cells, this marker provides signal for the fc-specific test FC compound sample.If those markers were present in the lipoid film of liposome originally, then these markers are distributed in the okioplast film of recipient cell now.The surface (panels) of the accessible monoclonal antibody specific of antigen of this position.Originally the rare earth ion that was present in special liposome aqueous phase is present in the recipient cell tenuigenin now.Fusion steps also can add the liposome marker of total recognizing cells, and these cells are former to be recipient cell, does not participate in the excessive cell that liposome merges from those.Marker can be a fluorophore of shifting to the recipient cell after birth from liposome membrane.

Next step, cell, the cell that merges with indivedual liposomes and the mixture of any one liposome that does not merge are with exogenous ligand incubation (as under the situation of test antagonist) or do not add any material and carry out incubation (as under the situation of test agonist).Use limiting concentration and the control compound of incubation time to determine the time of this incubation.

The preferred FACS of use selects institute's compound of interest (cell).For example can use front or ambient light scattering sorting cells (recipient cell or non-recipient cell) from the liposome that does not merge arbitrarily earlier.Big cell can be separated from small liposome very soon.Next step can not be that sorting is the cell of liposome acceptor the excessive cell of liposome acceptor from those, the cell that is acceptor has total liposome deutero-fluorescent mark, but not these marks that recipient cell has are non-fluorescence, certainly this step is selectable, if but it is operated as pre-sorting step, can obtain the separation of common most cells, these cells are incoherent with measuring as the subsequent analysis of a few cell of acceptor.Characteristic for the conclusive evidence antagonist, can on the radiative basis of report albumen (as beta-galactosidase enzymes or my plain enzyme), carry out sorting, isolate most positive fluorocytes (suppressing these positive fluorocytes) by the exogenous part of early stage adding from negative fluorocyte of minority or low fluorocyte.The two kinds of cell types in back are produced by the supposition antagonistic effect of the compound that is encapsulated in the specific lipid body, and these specific lipid bodies merge with these individual cells.Be the conclusive evidence agonist, can carry out sorting on report albumen is penetrated the basis of light, isolate most negative fluorocytes from the positive fluorocyte of minority, latter cell results from the exciting effect of the supposition of liposome derived compounds.

In some experiments, the cell of all sensitization interests can be carried out sorting as a colony, and it is individual as the clone to collect the randomness cell with standard FACS method according to above-mentioned standard.These individual cells can be analyzed as single cell, be analyzed the particular marker that it has, accurately confirm the special liposome of the required effect of mediation.In other application example, but the assay determination marker whole by the distribution in the sorting positive cell colony, and according to the experimental design scheme and insert from the particular marker in the sample in different time/place/storehouse, for the first time by the time can confirm the diversity of marker type in the whole positive cell group.

Can analyze individual cells or the cell mass collected by the method that is suitable for used special marking combination, can analyze fluorescent marker with FACS and/or traditional spectrophotometry.The adding and the ELSA of the monoclonal antibody by suitable mark, FACS, radio isotope or luminous assistant analysis can carry out assay determination to antigenic label.But by atomic absorption spectrum assay determination metal ion marker, after the marker decoding, only with for the first time by the time produce those liposomes of positive findings mixture repeat to estimate, or use in the isolated cells sample, add each member's interested pure liposome carry out repeated test.

These and other method of the present invention can the automatization form be carried out, and is to implement operation of the present invention, as follows.VII. instrument

In certain embodiments, the coupling step of some monomers (as amino acid) can require the long relatively incubation time, reaches other reason for this reason, and it is desirable making the system of the parallel adding of many monomers.The present invention relates to self-reacting device, be used to produce and screen the synthetic molecules library that is encoded, a kind of preferred instrument, can carry out 50 to 100 or more a plurality of parallel reactor simultaneously, be described in the U. S. application No.08/149 that submitted 1993 November 2, in 675, draw here and be reference.This device can be under time variable control, and the slip of the solid carrier of reaction mixture or building-up reactions is distributed in each passage, collects, and mixes and heavily distributes.

Yet the specialized equipment of the synthetic library of production tagged molecule generally speaking as peptide synthesizer, all needs complicated pipeline system, also needs large batch of monomer pond/and marker simultaneously, and often follows various linked reactions.Since the distribution capability of marker, the distribution that it is converted into some simple instructions suitable probe mixture signal and therefore instructs compounding substances.By monomer synthetic product, as required, in specified mixture, distribute.The startup of reaction, the temperature and time controlled function provides by system.Through suitable design, this product also can be used as the multipath peptide synthesizer, can produce 1～50 milligram (thick product) and reach the usefulness of 100 kinds of different peptide sections for analysis.Referring to patent documentation 91/17823, quote only for reference once more.

Typical this instrument mainly consists of the following components.(1) various storages, mixed and transport the device of synthetic product (as peptide section and oligonucleotide): the reaction of various complexity takes place in the reaction chamber of (2) sealings, various reactants in this inactive environment; (3) closed reaction vessel template dies; (4) the guiding reactant flow is to the device of each autoreactor; (5) collect and the device that separates miniature bead (0.1～100 micron); (6) device of bead in each chemical reaction finishes this reactor of post-flush.The template die of reactor can have several designs.Can put such as, reactor and to cause it can be in the collar, also can be placed in-12 * 8 the template die (the dull and stereotyped form 96-well of 96 hole microtitre microtiter plate format) according to a central shaft rotation (being equivalent to a whizzer).This any design all will make the automatization of the transportation of reactant and product thereof, suction (aspirstion) become simple, and propagation function (transfer functions) also has very high using value in some aspects.

Also there are several designs in the collection and the reallocation system that are used for particle.For example, bead can be suspended in the solvent of a suitable surface tension and concentration so that automatically liquid sucking device (the pipetting in strument) bead that can will dissociate be converted into the reactor of an associating.After the mixing, these beads can be redistributed in the reactor by same automatic liquid sucking device.Equally, bead also can be by a specific reaction chamber cross-linking that valve is arranged.Solvent flows into during valve open, causes bead to become a crosslinked container.After the mixing, again by causing bead to separate again to anti-solvent streams with the aforementioned flow direction.

In another embodiment, bead also can be by open-topped compression (closely spaced) reactor cross-linking.Soaking reactor also can make bead mix.If zona glomerulosa is magnetic, use certain magnetic field so, make bead again by the bottom of reactor, can make them obtain again separating.Unmagnetized bead can make bead make it obtain again separating by reactor bottom by vacuum take-off (vacuum suction).In other embodiment, be exactly with on the bead horizontalization plate, cover the device of one " cutting knife " (" cookie-cutter ") shape then in the above so that its each several part obtains separating, below more detailed description will be arranged.

Wash the ball system and also several designs can be arranged.Bead can be cleaned by infusion set (liquid delivery) and suction channel system.Each reactor all is equipped with the set of tubes system of oneself, perhaps possesses a pipe blow-through system that is fit to various reactors.Under latter event, move on the arm that liquid and suction channel system can be installed in an automatization so that each reactor can be worked separately.Can use centrifugally, magnetization bead or certain means such as magnetic field are made spherical corpusculum with the bead in each reactor.The diapire of reactor can plate one deck inert film so that reactant and product vacuum available resorber thereof are removed it.Equally, use a kind of reactor that allows a lasting fluid to pass reactant and scavenging solution, promptly the reactor terminal is provided with the road and joins formula device (1uer fittings) and filter membrane, also can make reaction content be able to smooth removing.

Any automatic multifunctional instrument all will be subjected to some very important restrictions in the use.Work because this system relies on each independent reaction chamber, each reaction chamber all must with the moth-er pot of transport system and " parent jar " (" ") link to each other.Bead is assigned to each reaction chamber after being pumped into and mixing in the jar again, is able to carry out without any confusion so that later monomer adds.Because monomer or some other construction unit quantity are huge, and in a huge reactive system, separate bead and reactant and all compare difficultly,, make wherein produced simultaneously reaction less than 100 so this quasi-instrument has been subjected to limitation in actual applications.

The present invention avoided because of mixed and reallocation bead with its necessity at the indoor pump back and forth of differential responses, simplified transport thing transportation formality, and the accurately and easy row of the separation that makes a small amount of bead.The most basic design mainly comprises a flat board, has a series of reaction " site " on the planar surface; This plane or level, perhaps have a series of apertures to form reaction site.Such as, reaction surface can be designed 256 reaction site, arranges with 16 * 16 dot matrix.Each reaction site be exactly on the planar surface a bit or an aperture, a small quantities of synthetic bead will be adsorbed on herein.Adsorptive power can be by magnetic force, vacuum filtration (vacuumfiltration), follow gravity or some other simple means of passive machinery classification to obtain.At first the bead suspension of dilution is painted skim at planar surface equably, with certain adsorptive power bead is focused on each reaction site then.

After bead is focused on certain reaction site, use isolation technology each reaction site is made an isolated reaction chamber (temporary), as shown in figure 29.By a kind of flexible means, disrupter can permanently be adsorbed in planar surface, to form shallow reaction aperture.Reactant is transported to each reaction chamber, and bead is discharged in the suspension that is formed by reactant, and reaction will be carried out.When condition was desirable, bead also can be adsorbed onto planar surface again and reactant is removed.After all linked reaction circulation steps were finished, the reaction chamber isolated system was also removed thereupon, and bead is collected in the Xiao Chi of reaction site top and preserves.

The mixed of bead is accomplished by induce the generation convection current in reaction tank, and bead is adsorbed onto planar surface again to carry out the new linked reaction of next round then.Step subsequently comprises that rinse step finishes by similar fashion.The interpolation of reactant and removing are united by automatic imbibition and tubing system and are realized.The available automatic multi-function pipettor of the interpolation of specific reactants (as: monomer) (multi pipettors) is finished.The interpolation of common response thing and removing can be finished by a restrained line system.This system need not valve at each reaction chamber.Before the reaction chamber disrupter is installed or after removing, some conventional operations such as matting can directly be carried out (on the beads en masse) on bead.

The use of monomer in enormous quantities and other structural unit has brought extra burden to coding work.In an oligonucleotide marker cataloged procedure, 1000 basic sequence monomers need one 5 base sequence to come each reactions steps of mark; Surpassing 1024 sequence monomers just needs 6 base sequences ten to encode for it.Complicacy (promptly reduce specific reaction and add step) for reducing the synthesizer tubing system the invention provides a kind of specific coding strategy.Be explanation this method, existing is that example is illustrated with 16 * 16 Sptting plates.This arranging can make 256 different reactions carry out simultaneously.Using the coupling of polybase base is a very difficult task for the independent coding of each reaction.

This dot matrix is arranged, and tool 16 is gone, 16 hurdles, and each point all has a unique geographical position.Every capable site can be labeled as one group, and all 16 can be encoded with 2 base codons (" inferior codon ") by characteristic ground.One stripe board or flute profile piece can be used as the substrate that 2 bases are added, to form 16 reaction chambers (notice that the link coupled base is as monomeric phosphinylidyne aminate (phosphoramidites), rather than as dimer).Referring to patent documentation No.WO93/09668, quote only for reference once more.This reaction site positioning form and other is similar; For example, available optical means mark bead, referring to U.S. Patent No. 5,143,854, bar shaped cladding process wherein (striped masking process) is quoted only for reference here.If use flat board or flute profile piece, so flat board must reduce to the same horizontal plane of grid synthetic surface on so that in the process of synthetic molecules library, each reaction is separated.

In the labeled reactant process, bead can not discharge into.Yet their spatial isolation will be maintained to till next small step finishes always.After each reaction site of going is by inferior codon mark, plate is risen and half-twist, reduce the band that covers the hurdle to form then.16 hurdles are again by the inferior codon mark of 2 bases, and like this, 256 reaction site are all by on 4 bases " ultra dense numeral " (" supercodon/) the characteristic ground mark.By position, recognition reaction site, ultra dense numeral just can provide the monomer that each reaction chamber will add.

VIII. parallel coupling building-up reactions instrument (Apparatus for Parallel ouplingSynthesis Reactions)

On the whole, the present invention provides a solid substrate (solid substrate) for synthetic diversified material.For example, this instrument has used bead recited above.Below just synthesize the basic condition that example is introduced this instrument with peptide.

At first it will make substrate diversification (plurality), and with " S " expression, building-up reactions can be carried out on the diversification substrate.Help power at the contact molecule " L " that linked reaction takes place can provide substrate selectively.Substrate is assigned with the back and reacts with various monomers, forms following substrate form such as reaching " B " monomer reaction with " A ":

S-A and S-B then, substrate makes up again, mixed and sub-distribution again.After mixing and allocation step, just form two or more substrate forms, not only comprise S-A but also comprise S-B in every kind of form.

Remaining linked reaction just can be carried out like this.For example, above-mentioned polymerisate can react with the product form below the form with monomer C and D again:

1.S-A-C?????S-A-D

2.S-B-C S-B-D just can find out that by top simple example this synthetic technology can obtain a large amount of different substrate forms at short notice.By to the careful design of the synthetic multiple gleanings of this quasi-molecule with (or) by providing parallel synthetic method to this substrate marker, as mentioned above, these substrates will have high application prospect.In a special embodiment, this invention provides some can effectively produce the equipment and the method for all kinds of purposes substrates.

A. generalization (General)

Shown in Figure 1 is one can synthesize the equipment of multiple gleanings molecule.This equipment comprises a parent mixing tank 200, by a general terms formula manifold (topcommon manifold) 212 and 215, and a 221-229 pipeline and a diversification reactor 201-209 coupling mutually.212 casings are managed couplings mutually with 221-229 and 215 again.Reactor 201-209 also by 111-119 valve and 241-299 pipe optionally with add monomeric reactant supply reservoir 231-239 coupling mutually.Pressure delivery system (pressurized dilivery systen PDS) 265 is respectively by pipe 260 and 256 and parent mixer 200 and reactor 201-209 coupling mutually.

When the bead suspension when parent mixing tank 200 is transferred to reaction tubes 201-209, building-up reactions promptly begins.When 129 valves were opened (all other valves cut out), the bead suspension entered top formula casing 212 from parent mixing tank 200 by 215 pipes.The bead suspension distributes in the 201-209 reactor by the 221-229 pipe then.The reactant of selecting from monomer storehouse 231-239 enters each autoreactor 201-209 by pipe 241-249 separately.Like this, in the 201-209 reactor of bead was arranged, linked reaction just took place.

Pressure delivery system 265 shown in Figure 1 is by pipe 260 and parent mixing tank 200 coupling mutually.When valve 10 and ventilation valve 90 were opened, this system transported to parent mixing tank with the reactant of pressurized from system by managing 260.

Pressure delivery system 265 is also by managing 256, seperating vale 100, low level casing valve 101-109, low level pipe 271-279, introduction valve 111-119 and pipe 241-249 and reactor 201-209 coupling mutually.For reactant is optionally transported to reactor 201-209 from PDS265, go into pipe 256 and one low level manifold 214 during 100 valves of the reactant of pressurized by opening.Then, the reactant of pressurized rises to pipe 271-279 pointedly under stress by selectively opened 101-109 valve.Reactant enters separately reactor 201-209 by selectively opened 241-249 pipe more then.

For example, be transported to reactor 201 for making reactant from a given monomer pond 231, a certain amount of pressurized solution is pressed towards pipe 256 from PDS265, enter low level manifold 214 then, and then enters pipe 271.A suitable moment, a certain amount of monomer reactant is expelled to the solution stream that flows to pipe 271 from monomer pond 231.After the injection monomer, flow to the linked reaction of reactor 201 below participating in by 241 pipes from reaction tank 231 effusive mixed solutions.

For with the bead among the monomer selectivity ground mark reactor 201-209 among the monomer pond 406-412, through the valve 4-7 that opens, a pressurized flow to coventional type manifold 255 from storehouse 406-412 labeled monomer reactant solution into-PDS265.Then, pressurized marker monomer and a suitable pressure solvent stream enter low level manifold 214 by 100 valves and 256 pipes of opening.The monomer marker of pressurized and appropriate solvent stream thereof rise to 271-279 pipe separately, enter reactor 201-209 by the 101-109 valve of opening separately then, at this bead labeled reactant will take place.

After ideal monomer and/or marker interpolation reaction, the bead suspension of reactor 201-209 can be recycled to parent mixing tank 200 for regrouping and mixing.For making the bead suspension be recovered to parent mixer 200, can to the bead suspension, be blown into argon gas so that its supercharging from managing 250 by 122 and the 101-109 valve of opening from reactor 201-209.100 and 110 valves cut out, Gu this pressurized presses the bead suspension just to flow to pipe 221-229, enter coventional type manifold 212 then, enter 215 pipes by 129 valves of opening again, finally enter parent mixer 200.In 200 mixers, the bead suspension is mixed again, and then is re-assigned to reactor 201-209, thinks that further synthesizing other molecule gets ready.

In another kind design, the product of each reactor of 201-209 and and parent mixer 200 between, can install a multi-functional three-way valve (plurality ofthree-way vessel).This valve preferably is installed in the common top formula manifold 212.In this way, some reactors can separate with parent mixing tank 200.This is to redistributing the bead suspension with particularly useful for further synthetic.For example, bead is assigned to reactor 201-209 at first and is used for building-up reactions, be recovered to then in the parent mixer 200, in this re-allocation process, only a part of 3 channel valves are opened, such as like this, only reactor 201,205 and 209 can enter the bead suspension for further synthetic usefulness.

Fig. 1 has also shown non-concentric agitator (nonconcentric agitators) 280 and 285, respectively with a top formula reactor carriage 290 and end formula reactor carriage 295 couplings mutually.Top formula reactor carriage 290 is Immobile, but end formula 295 can produce whipping force thereby make among end formula reactor carriage 295 and the bottom reactor 201-209 with non-concentric agitator and volute motor (vortexing motor) 300 associatings.Because the pipeline between the carriage is very flexible,, and therefore promote the carrying out of building-up reactions so whipping force makes the reactant among the reactor 201-209 produce eddy current.

The common manifold 212 in top at one end links to each other the pipeline that so just provides material to transport with 215 pipes between parent mixer 200 and manifold 212.At the other end, manifold 212 links to each other with 126.Pipe 216 feeds the high pressure argon gas by 121 valves in casing 212, the while also makes the casing 212 can be to its internal ventilation by 120 valves.

Two

powerful transmitter

90S and 99S are installed in the outside surface of parent mixer 200, to survey liquid level wherein.If fluid is present within the useful range (detection envelope) of big sensor, transmitter will be opened.Otherwise transmitter will cut out.

Transmitter 101S-119S is an optical pickocff, mainly whether fluidic in the translucent tube is existed and monitors.When a fluid streams is present in the pipe, transmitter will be opened.Otherwise transmitter will cut out.

Similarly, an acoustic sensor 120S also monitors the fluidic existence.Fluid comprises the bead suspension, will be monitored by it and transmitter be opened when flowing through pipeline.On the contrary, no fluid exists in the pipeline that acoustic sensor is housed, and transmitter 120 will cut out automatically.Because optical pickocff can not be distinguished the polyfluortetraethylene pipe of translucent sky reliably and contain the same pipeline of bead suspension under some condition, the application of acoustic sensor has then remedied this deficiency.In addition, though transmitter 120S has used acoustic sensor, any other can be distinguished blank pipe and contain fluid or the transmitter of bead suspension pipe all can use.Because acoustic sensor compares to other type sensor, want much expensive as optical pickocff, so acoustic sensor of 120S only in every synthesizer.

The design in reactor storehouse (bank) makes and only needs one or valve still less between parent mixer and each reactor.This design has its original superiority.Because the machinery of valve opens and closes regular meeting and causes fragile bead and synthetic polymeric infringement, this design greatly reduces the damage intensity of this process.Equally, if the bead volume is excessive, they are normal blockedly to cause system's running to be obstructed around valve.In addition, the pass back and forth of numerous valves is opened and will system temperature be raise heat production, and this polymerizable molecular to some temperature sensitivities will produce bad side effect.Therefore, reducing the valve number that the bead suspension passes through is a very favourable behave.In if 129 valves are not included in, can utilize supercharging technology to prevent fluid flowing between parent mixer and reactor storehouse (reaction vessel bank).

As previously mentioned, said valve is all closed as in working order the time in the last example.The OPEN that none is specific, valve always are in idle closing condition.

B. mechanical file

From mechanical angle, this automatization synthesizer can roughly be divided into three systems: reactor storehouse, parent reactor and pressure delivery system.As mentioned above, coupling building-up reactions is carried out in each reactor in reactor storehouse.The parent reactor is responsible for holding, collecting and mixed work from each reactor bead.Pressure delivery system is responsible for rationally transporting of appropriate solvent and/or appropriate reaction thing solvent strength solution, and they are delivered to parent reactor or each reactor storehouse at the certain phase branch of building-up reactions.In addition, whole system is worked under closed state.Supercharging in case of necessity is achieved as argon gas by feeding rare gas element often.Argon gas is owing to obtaining easily and unreactiveness, so be a kind of ideal pressurization gas.

In order to narrate conveniently, existing is that example is illustrated with a specific automatization synthesizer.This example will be referred to a synthetic cover polypeptide on bead.Bead is labeled with easy to identify, then is the linked reaction of each amino acid and four base monomer: A, T, C and G.

Yet must recognize that automatic DNA synthesizer DNA both not only had been confined to specific polymer synthetic described in the above-mentioned special case, also not only was confined to the synthetic of mark polymer.Synthesizing and react with above-mentioned Nucleotide mark bead with polypeptide in below describing is example, and 9 reactors are contained in a reactor storehouse (reaction vessel bank) in this example.But this is a special case, and the number of reactor does not have the fixed bound in the retort.In fact, the modular of synthesizer (modular con-structien) makes the interpolation of retort become simple and easy to do.In addition, many other molecules and marker also can synthesize on bead, perhaps existence that need not marker in the reaction system.

1. pressure delivery system

Aforesaid pressure delivery system 265 is to finish the synthetic of polypeptide through particular design, and it can be subdivided into again: polypeptide synthesizes pond, bead mark pond and transports valve.

A. polypeptide synthesizes the pond

Except that the raw material of needs 9 seed amino acid monomers as synthetic polypeptide, other several " routines " (common) reagent are also used in synthetic in this example.For example, in a typical polypeptide building-up reactions, mainly use following reagent:

The DMF solution that table 2 functionalized chemical composition deprotection (Deprotection) contains 10% piperidines activates (Activation) 0.2M HBTU and 0.6M DIEA

Be dissolved in the THF solution of DMF/DCM mixed solution cap envelope (Capping) diacetyl oxide of 3: 1

The THF solution scavenging solution DMF of n-methyl imidazole

THFb. mark bead storehouse

In this embodiment, bead is by the selectivity ground mark.In another routine peptide building-up reactions, bead is by on four kinds of Nucleotide A, T, C and the G mark.Except four kinds of thuja acids, in marker is synthetic, must use following solvent and reactant.

Table 3 functionalized chemical is formed DCM liquid oxidation iodine, the trimethylpyridine of deprotection Tricholroacetic Acid, and water and MeCN activate 0.5M tetrazolium MeCN liquid cap envelope diacetyl oxide THF liquid

N-Methylimidazole THF liquid cleans NeCN

These Nucleotide reactants are loaded among the Xiao Chi of enough large volumes and quantity so that automatic DNA synthesizer DNA is finished the synthetic of marker.

Representational 400 ponds hold the MeCN in the table 3 as shown in Figure 2.Table 2, two ponds of table 3 all are connected with 2 conduits.As shown in Figure 2, in the pipe 450 the high pressure argon gas is housed, content among the Xiao Chi is pressed to 452 pipes.In some design, reaction tank always is in high pressure conditions.And in other designs, only when needs mediocre person matter, the more logical specific controllable valves of tunger tube boost to wherein blowing.

C. transport valve

Fig. 3 has described logical (3-port) solenoid valve of a typical 3-in detail.As, the 2-110-900 type valve that the General Valve Corp.of Fairfield of New Jersey produces.In addition, 3 logical valves shown in Figure 3 transport reactant such as valve 4 from 412 ponds.The PDS265 of Fig. 1 is also with the three-way valve that is.Used three-way valve comprises first channel 454 and second passage 456 in this example, also has a passage 458 that runs through itself, with a passage 454 and two passages 456 material exchange is arranged all, and can make fluid in one and two passage unrestricted flows.As forming coventional type manifold 462, so, this side says that two passages 456 are with regard to first channel 454 coupling mutually necessary and another three track valve.Other triple channel valve also has valve shown in Figure 15.A solenoid coil of its inside is by 190 pairs of control signals of line.

Make and replying, and optionally open third channel 460, make itself and 458 passage generation exchange of substance.The third channel 460 of

valve

4 and 412 Xiao Chi coupling mutually among Fig. 1.

Flow to manifold 462 in order to control reactant well from 412 Xiao Chi, the spool 464 that is loaded with the reaction under high pressure thing links with the third channel 460 of valve 4.In due course, solenoid coil is opened, and causes

third channel

460 and 458 passage generation exchange of substance, nationality so that the reactants in 3 passages 460 of pressurized inject 458 passages of 4 valves and and then enter coventional type manifold 462.

Shown in Figure 4 one typical switching mode 2-passage solenoid valve 10.Only be suitable for this such as the 2-17-900 type valve of newly translating western General Valve Corp.of Fairfoield production.As shown in Figure 4, valve 10 comprises 468 and 470 2 passages.Valve 10 also has a scroll pipeline to run through wherein, makes itself and the first energy road 468 and second passage 470 that information interchanges take place, thereby liquid or gas can be flowed between two passages.Solenoid coil in the valve 10 is made control signal by 472 lines and to be replied, nationality so that first channel 468 selectivity exchange with second passage 470.When a passage of valve 10 connected a pipeline that has high pressure gas or liquid, valve 10 just can be used as the control fluid and flows to the wherein switch of other passage from this passage.

Fig. 5 has more at large described a pressure delivery system 265, wherein has very consistent with the present invention on the one hand.Fig. 5 has expressed 24 valves a manifold 0-23 number.First and second passages of 2 passages and triple channel valve strand is connected (daisy-chained) to be flow through by reactant to form the coventional type manifold.Triple channel valve 1-7,9-12 and 14-22, for example structurally with Fig. 4 in valve 10 comparing class seemingly.The following several parts of coventional type manifold constitute, and tool runs through the pipe coupling between three track valve, switch (on/off) valve and the adjacent valve of pipeline.As preceding described, the third channel of triple channel valve is controlled by the solenoid coil in the valve.The third channel of triple channel valve links to each other with the conduit of reactant or solvent cell, thereby reactant or solvent can be transmitted in PDS265 back and forth.Equally, third channel also can be used as an outlet, such as, it can be transported to reactant the valve 100 in the set of reactors.Two channel valves are mainly used in isolates and the feeding argon gas.

As shown in Figure 5,24 valves are installed in three isolating set of reactors to save the space.Pressure haulage system among Fig. 5 expresses a conduit 480 set of reactors on the valve left side and the set of reactors at middle part is linked.Table 4 has been listed valve used in the haulage system, and to some typical valve types and be subjected to being illustrated corresponsively and explaining of valve control.

Pond, table 4 valve model pond inclusion 0 ON/OFF-) DCM solution 3 FWO60 404 tetrazoliums 4 FWO30 406 C5 FWO30 408 T6 FWO30 410 G7 FWO30 412 A8 ON/OFF 9 FWO30 414 refuses 10 FWO30 416 parent reactor bottoms 11 FWO30 100 RV storehouses 12 FWO30,420 refuses, 13 ON/OFF, 14 FWO60,422 top parents, 15 FWO60,424 iodine of argon 1 FWO60 400 MeCN2 FWO60 402 1% trichloroacetic acids, collidine, water, THF solution 18 FWO60 430 of the THF solution 17 FWO60 428 N-methylimidazoles of acetonitrile 16 FWO60 426 acetic anhydrides send pyridine 19 FWO60 432 HBTU20 FWO60 434 DIEA21 FWO60 436 acetonitriles 22 FWO60 458 DMF23 ON/OFF argons

For example, valve 22 is shown it is a kind of FWO60 valve, and what promptly have 60/1000 inch current pipeline gets (FWO) three track valve express developed.And valve 22 controlling a kind of reactant in the pressure tank 438, and as shown in table 4, DMF is contained in this pond.For another example, valve 23 is a kind of switch-valves, the flow of compression argon it is controlling from argon protactinium Ying Yuan (not providing) to the general casing of PDS265.

The confession that Fig. 5 represents to link to each other with valve 0 is from the pipe 486 of an end to the general manifold pressurization of PDS265.Another pipe 486 is to link to each other with valve 23 and pressurize for the general manifold of PDS265 from the other end with argon.

As an illustration, the operation of the pressurization delivery system 265 in a polypeptide synthesis and degradation circulation is described as follows.For the degraded of polypeptide, the DMF solution of 10% piperidines is transported in the reactor in reactor (RV) storehouse.Table 4 shows that valve 18 allows the piperidines stream from pond 430.Therefore, valve 18 needs outlet and flow in the general manifold of PDS265 from the piperidines of pressure tank 430 allowing.In order to force solution to enter RV storehouse valve 11, shut-off valve 8 and open valve 13 and enter open RV storehouse valve 11 to force the pressurization piperidines.

Shown in Fig. 5 and table 4, RV storehouse valve 11 and parent reactor valve 10 are positioned in the central position of valve string.This being mounted with is beneficial to the length that reduces position between interior these valves of manifold and a certain particular agent valve.Therefore, need reagent in a small amount to fill this manifold position.Segregaion valve 8 and B can be closed to prevent that reagent from spraying into RV storehouse valve 11 or parent reactor valve 10 or unnecessarily entering a position of general manifold from an end of manifold.

Table 4 shows that also valve 4-7 and 9-12 have 3 channel valve that size is 30/1000 inch current pipeline.Comparatively speaking, to have size be 60/1000 inch current pipeline to all the other valves of manifold.The minimizing of the current crossover sites of pipeline has more reduced the manifold volume at position separately.Thereby the reagent that need be used for filling manifold still less.

For example, Nucleotide A, T, C, G are relatively very expensive.Therefore the very worth agents useful for same amount that allows remains on required minimum.It is very short thereby required amount of reagent is low with the distance that keeps 11 of Nucleotide valve and RV storehouse outlet valves that Nucleotide valve 4-7 is positioned in place near RV storehouse outlet valve 11.The crossover sites of the manifold on the path from any Nucleotide valve to RV storehouse outlet valve 11 also keeps very little so that further reduce to be present in nucleotide reagent amount the manifold.In fact, position between Nucleotide valve and the segregaion valve 8 has 30/1000 inch the crossover sites that has been reduced in the pipe 480 of Fig. 5 and the manifold.

C. reactor storehouse

Fig. 6 has represented a simplification set of reactors 500 according to one aspect of the invention.For convenience of explanation, the pipeline of solution stream warp has partly been left out.Reactor storehouse 500 comprises 502, two side holder 504 of a top support frame (bracket) and 506 and two bases 508 and 510.Top reactor carriage 290 is connected on side holder 504 and 506.An eddy current motor 300 is positioned on the top reactor carriage 290 so that power is provided for reactor 201-209 complex body by transmission belt 521.The bottom of reactor 201-209 is positioned on the bottom of reactor carriage 295.Carriage 502,504,506,290 and

base

508 and 510 can be made by any suitable material.Consider machinofacture, intensity and light weight, make the above-mentioned carriage of carrying with aluminium in the present invention.

Bottom reactor carriage 295 is fixed on the top reactor carriage 290 by two in the axle sleeve 520 non-concentric shafts 280.Non-concentric shafts 280 rotatably is mounted by the hole (not indicating) on the top reactor carriage 290.By transmission belt 521 with non-concentric shafts 280 and eddy current motor 300 coupling mutually.As will be discussed, non-concentric shafts 280 revolving force that eddy current motor 300 is provided translates into motivating force so that bottom reactor carriage 295 moves with ring-like form.This circulatory motion produces eddying effect to the inclusion among the reactor 201-209.Because each among the reactor 201-209 all links to each other with bottom reactor carriage 295 in bottom separately, so all reactors all simultaneously and are as one man driven.

Fig. 6 has also shown bottom bracket 522.It is to be connected on

side holder

504 and 506 that the bottom holder adds 522, and also can be made by any suitable material, comprises aluminium.Amino acid pond 231-239 is installed with bottom bracket 522 times.Amino-acid reagent among the 231-239 of amino acid pond is used as the basal component of respectively overlapping polypeptide in the synthetic particular.For synthetic polypeptide the present invention considers to use nine kinds of different amino acid monomers.

Fig. 6 has also shown the segregaion valve carriage 526 that is positioned on side holder 504 and 506.Segregaion valve carriage 526 comprises the pipeline 530 of low manifold valve 101-109 of generation arrangement.As shown in Figure 6, each low manifold valve 101-109 is fixed in the pipeline 530 in the present invention.Yet low manifold valve 101-109 also can use fixing metal utensil available on the market or other fixing means to be fixed on the segregaion valve 526.Low manifold valve 101-109 is the triple channel solenoid valve and is identical with associated 3 channel valve of Fig. 3 with former discussion.As employed in the set of reactors 500, the current pipeline among the low manifold valve 101-109 is coupled at together to form a general low manifold 214, comes the solution in the self-pressurization delivery system 265 to flow by it.

Three 2 channel valve 100,110 and 122 have also shown in Fig. 6.Nine valve 101-109 are controlling solution flowing from low 214 to nine reactor 201-209 of manifold.Segregaion valve 110 at first end of general low manifold 214 is opened on a refuse line (showing) in Fig. 6.Optionally forbid or allow solution to flow to all the other positions of casing 214 from PDS265 at the segregaion valve 100 of second end of general low manifold 214.The each several part of autoreactor group 500 is transported to or transported to the local argon pressure of alternative segregaion valve 122 supplies to assist solution.

In another embodiment, these 11 segregaion valves 100～110 and 122 forms with one 11 valve group provide, as by the model P/N601374 that ABI provided of Li Funiya state Foster City.This valve group has been assembled in advance thereby has been simplified the establishment program.It is described equally that in fact the function of 11 valves of this 11 valve group catches up with face.

A jet valve carriage 532 that is made of suitable material such as aluminium is fixed on side holder 504 and 506.Jet valve group 111-119 is installed by the hole on the jet valve carriage 532.Fig. 6 represents to have 9 jet valve 111-119 and controls the injection of amino acid from nine amino acid pond 231-239.Jet valve 111-119 is 3 passage solenoid valves and is the same as far back as 3 channel valve described in Fig. 3 together.The first part of each jet valve and reactor 201-209 coupling mutually.And the third part coupling mutually of second section and low manifold 101-109.This coupling is to realize with the pipe of suitable size, as the 1/16 inch teflon tube that adopts in the present embodiment.Understand that as preceding the current pipeline of each jet valve 111-119 allows the unrestricted flow of solution between reactor 201-209 and low manifold valve 101-109.The third part of each jet valve 111-119 links to each other with amino acid pond 231-239 so that optionally forbid or allow amino acid pond 231-239 to link to each other so that optionally forbid or allows amino acid to be launched in the solution stream of hanging down between manifold valve 101-109 and reactor 201-209.

Fig. 6 has also shown a general manifold 212 in top.This casing comprises nine manifold passages 542 so that this case links to each other with nine reactor 201-209 entirely.In the present embodiment, 1/8 inch elasticity teflon tube is used to make the top coupling mutually of casing passage 542 and reactor 201-209.The general manifold 212 in top also contains one first end channel 544 so that it links to each other with parent reactor (not shown), and wherein the bead from each reactor 201-209 has mixed.Second end channel 546 links to each other the general casing 212 in top with 3 channel pressures/breather valve (not shown).This pressure/breather valve and second end channel 546 provide another pipeline, can be supplied in the top casing 212 by this line pressure argon, solution and reagent etc.On the other hand, this pressure/breather valve and second end channel 546 provide another pipeline, can be discharged into the suitable pond from casing 212 by this line pressure argon, solution etc.

Fig. 6 A has shown the another kind arrangement to pond 231-239.It does not provide single pond group, as pond 231-239, and provides, and cohort is as 231a-239b, 231b-239b etc.These are positioned in the rotatable travelling belt 1000.They all are opened on the end face 1002 of travelling belt 1000 so that the reagent in the pond can be estimated from end face 1002.Travelling belt 1000 is positioned in the forcer (not shown) so that each pond all is subjected to uniform pressure in forcer.For reagent in the pond being transferred to reaction 201-209, a nest of tubes that links mutually with pipe 241-249 is positioned in the forcer.Pipe in the forcer is placed on one and selects in the pond of pond group, as pond 231a-239a.Move in the pipeline and can be placed in pipe in the storehouse by pipe is moved in the pond or travelling belt 1000.Travelling belt 1000 is rotatable so that these pipes are consistent with selected pond group.Pressure in the forcer can make between pond and pipe 241-249 and have a pressure gradient.When these pipes are positioned in the pond or selector valve 111-119 when being opened, the reagent that pressure gradient is ordered about in the pond just is transported among the reactor 201-209 in pipe 241-249, and this is as preceding described.Therefore by allowing selective reagents from various different reagent, for example, allow to use and respectively overlap basic raw material in each synthesis step, travelling belt 1000 provides handiness to synthesizer.

Fig. 7 shows interconnecting between amino acid pond 231-239, jet valve 111-119, low manifold valve 101-109 and reactor 201-209 in greater detail.Low manifold valve, for example valve 101 (it is a kind of three track valve) links to each other with general low manifold 214, so that solution unrestricted flow between first channel that hangs down manifold valve 101 and second passage.The third channel of low casing valve 101 links to each other with the first channel or the second passage of 3 channel injection valves 111 by pipe 271.And first can the road with second passage in another passage link to each other with an end of reactor 201 by managing 241.The other end of reactor 201 links to each other with manifold passage 542 in the general manifold 212 in top (not showing in Fig. 7) by pipe 221.Pipe 271,241 and 221 all is to be made by chemical resistance material such as teflon.In fact, translucent PTFE and FET teflon tube have been adopted in the present embodiment, because it has the optical signature of low reactivity and translucent teflon material with many cross sections.

Amino acid pond 231 utilizes rare gas element such as argon to pressurize through pipeline 562.Pressurization amino acid solution in the amino acid pond 231 has entered the third channel of channel injection valve 111 by pipeline 560.After receiving a suitable instruction, jet valve 111 is opened and goes with the current pipeline that allows the pressurization amino acid solution to enter into jet valve 111

Fig. 7 shows that also existence that two optical pickocff 111S and 101S optical sensing equipment 111S and 101S survey the liquid in translucent teflon tube 271 and 241 whether.As shown in Figure 7, optical sensor 111S is positioned under the jet valve 111 and optical sensor 101S is positioned under the reactor 201.Data from optical sensor 111S and 101S are sent to a control computer (not showing among Fig. 7) with each stage with a control building-up reactions.

Fig. 8 shows the non-concentric agitator 280 among Fig. 1 in greater detail.Non-concentric agitator 280 comprises with cylinder opens hook 566 link coupled two

cylindrical shafts

564 and 568 mutually.Axle 564 is with the vertical alignment of the radial axis of cylindrical hook 566 and in the coupling mutually of one of its two plane.Axle 568 is in another plane phase coupling.And be offset to the radial axis of cylindrical hook.In one embodiment,

axle

564 and 568, cylindrical hook 566 are made by same the full planing bench that belongs to.

When cylinder axis 564 is fixed rotation upholder such as roller bearing one, (it is offset to the axle center of cylinder axis 564 and does circulatory motion cylinder axis 568.But coupling is more than one non-concentric agitator 280, for example by being with a wheel construction to allow one group of non-concentric agitator 280, for example by being with a wheel construction to allow one group of non-concentric agitator 280 to move together in the operation.In the present embodiment, the axle 568 of two non-concentric agitators 280 link to each other with single bracket in case when axle 564 rotates with the ring form movable support bracket.And axle 568 and eddy current motor 300 are designed with per minute about 1500 changes the bottom of moving each reactor in circular orbit.In order to prevent the damage of bead, the circular orbit in the present embodiment preferably is limited to radius and is about 3.5mm.

Fig. 9 shows the upper section of set of reactors 500 in greater detail, and it comprises reactor 201-209 and eddy current motor 300 among Fig. 6.With discussed among Fig. 6, set of reactors 500 comprises the set of reactors 201-209 that links to each other with top support frame 290.Top support frame 290 has the one group of hole 630 that links to each other for reactor 201-209.Link to each other with end on each reactor 201-209 in hole 630 from the teflon pipeline on this hole (not shown), its mode of connection allows liquid unrestricted flow between teflon tube and reaction tubes 201-209.

The low-end of reactor 201-209 links to each other with low reactor carriage 295.Figure 10 A and 10B show the bottom view of the low reactor carriage 295 among Fig. 6 in greater detail.Figure 10 A is the amplification bottom view of low reactor carriage 295-part.

Figure 10 B is presented at a group pipeline 650 of low reactor carriage 295.Elasticity and translucent teflon tube (omitting from Figure 10 B so that demonstration) stretch out and are positioned in the pipeline 650 from the low-end of each reactor 201-209 (also saving from Figure 10 B).Link to each other with each pipeline 650 for the ditch 652 of laying optical sensor.Ditch 652 is shown by clear in Figure 10 A.

Figure 10 B has shown optical sensor has been fixed on a group open holes 654 on the low reactor carriage 295.Figure 10 B has shown that also a group selecting hole 656 is with weight reduction.As previously discussed, low reactor carriage 295 along with the circulatory motion of non-concentric agitator the inclusion in the Vortex reactor.Selective hole 656 also can be made alleviating the quality of carriage by low reactor carriage 295, and then reduces the required power of movable support bracket.

Near each end of low reactor carriage 295 current hole 658 is connected to a non-concentric agitator 280 on the low reactor carriage 295.The low-end of reactor 201-209 moves along with the circulatory motion of low reaction carriage 295, and they are to stretch on the current pipeline 650 that is positioned on low reactor carriage 295 from elasticity teflon tube 241-249.When low reactor carriage 295 during with circulatory motion, the inclusion among all the reactor 201-209 in the set of reactors is all done vortex motion with correlation method.

Fig. 9 has also shown for the optionally non-concentric agitator cover 520 that non-concentric agitator 280 is installed.This agitator cover round the non-concentric shafts in hollow round column cover with prevent when non-concentric shafts is moved to the user may injure and to the damage of equipment.

The present embodiment uses level type motor (the PX245-01AA model that Jia Nifuniya Oriental MoforU.S.A Corp.of Torrance provides) and level type motor controller (the RD122 model that Jia Nifuniya Semix Corp.of Fremont provides) to provide revolving force to give non-concentric agitator.As shown in Figure 9, three pulleys 670,672 and 674 with eddy current motor 300 and two rotating bands 521 and 676 operations so that in non-concentric agitator cover 520, two non-concentric agitators 280 are rotated together.Although the present embodiment has been used level type motor and group type motor controller, revolving force can be by the motor supply of any other model, and this comprises other electronic or pneumatic motor.And the power that is provided by vortex motor 300 can comprise the chain and the gear teeth, pulley and band, gear etc. by any suitable transfer system, is delivered to non-concentric agitator 280 and goes.

Figure 11 A has shown and has been used to survey the typical light transmitter 680 whether translucent teflon liquid in pipe exists.Optical sensor 680 is identical with optical sensor 101S-119S in the present embodiment.Optical sensor 680 (Yi Li Louis state Omron, the EE-SX671 model that Inc.of Schaumburg provides) comprises for two furcated end 682 that hang light converyer and condensing apparatus and 684.Optical sensor 680 also have one 686 Room for lay suitable circuit with the light sensation data transfer in main control computer and also for optical flame detector 680 is satisfied with on the carriage.

Figure 11 B shows the

furcated end

682 and 684 of optical sensor 680 in greater detail.Be installed in being and being roughly rectangular converyer 685 of terminal 682 internal surfaces of fork, for light being delivered to being roughly in the rectangular condensing apparatus (showing among Figure 11 B) of arrow 688 directions.Condensing apparatus is positioned on the internal surface of furcated end 684 and is to be roughly rectangle equally.In order to survey the existence of roughly translucent teflon liquid in pipe, teflon tube is positioned in the slit between two furcated end 682 and 684.When having liquid in roughly translucent teflon is swelled pipe, condensing apparatus is initiated, and indicating to detect has a kind of liquid in the teflon tube.

In practice, found when being empty that roughly translucent teflon material can not cause that optical sensor 680 causes.In order to use the general light transmitter to experience the existence of translucent liquid in pipe well, just used a kind of novel optical calibration light-blocking matter.Figure 11 C shows optical correction's light-blocking matter 690 in greater detail.It is by being enough to stop that any light-proof material that is sent by converyer 685 makes.It comprises two be in the

light wall

692 and 694 of first surface 696, and they are for light-blocking matter 690 frictionally is attached on one of the furcated end of optical flame detector 680, and optical correction's light-blocking matter 690 is fixed on the furcated end.In one embodiment, be in the light on the furcated end 684 that

wall

692 and 694 is designed to make condensing apparatus adhere to Figure 11 B.

Optical correction's light-blocking matter 690 also contains build second surface 700 the pipeline 698 that is in the light in.Second surface 700 of being in the light is surfaces back to above-mentioned first surface 696.The axial line of pipeline 698 is perpendicular to retaining wall 692 and 694.The size of pipeline 698 makes it grip teflon tube just.Therefore, teflon tube is to build in admittedly in the pipeline 698, and it is perpendicular to above-mentioned converyer band 685 when optical alignment light-blocking matter 700 is positioned on divergent ends 682 and 684 s' slit.

Figure 11 D has shown a hole 702 that is positioned at pipeline 698 medullary rays.Hole 702 allows a little light to pass optical alignment resistance light thing 690 along the hole of 700 of first surface 696 and second surfaces.When roughly heavy Guan Ruguan 241-249 of translucent teflon or 271-279 were secured in the pipeline 698, the center of special fluorine pipe was passed in the axle center in hole 702.The shape in hole 702 and size are the optical signature functions of a pipe.

When optical alignment resistance light thing 690 is installed between

furcated end

682 and 684 in mode shown in Figure 11 A, pass a roughly transparent tube 704 from the light of the converyer 685 in the furcated end 682.Most of light is stopped by the optical alignment light stopper after passing pipe 704.Some light that pass pipe 704 arrive holes 702 (being blocked) and arrive condensing apparatus in the furcated end 684 along the eyelet in hole 702 in Figure 11 A.

Since the center of translucent tube 704 is passed in the axle center in hole 702, the light that passes die arrives the condensing apparatus position in the divergent ends 684.Since the light by blank pipe 704 is dispersed, do not cause susceptor with regard to there being enough light to arrive condensing apparatus.Heavily manage when having liquid in 704 when translucent teflon, the light that passes full packages is by wherein liquid optically focused.Entered hole 702 by accumulative light and cause condensing apparatus in the divergent ends 684 from the direction of divergent ends 682.When liquid did not exist, the optically focused effect was very little.Therefore, light seldom enters hole 702.In fact, when not having liquid in the teflon tube 704, do not cause condensing apparatus in the divergent ends 684 with regard to there being enough light to pass hole 702.

As previously mentioned, the size of optical alignment light-blocking matter 690 is to grip teflon tube 704 just.When optical alignment light-blocking matter 690 was contained between

divergent ends

682 and 684, shown in Figure 11 A, teflon tube was firmly clamped by optical sensor 680 and light-blocking matter 690.By optical flame detector 680 being fixed on the carriage, teflon tube 704 is also along with being fixed on the carriage.With the same manner, the teflon tube 241-249 that the bottom of the present embodiment reactor 201-209 is stretched also is fixed on the bottom reactor carriage 295.

As shown in figure 12, the reactor of reactor in the present embodiment 201 and so on is made of one section FEP teflon tube 710.The external diameter of pipe 710 is 1/4 inch, and internal diameter is 0.19 inch.As the back more detailed description, pipe 710 diameter is bigger for what also can do for the purpose of using, can mix in reactor 201 simultaneously at these two or more reagent.Manage 710 closure ground and elasticity teflon tube 271 coupling mutually.Elasticity teflon tube 271 is fixed on the low reactor carriage 295 of set of reactors.When low reactor carriage 295 was done circulatory motion, described circulatory motion was done along with bottom reactor carriage so that manage 710 interior inclusion vortex motions in the bottom of this elasticity teflon tube 271.

In this embodiment, pipe 271 external diameter is 1/8 inch and internal diameter is 1/16 inch.Figure 12 has shown a pipe jointer, and it is made up of so that the pipe of different cross sections closely connects together with the 3rd coupling mechanism 718 first coupling device 714, second junctor 716.The aforementioned tubes junctor can be from Norton, and Inc.of Akron buys.Bottom at pipe 710 has a filter or medium 1102 (being blocked among Figure 12) to enter flexible pipe 712 for the lane matter that prevents to manage in 710.For example, this filter can be 2 microns titanium filters.

At pipe 710 the other end, one the 4th coupling mechanism 720, the 5th junctor 721 and one the 6th coupling mechanism 722 closely will manages 710 and be connected to and manage on 221.

Coupling mechanism

720 and 722 and junctor 721 be essential because in the present embodiment there be from pipe 710 different crossover sites pipe 221.Pipe 221 is connected on the manifold passage 542 of top manifold 212 and (shows among Fig. 6).

An elasticity O-ring 724 (is shown with the cross section in Figure 12) in the hole 726 in the peace pressure bracket 290.

O-ring 724 is flexibly clamped coupling mechanism 722, thus flexibly with reactor 201 firmly on carriage 290.When the bottom of reactor 201 was driven, the O-ring made the top of reactor 201 quite motionless to increase the vortex effect as a fulcrum.

Shown in Figure 12 A and 12B, the reactor 201 in a particular optionally has control cover 1100 temperature with controlling reactor 201-209.Temperature control cover 1100 in this embodiment comprises that a fittings 1104 is to be connected to reactor 201 221 lines and 241 line (not shown)s.

Advance/go out passage the 1106, the 1108th, go for thermal conductance liquid is fed in the reactor 201.This thermal conductance liquid can be used to reactor 201 heating or cooling.In this example, reactor 201 is to be formed by a teflon tube 1110, and preferably its external diameter is 1/4 inch, 1/32 inch of wall thickness.On the space from pipe 1110 be an outer tube 1112, it preferably is made of teflon and has 3/4 inch of external diameter, 1/32 inch of a wall thickness.Outer tube 1112 surrounds fittings 1104 and forms an annular space 1114 for receiving thermal conductance liquid.O-

ring

1116,1118 between outer tube 1112 and fittings 1104 to form a liquid closed district.By this way, thermal conductance liquid can be imported into annular space 1114 with reactor 201 heating or cooling by one of passage 1106 and 1108.

Passage

1106 or 1108 also allows thermal conductance liquid to continue circulation by annular space 1114.

Another embodiment that reactor 1120 has an integral form temperature control cover 1112 has shown in Figure 12 C and 12D.Except reactor 1120 preferably was made of glass, the function of reactor 1120 cardinal principle was identical with reactor 201.Temperature control cover 1122 also preferably is made of glass.Such structure permission reactor 1120 and temperature control cover 1122 are integrated unit as one and are formed.Reactor 1120 comprises strainer 1124 and is connected 1126,1128 to be connected on 221 lines and the 241 line (not shown)s.Advance/go out passage the 1130, the 1132nd, in order to make the annular space internal recycle of thermal conductance liquid at cover 1122 and 1120 of reactors.Hose barb the 1136, the 1138th is linked on the suitable fluid supply for the ease of advancing/go out passage 1130,1132.By this way, thermal conductance liquid can be at annular space 1134 internal recycle to give reactor 1120 heating or cooling.

D. parent reactor

Figure 13 shows the parent reactor of embodiment of the present invention in greater detail.For example, the volume of parent reactor 200 is about 30 milliliters.Pipe 260 closely links to each other so that reagent or argon are transported to or transported from throwing the PDS of system 265 (Figure 13 does not show) with the bottom of parent reactor 200.One filter filtrate 746 manages 260 to prevent that matrix from entering near being positioned on parent reactor 200 bottoms.Removable hood 743 is housed for adding or removing material on the top of parent reactor 200.Pass and cover pipe 215 transferred material between the general manifold 212 in the top of parent reactor 200 and set of reactors (not showing among Figure 13) of 743.Pipe 215 passes from covering 743.Article one, alternative flushing pipeline 281 is connected to solvent source and washes this inwall with the inwall of pressurized solvent being transported to parent reactor 5.

Figure 13 has shown two capacity transducers 905 and 995 (ElectromaticControl Corp.of Hoffman Estates, the model 18-08 of Illinois), and they are positioned near the outside of parent reactor 200.Capacity transducer 905 or 995 is surveyed near there is situation and sensing data being delivered to a control computer (not shown) by line 756 and 785 respectively of its liquid.Capacity transducer 995 is used for surveying the reagent that adds to parent.Capacity transducer 905 is used to survey the spherolite suspension of redistribution.According to bead amount and used two kinds of transmitters of reaction vessel adjustable number joint.The data of capacity transducer are used by software, with each circulation of control building-up process.

E. Controlling System

1. control computer

Automatic DNA synthesizer DNA uses control computer to obtain data by transmitter, and in each circulation step of building-up process by-pass valve control and eddy current motor.When being connected with automatic DNA synthesizer DNA when using, any computer comprises the microcomputer of common general knowledge, minicomputer, and workstation, large scale computer or the like can be used for process sensor data and issue an order by-pass valve control and eddy current motor.

And the data presentation device of the use general data display packing that can sell by any merchant from the sensing data of synthesizer transmitter obtains.Equally, sell i/o controller, respond suitable computer command with the merchant, but also Be Controlled of valve and eddy current motor.

In one embodiment, IBM-compatible microcomputer (also thinking Personal Computer or PC) is used as control computer (Model Gateway 200.4DX2-50V，by?Gateway?200Inc.of?No.Sioux?City，South?Dakota)。In PC, the complicated expansion slot that allows to add various expansion boards is arranged.The total line source of these wiring boards and PC is head and allow Circuits System UNICOM on PC and the card to finish electric function then.Some expansion boards also allow PC and peripheral equipment and Circuits System UNICOM.For example, a common general knowledge be that the wiring board of modem boards inserts the expansion slot on the PC, and allow PC and another have the computer UNICOM of modulator-demodulator unit.Using expansion board with Personal Computer is general technology knowledge.

In one embodiment, automatic DNA synthesizer DNA and PC by multi-channel digital I/O wiring board UNICOM (Model PCTIO120-P by Industrial ComputerSource of San Diego, California).The description detailed of PCDIO120-plate is in Product Manual No.00431-050-20A, and it also can be obtained by In-dustrial Computer Source.

Each PCDIO120P provides 120 buffering I/O (I/O) passages with one group in 24 passages.Each 24 road group is by program utility appliance joint (PPI) 8255A chip controls.These passages selectively with 8 passages be one group in order to input or output.

Figure 14 has shown the simplified illustration of control hardware part.Computer 760 with complicated expansion slot is connected with keyboard 764 with display monitor 762 as PC.PCDP120-PI/O plate 766 is inserted into makes PC and five controller line 7688 UNICOMs in the expansion slot.Each controller line 760 is by conductor channel and I/O plate 766 UNICOMs.In the present embodiment, conductor channel 770 comprises that one has service in the 50-conductor belt of the ability of 24 passages.

2. controller line

The output signal of I/O plate 766 typically is 15mA source electric current and 24mA receptor electric current, is not enough to the actuating magnetic valve.So controller circuitry 768 will be converted into power signal from the output signal of I/O plate 766, it has the actual actuating magnetic valve of enough power.Controller line has the ability of reception from 24 output signals of I/O plate 766, and order is exported the various devices that 24 power signals 771 are controlled synthesizer.24 power lines 771 of each controller line 768 are seen Figure 14.

Each controller line 768 also provides a center physical memory cell, and the sensing data of 24 sense lines of each transmitter as many as is concentrated there.Data from 24 different sensors of as many as can be received by the sensor port 772 on each control circuit 768.

Therefore, each control circuit 768 can be served as many as 48I/O passage on the I/O plate 766,24 inputs and 24 outputs.Figure 15 has shown the synoptic diagram according to the controller line 768 of one aspect of the invention.50-grafting stem 781 is connected controller line 768 with I/O plate 766 (not showing) in Figure 15.Stem 781 is input stems, and is connected by bus 780 with sensor port 772.Sensor port comprises stem or is used for connecting controller line 768 and the input of 24 of as many as is provided with joint as transmitter.Each conductor on the bus 780 passes to input stem 781 with signal by a transmitter.Figure 15 has shown that also a selectable LED conductor signal of autobiography sensor port 772 in the future of being used for is sent to the LED bus 784 of a selectable photodiode (LEDs) 786.Each conductor that can select LED bus 784 is delivered to a LED in the storehouse 786 with a signal.

Figure 15 has shown that also another is used for connecting another 50-grafting stem 788 of controller line 766 and I/O plate 766 (not showing) in Figure 15.Stem 788 is one to be used for receiving the output stem from I/O plate 766 output signals.Bus 790 is carried to 8 output signals from stem 788 of as many as on the scale-of-eight phase inverter 74LS240 chip 792.Scale-of-eight phase inverter 74LS240 chip 792 is by Texas Instiuments, Inc., and Dallas, Texas makes.Bus 794 is carried to a scale-of-eight latch with the buffer output signal of counter-rotating by chip 792 and tends to act on the device 796.The latch device chip 796 of tending to act is Model MIC59p50, by Micrel, and Inc., San Jose, California makes.Be connected by bus 800 with an output port 798 from each output signal that latchs the device chip 796 of tending to act.Selectable LED line 802 of determining will be delivered to selectable photodiode (LEDs) 804 storehouses from the LED conductor signal of chip 796.But can select the LED of signal of each conductor 802 carrying to the storehouse 804 in the LED bus.

3. valve

Be used in magnetic valve in the example of the present invention as, valve 4-7 for example, 10,14,90-91,100-121 and 129 are normally closed, unless order it to open, so the system intialization state is closed in the synthesizer.Because when synthesizer place system intialization state, do not have material to allow to flow, therefore ensured security.When starting, valve has used power and thermalization.Except that the obvious loss of power system, thermal valve can influence chemical substance unfriendly by its gangway.Because with not closed comparing of opened valve of maintenance, opening a magnetic valve generally needs bigger power, a kind of striking relay, and for example by Crydom, Inc., the DID20 type that Long Beach, California provide is used to the actuating valve.Striking relay such as DID20 in specified time, typically are 100 milliseconds, to valve provide+12 volts open the valve of closing.The striking relay provides+and 12 volts time slice can be specifically by software control, and striking is worked by an I/O passage.After this, the striking relay provides a voltage that reduces, and typically is half of voltage rating or is approximately 6 volts in example of the present invention, keeps the open mode of magnetic valve.Thereby need less power to come actuating valve and the more a spot of heat of generation.

The invention provides four kinds of other power supplies of branch.First power supply is exported+5 volts with the 77L chip of tending to act, as the chip on those controller lines 768.Second power supply output is used for step motor for+32 volts.The 3rd power supply provides+12 voltaic open electromagnetic valves.Can select the 4th kind of power supply also provide+12 volts be used for transmitter.That separates provides the 4th power supply assurance of power to work as valve open and close the not disturb sensor work of any noise that is produced to valve.

At its system intialization state, all valves are all closed.As additional security measures.Synthesizer further provides a solid-state striking watch-dog, can close all valves when control computer breaks down.The SM-WDT5 type that solid-state watch-dog such as Brentek Inter-national provide (by Industrial Computer Sourceof San Diego, Califonia obtains) is installed between control computer and the valve power supply.The pulse that software produces is delivered to a striking watch-dog on the I/O passage by control computer.Disappear in arteries and veins, for example the CPU mistake is locked key or latch-down key mistake, the power supply of striking watch-dog shut-off valve, thus close all valves.

F. control software

Now the flow sheet with reference to Figure 16-24 goes through control software.It is to finish the relevant phase of synthetic method and issued command that these flow sheets have illustrated by control computer.For simplifying following discussion, suppose that in all correlation time the valve of pressurized feeding system has been received the correct order of control computer, required reagent has been delivered to reactor storehouse valve.

To be the explanation control computer be the flow sheet of the combination of the program of draining reactor 201-209 inclusion and issuing to Figure 16.Before drainage, reaction vessel 201-209 contains liquid or bead suspension.The argon gas steam supply valve 121 that is connected with top Common Ducts 212 is received open command and is opened, and gives the top Common Ducts 212 pressurizations with argon gas.Simultaneously, selection a port valve 101-109 open and to make liquid flow into low pipeline 214 by reactor 201-201.Only selectable 201-209 valve open is not because be to use all reactor 201-209 in some linked reactions.If a reactor is empty a step of reaction, then need not drain its inclusion.Eduction valve in the bottom pipe 214 is opened and is allowed liquid flow out.The different result of pressure makes argon pressure order about liquid by reactor 201-209, by low pipeline 214, is flowed out by eduction valve 110.110S closes when transmitter, and showing does not have remaining liquid to drain, and valve still is held open 2-5 and is discharged by the reactor storehouse to guarantee all liquid second.

Figure 17 represents the combination of low pipe 214 issued command of computer control removing.In initial step, retained material at low pipeline 214.After this, seperating vale 100 is opened and is allowed the pressurization argon gas enter low pipeline 214 by PDS265.Simultaneously, eduction valve 110 is opened material is discharged from low pipeline 214.Material is discharged up to there not being the material residue by low pipeline 214.When optical pickocff detected in the waste pipe no material, valve was got back to its system intialization state again.

Figure 18 illustrates a cover control computer mixing parent container 200 inclusion issued command.This system goes into parent container 200 with argon gas by bottom 31 and mixes inclusion.Argon gas bubbles has stirred the inclusion of parent container 200 when hold holding the bottom from parent when rising to inclusion the parent container surperficial.Do not expressed and order all valves that are held open all to be in its system intialization state.Valve 100 is opened and is made the pressurization argon gas stir wherein inclusion by the bottom that PDS265 enters into the parent container.Valve 90 is also opened from the parent container and is discharged argon gas.Approximately 15-30 is after second, and

valve

100 and 90 is got back to its system intialization state.

Figure 19 A and 19B have explained the command program that control computer writes for each reactor by the parent container allocation to bead suspension.This program has implied the technology of filling the measurement volumes of each reactor with bead suspension.Use this program, the filling of reactor does not almost have the flow velocity dependency.Therefore will see that no matter bead suspension is the reactor that enters and the distance of the distance between the pipe end, bead suspension is distributed evenly in each reactor.When assignment period began, argon gas was only contained in the reaction vessel storehouse, and the parent container contains bead suspension in step 852.

In step 854, before the reallocation stage, the reaction vessel storehouse partly is equipped with DMF and is come the argon gas that exists in the surrogate response container storehouse.Seperating vale 100 is opened and is made pressurization DMF enter low pipeline by pressure-flow system.Valve 101-109 opens and send DMF on reactor.Valve 120 is opened from the top shared pathway and is discharged argon gas.After the pre-program time cycle (about 3 seconds of per stage), valve 100,101-109 and 120 gets back to, preferred sequence ground, its system intialization state.The mixed removal bubble of reactor 201-209 then.Valve 100-109 opens about 3 seconds again.When pre-program time was expired, each the DMF content of managing among 241-249 that leads to reactor should be near top Common Ducts 212.

Then in step 858, reactor storehouse and parent hold along 200 DMF that pack into.Valve 100 is opened and is made pressurization DMF enter the reactor storehouse by PDS265.Valve 101-109 opens and continues to add DMF to the reactor storehouse.Valve 129 is opened and is made the DMF that flows through the reactor storehouse enter parent reactor 200.Valve 90 is also opened from parent reactor 200 and is discharged replaced argon gas.Continue to insert until transmitter 99S and detect its DMF in its sensing range the time, content liquid rises to the level of upper limit capacity sensor 99S in the parent container 200.The reactor storehouse is finished and is inserted then.The about second capacity sensor 99S level of parent container 200 weighting material as many as.In step 860, valve 90,100,101-109 are got back to its closed attitude again then.

Next step, in 862 steps, the top shared pathway is eliminated DMF.Valve 121 is opened, and gives top Common Ducts pressurization with the pressurization argon gas.Valve 129 is opened and is allowed DMF enter parent precursor reactant device by the top Common Ducts.Valve 90 is opened by the parent container and is discharged replaced argon gas.Thereby DMF is transferred to the parent container from the top Common Ducts.The parent container is designed to have enough volumes and holds the DMF that adds in addition that does not flow through.The all after dates of for example about 5 seconds pre-program time, valve 90,121 and 129 is got back to its system intialization state again in step 864.The pre-program time cycle is variable, removes the required time of top shared pathway DMF but necessarily equal or exceed.

If do not pack DMF in the reaction vessel into before entering bead suspension, if promptly reactor is empty, then the distribution of ball suspension will not be uneven.If reactor is empty, then send into by the parent container and the pipe go side nearest reactor of mouth that passes through at first is loaded into from bead suspension.Several vessels is excessive packs into if allow, and then may some reactors not had remaining bead suspension.

The present invention has used a kind of new controlling reactor to receive the method for bead suspension vol.At first, in step 866, a pillar argon gas is introduced into the top that makes each pipe that reactor is connected with the top Common Ducts.For producing argon gas bubbles, valve 121 is opened and is allowed the pressurization argon gas enter the top Common Ducts.The valve 101-109 that selects opens in proper order and allows some DMF drain into low pipeline by reactor.Valve 110 is opened and is allowed replaced DMF be discharged by bottom pipe.After about 0.3 second, a pillar argon gas appears at the top of the pipe that reactor is connected with the top shared pathway.Get back to their system intialization states again at step 868 valve 101-110 and 121 then.

In step 869, in preparation, the bead suspension in the parent container is mixed to distribute between reactor.The discussion that the order that interrelates of step is relevant with Figure 18 is

similar therewith.Valve

10 and 90 is got back to its system intialization state again in step 870.This step guarantees that even bead suspension is distributed evenly between selected reactor.

The part of bead suspension is introduced into top Common Ducts, step 871 then.This step is according to pre-program time cycle timing, so that the bead suspension that enters in the Common Ducts of top replaces the major part of the argon gas in the Common Ducts of top and do not flow through pipe end.This port and last reactor tube, the reaction tubes that for example is connected with transmitter 109S connects.Found that be gratifying about 0.5 second pre-program loop.For this part bead suspension is sent into the top Common Ducts, valve 91 is opened with argon gas and is given the pressurization of parent container.Valve 129 is opened and is made bead suspension flow to the top Common Ducts.Valve 120 is opened and is discharged the argon gas that remains in the Common Ducts of top.After the previous pre-program time period expires of discussing, valve 91 and 120 is got back to its system intialization state again in 872 steps.The more important thing is that valve 129 continues to open to prevent bead and polymkeric substance because the closing movement of valve and damaged.In embodiment of the present invention, 2-port valve 129 continues to receive the control computer command signal and is held open in the above described manner.But 129 can be a valve, and one receives that the control computer command pulse just is socketed between the open and close state.If valve 129 is valves and has opened that then control computer does not need issue an order to keep valve 129 to open.

Figure 19 B is the continuation of Figure 19 A.After part argon gas in being present in top Common Ducts 212 was replaced, residue bead suspension was transferred to top Common Ducts 212 in 874 steps.Valve 91 is opened with argon gas and is given the parent container 200 pressurizations.The valve 129 that is held open makes pressurization bead suspension enter top Common Ducts 212.Selectivity valve 101-109 opens the DMF that makes in the reactor 200 and flows out to low pipeline 214.Valve 110 is opened from low pipeline 214 and is drained DMF.Part is owing to the resistance of bead suspension, and DMF backs off along selective reaction pipe 201-209 relatively slowly.Alternative DMF from the selection pipe 221-229 that selecting reactor is connected with top Common Ducts 212 is substituted by bead suspension.The blistered DMF post that suspends slowly advances downwards to low pipeline 214.

Early introducing blistered post also can promptly provide a control computer to measure the approach when each reactor has received q.s bead suspension advantageously as a volume markings.Because the surface properties of tetrafluoroethylene and the high contact angle between DMF and the tetrafluoroethylene, the foaming post is trapped between DMF post and the column of suspension.As discussed above, the optical pickocff that uses in embodiment of the present invention can detect the existence of liquid column in the tetrafluoroethylene of substantially transparent or not exist.Replace because DMF is suspended by downward a large amount of beads of progressive, bubble is moved down into reaction tubes and with the pipe of reactor with low pipe connection.Move downward with fluorescence detector 101S-109S detection argon gas bubbles this moment, and detector moment closes.Sensing data, as discussed above, pass to control computer, control computer each valve of signal at stop 101-109 that gives an order immediately.After all valve 101-109 closed, bead suspension shifts thing to be begun and continues this step again in 878 steps and detect in the pipe that is connecting parent container and top Common Ducts until acoustic sensor 120S and do not have liquid.In step 882, all valves except that 129 are got back to its system intialization state.As discussed, valve 129 continues to open to avoid damaging bead and valve 129.This closes and is carved into instrument contains suspension in its reactor.And having some bead suspension remains in the parent container.For guaranteeing that all beads have all transferred in the reactor, use at least one drip washing round-robin lessivation.

This drip washing circulation is sprayed the parent container inner wall with the loose bead suspension resistates that sticks on the wall from 890 steps with DMF.Valve 14 is opened sprinkling DMF and is washed wall from top to bottom.Valve 90 is opened to discharge from the parent container and is substituted argon gas.Continuation is sprayed inwall with DMF and is risen to level than lower volume transmitter 90S up to parent container DMF level, and it is closed.Control computer receives sensing data and gives an order immediately and allows except that valve 129 all valves get back to its system intialization state in 892 steps.Mode with above-mentioned discussion allows argon gas bubbles stir the parent container contents then.

Perhaps by reinstalling DMF and mixing the bead suspension resistates that adheres on parent container inner wall or the frit with loose.At first, in step 884 with the fresh DMF parent container that recharges.Allow DMF enter the parent container by open valve 10 and finish this step from pressure-flow system.Valve 90 is also opened and is allowed replaced argon gas be discharged by the parent container.The DMF liquid level rises to top capacity sensor 99S level in the parent container, and top capacity sensor 99S connects, and control computer receives sensing data issue an order immediately allows all valves except that 129 get back to its system intialization state in 886 steps.Use mode discussed above, mix the parent container contents in step 888 by argon gas bubbles being imported the parent container.

Transfer to top Common Ducts to the pressurization of parent container and with mixture by valve 129 by open valve 91 usefulness argon gas then, in step 894 mixture of DMF and bead suspension is transferred in the reactor, wherein valve 129 stays open state in whole lessivation.Selector valve 101-109 opens and allows liquid by the extremely low pipeline of reactor stream.Valve 110 is opened DMF is discharged from bottom pipe.The frit of reactor bottom has been discharged all beads in the reactor.Last parent container is drained.When connecting parent container and top and do not have liquid in the pipe with pipeline altogether, transmitter 120 cuts out.Sensing data is passed to control computer and is presented at and does not have remaining liquid to shift in the parent container.Control computer continues open valve 915-10 and makes any mixture that retains enter reactor for top Common Ducts pressurization second.5-10 after second except that 129 all valves get back to the system intialization state.Finished a drip washing circulation.

As discussed above, can carry out repeatedly drip washing circulation to guarantee that all basically bead suspension is transferred to reactor by the parent container.Find that 2 to 3 drip washing circulation is gratifying.

When finishing all drip washing circulation times, comprise that all valves of 129 are got back to the system intialization state in 896 steps.Notice that valve 129 in whole bead suspension redistribution process, comprises lessivation, stay open, to reduce damage ball and polymkeric substance.Method with previous discussion is discharged all liquid in step 898 from the reactor storehouse.

From the step of Figure 19 A-19B, use argon gas bubbles that bead suspension is evenly distributed between reactor as can be seen, and do not consider the flow velocity of each stream between parent container and reactor.As long as it is stable that argon gas bubbles keeps between bead suspension and DMF reagent, just provide one to have mark, make the situation of sensor determination reactor.As above to stablize the ability of argon gas bubbles be because the good physical properties of DMF, the i.e. high contact angle of DMF and tetrafluoroethylene in the maintenance of being mentioned.

But when when reactor transmits bead, also wishing to use the liquid that is not DMF.This may have problems, and especially can not produce the physical properties of stablizing argon gas bubbles when the liquid that replaces does not have.For example acetonitrile (MeCN) just can not produce stable bubble as DNA synthetic solvent.Therefore need diverse ways to distribute bead in reactor.

Figure 19 C-19D illustrated when can not producing when stablizing argon gas bubbles, and control computer is mixed the parent container contents and the selectable cover order of issuing.

When only using the minority reactor, for example at the most 4 to 5 the time, this technology is particularly useful.

Step 854a～860a is similar to step 854～860 among Figure 19, and just minority valve 101-109 opens, and promptly only opens corresponding to those valves of selecting reactor.Solvent is housed, as MeCN, to substitute the argon gas that before the reallocation stage, is present in the reactor storehouse with selecting container portions.After this, the MeCN that packs in selecting reactor and the parent reactor.When the amount of MeCN in the parent reactor 200 rises to the second capacity sensor 99S level, stop MeCN stream, show that filling finishes, after finishing, selector valve is got back to its closure state.

Then, step 872a, the bead suspension in preparation in the mixing parent container is to distribute to selecting reactor.At step 874a, open valve 91,129 and selector valve 101-109 bead suspension enter the top Common Ducts and are distributed in the selecting reactor.These valves continue to open to detect in the pipe that connects parent container and top Common Ducts up to transmitter 120S and do not have liquid.Owing to do not produce argon gas bubbles, do not use transmitter 101S-109S.Because have only the minority reactor to receive bead, so bead arrives each reaction and takes time and approximately equate, thereby guaranteed the distribution that is equal to of each reactor usually.

At step 890a, use lessivation, discussed at Figure 19 A-19A, be transferred to reactor to guarantee all beads.After lessivation is finished, comprise that all valves of 129 are got back to the system intialization state in the 896a step.At step 898a, all liquid has been discharged in the reactor storehouse.

Figure 20 is the flow sheet of control computer for the reactor issue an order order of packing into the required reagent of delivery system.Step with reference to Figure 20 discussion also is described in Figure 21 A-21D generally.In the low pipeline of this method hypothesis inert argon (step 900) only is housed.Figure 21 A illustrates the relevant portion in the reactor storehouse with vacant duct.Low pipeline is at first at step 902 reagent of packing into.Valve 100 is opened and is allowed reagent enter low pipeline, and valve 110 is opened with low pipeline and discharged substituted argon gas.When reagent was detected by transmitter 110S, all valves were got back to the system intialization state in 903 steps.

In some instances, transmitter 101S-109S may be closed by accident or prematurely when loading the pipe of ligation device and low pipeline.For example, transmitter may be dripped driving by loosing of reagent before the actual arrival of reagent.Can cause not having the reagent of q.s to be present in the pipe that reactor is carried like this.

In order to reduce or to get rid of and the relevant problem of transmitter driving too early, control computer is opened certain hour for preloaded Guan Erke selectively orders arbitrarily with valve 101-109 in 904 steps.Generally limiting time reaches about 75% so that load pipe before being detected by transmitter 101S-109S.As described, this step does not rely on the use of transmitter 101S-109S.Even therefore preloaded guarantees that transmitter is driven too early, also can exist q.s reagent to come injecting reactor in the pipe at least suitably to mix.In step 905, after the scheduled time expiration of loading pipe, valve is got back to its system intialization state in preparation.

In step 906, the ligation device is loaded into the transmitter level with the effective reagent of low pipeline.Loading process can parallelly carry out to save time or to carry out in proper order.Valve 100 is opened and is made the pressurization argon gas enter low pipeline by pressure-flow system.Selector valve order or parallel opening enter in the pipe of ligation device and low pipeline reagent.Go up in limited time when pipe pilot scale agent content reaches optical sensor 101S-109S, optical sensor is connected, and control computer moment is closed relevant valve 101-109.When all the sensors 101S-109S lamp was connected entirely, all valves were all got back to the system intialization state in step 908.Result after Figure 21 B general idea explanation loading process finishes.

Remove bottom pipe with method discussed above in step 910 then.Figure 21 C has illustrated the relevant portion of the reactor with cleanser conduit and a post reagent, and wherein reagent is at top sensor for example 101S and valve for example in the tube portion between 101.After bottom pipe was removed, all valves were got back to the system intialization state again in 912 steps.Reagent in the pipe pours in reactor by frit.For making reagent enter reactor, argon gas valve 122 is opened and is made argon gas enter the low pipeline of pressurization by pressure-flow system.Eduction valve 120 is also opened.

In step 913, start the common stirred reactor of vortex motor storehouse one predicted time.This time cycle is enough to make reactor content to be mixed fully.Discovery was suitable in general about 4 seconds, but also can change according to reaction type.

During above-mentioned mixing period,, valve 101-109 is opened a pre-program time cycle so that small quantity of reagent stream and main reactor in step 914.Usually repetition loading and emptying reactor in building-up process.Each reactor discharging, wherein bead just becomes dry and the formation that flocks together " ball piece ".Step 914 makes the inclusion fluidization in the reactor and dissolves the ball piece.Because eddying motion bead or be the most effective during near the reactor substrate, so only need inject small amount of liquid.On the other hand, the bead piece may swim in reactor head, needs more times that it is dissolved.In step 915, after the expiration, valve is got back to the system intialization state at the fixed time.

In step 916,, make residue reagent partly pour reactor into by open valve 101-109.Loading each container does not have liquid to have the control computer shut-off valve up to respective sensor 101S-109S detection.When all the sensors 101S-109S closed, all reactors had all loaded.For improving mixed effect, stirred vessel storehouse when loading.As noted above, by once opening whole valve 101-109 or order is opened, but reactor design is loaded parallel container or order.

Perhaps, can not rely on transmitter 101S-109S and load reactor.For example, the pre-program time cycle open valve 101-109 of container to desired content can be enough to.The time cycle of having found 0.1～1 second is gratifying.But this time cycle can change according to used container number, promptly uses the container number many more, and required time is long more.Figure 21 D has shown the sketch that loads post-reactor.

Note length that the volume of a container that enters reactor also can be by improving pipe between valve 101-109 and the transmitter 101S-109S and diameter and easily change.This change is by replacing ligation device and fillup valve with a pipe with different lengths or cross-sectional dimensions, for example, and the pipe of valve 111 and easily realizing.

In some example, also can advantageously increase the diameter of reactor self.For example, perhaps wish to mix simultaneously bead and two or more reagent.Such mixing can be carried out after Figure 21 A-21D introduces first kind of reagent the step of reactor.Second kind of reagent can be guided in the reactor by the step that repeats Figure 21 B-21D then.But when doing like this,, therefore will remove the argon gas bubbles between the bead suspension and second reagent owing to before introducing second kind of reagent, there is argon gas to stay among pipe 241-249 and the 271-279.For argon gas bubbles is discharged from reactor, the reactor interior diameter can be reduced the height of bead suspension in the reactor greatly, thereby argon gas bubbles is overflowed from bead suspension easily.After removing Argon Bubble,, the bead suspension and second reagent are mixed together by carrying out vortex as mentioned above.

Figure 22 is the sequential flowchart of control computer for filling activatory amino-acid reagent issued command in reactor.Figure 23 A-23C illustrates the suitable step of amino acid filling process generally.Suppose that again low pipeline cleans when initial, promptly only contain argon gas.Reactor when Figure 23 A is initial, low pipeline, valve, the synoptic diagram of transmitter and relevant tube.

Another selectivity of container 231-239 is settled and is seen Fig. 6 A.

Have Guan Liecheng one row who selects groups of containers for the motion drive rotating-disk travelling belts of organizing containers being specialized more, being transmitted at step 917 rotating-disk.In case selected suitable containers, just with the DMF of activation of amino acids agent such as 0.2MHBTV and 0.6M DIEA and DCM ratio 3: 1 solution flow in pipes in 918 steps.With the method carrying conduit of the discussion relevant with Figure 20, promptly

open valve

100 and 110 is connected up to transmitter 110S.After this all valves are all closed in 920 steps.

Activator enters in the pipe of ligation device and low

pipeline then.Valve

100 and 120 is opened and is allowed the pressurization activator go into low pipeline in 922 steppings.The selector valve of valve 101-109 is opened up to the low transmitter 111S-119S that fills and is shown and have liquid.In step 924, all valves cut out again.Figure 23 B illustrates the existence of liquid in this initial implantation step post-reactor storehouse relevant portion.

For finishing injection, in 926 steps,

valve

100 and 120 is opened again and is allowed the pressurization activator enter low pipeline.Selector valve parallel opening among the valve 101-109 allows pressurization activator post rise in aforementioned tube.Simultaneously, the related valves among the valve 111-119 is opened last setting prop and and the activator mix of amino acid being injected the pressurization activator.Figure 23 C illustrates this implantation step.

When each transmitter 101S-109S had detected liquid and exists, control computer was closed the valve 101-109 relevant with this transmitter.When all valves cut out in 928 steps, all valves in reactor storehouse were got back to the system intialization state.The mixture of amino acid and activator preferably allows to rest in the aforementioned tube about 2 minutes to guarantee in the suitable activation of step 930.

After this, remove bottom pipe with the method for discussing in early time relevant in step 932 with Figure 17.Mixture post between valve 101-109 and the upper sensor 101S-109S enters reaction tubes with the method for discussing relevant with Figure 20 at step 913-916 then.

Figure 24 shows that control computer is for shifting back the bead suspension in the reactor order of parent container issued command.During beginning, the assumed response device is emptying, and the dawn, low pipeline was filled with argon gas.At first reactor injects solvent in 936 steps with the method for discussing relevant with Figure 20.After this all valves are got back to the system intialization state.

Then the mixture at step 938 vortex bead and reagent gets a suspension.Perhaps vortex bead mixture when reactor injects.This method has improved to become the speed of suspension and help to prevent that bead is coalescent at the bottom of reactor.The reaction tubes inclusion is transferred in the parent container in step 940.Valve 122 is opened with argon gas and is given low pipeline pressurization.Valve 129 is opened and is allowed bead suspension flow into the parent container by reactor storehouse stream.Selector valve among the valve 101-109 is opened, and preferred sequence is opened, and allows argon gas promote each reactor content and upwards arrives the top shared pathway and enter the parent container.Therefore reactor content is transferred in the parent container successively.Although above-mentioned transfer also can be undertaken by while open valve 101-109 is parallel, sequential transfer makes the high Ar Pressure that keeps in the reactor storehouse, is preferred therefore.And each valve 101-109 preferably is held open about 4 seconds to guarantee that all given reactor content have been transferred to the parent container basically.In this course, valve 90,122 and 129 is held open state.

After all reactor content were gone to the parent container, the drip washing reactor also carried out another transfer process.Be the drip washing reactor,, repeat above step from contain reinjecting of DMF reactor with step 936.Shown in step 937 and 941, in a series of circulations, valve 129 is held open state to prevent to damage ball and valve 129.The parent container preferably has the volume of at least three transfer amount.Recombinant latter stage, the parent container is preferably by

open valve

110 and 91 dischargings, reaches the bottom than lower volume transmitter 90S and close this transmitter up to parent liquid in containers content.Found that three drip washing circulations are gratifying.

After this, comprise that all valves of 129 get back to its system intialization state in 942 steps.Stir the parent container contents to mix bead with the method for discussing relevant in step 944 from each container with Figure 20.If from the parent container, shift out bead, preferably drain the parent container in 946 steps and reach the bottom than lower volume transmitter 90S and close this transmitter up to parent liquid in containers content by open valve 10 and 91.In step 948, all valves are got back to its system intialization state successively.Can shift out the mixture that contains bead then from the parent container uses.

Perhaps, use the mode relevant with Figure 21 A-21B that bead is redistributed to reactor, after the reallocation, all valves are got back to it attitude are not taken place.

G. total software chart

Figure 25 is the source code flow sheet that this paper appendix I comprises.Module 950 is represented the user.Command interpreter 952 is received user's text command.Perhaps, the user can use menu system 953 input commands to start synthesizer.The set that menu system is accepted or change into command interpreter 952 spendable forms, or directly call support program supporting routine module 962.Command interpreter 952 is also analyzed the order that text enters or the user imports.After this, analyze the support program of command calls or execution support program module 962.And analyzing order is shown form 954 formats and is showing demonstration on 762.

Module 958 contains a large amount of macro instruction files.A macro instruction document definition, for example actual sequence of steps of synthesizing or making up the storehouse.In its basal level, a macro instruction file contains, and for example contains the macro instruction of the dispersion order system of control valve and read sensor information successively.Macro instruction can use other basic macro instruction to finish high-level function, as discharging reactor 201-209.

The macro instruction of receiving from macro instruction file 958 command interpreters 952 enters synthesizer storehouse control 960, and it is synthetic that synthesizer storehouse controller 960 call macroinstructions carry out reality.For example, a macro instruction can stipulate that the initial overall situation that must set up before the synthetic beginning changes.

Figure 25 illustrates the support routine module 962 of the various support metaprograms of operation.Such metaprogram is automatic filling, and it is to load reactor automatically up to the anastomosing metaprogram of transmitter.Support program 962 is received the input from command interpreter 952 or menu system 953.Option list 964 contains directives such as function.Figure 26 has provided the input and output of option list 964 and with command interpreter 952 with support the relation of routine module 962.

The original document 966 of grasping global change and overall situation setting is also arranged.Original document 966 is grasped, and for example, representative is given and opened the numerical value that a closed valve provides striking voltage time amount, or the like.Table look-up file 968 with synthesizer order library controller 960 carry out with, for example, allow monomer enter suitable selecting reactor.The file 968 of tabling look-up can contain, for example, and each reactor list, its respective markers monomer, the necessary monomeric list of synthetic required peptide institute.

Figure 25 has also provided log file 970.The input that log file 970 is accepted from command interpreter 952 and synthesizer library controller 960.Log file 970 contains the service data of diagnostic purpose.The entry of log file 970 contains, for example, and with the relevant information of institute's call macroinstruction.

Associated file 972 contains the name of each reactor and associated valve and transmitter.Associated file 972 is operated to simplify the task of each reactor of record and associated valve and sensor address with synthesizer library controller 960 and support program 962.

Digital command carries out driven in parallel device 974 by support program 962 outputs.Driven in parallel device 974 can be a PCDIO 120-PI/O plate 766 for example.Driven in parallel device 974 drives the network pipe valve by its I/O passage delivery valve control signal 976.Valve control signal, as discussed, controlled device circuit 768 is further handled.And driven in parallel device 974 output stepping motor controller signals 978 are controlled the vortex step motor.From the synthesizer optical sensor.Sonac, the transmitter input 980 of capacity sensor is also received by driven in parallel device 974, checks that by transmitter metaprogram 982 is by support program 962 processing.

Figure 27 has illustrated the data structure that control valve is required.Valve index VINDEX array 986 receives a valve number 984 and is input, and provides directive to VALVES array 988.The unit of each VALVES array 988 contains the directive of a digital byte 990.Each digital byte 990 contains 8 binary digits of valve numeral information.The binary digit that contains given valve valve data information by its data byte 990 the address and representative data byte 990 in the movement value of relative position of binary digit, be accessible.A valve can controlledly be connected or close to each binary digit of data byte 990 suitably.The valve data information of each binary digit is by the corresponding valve of output port 992 controls.

Figure 28 illustrates that reception is from the essential data structure of input port 994 transmitter information.Represent the information of each transmitter scale-of-two attitude to be stored in the bit of data byte 996, be the information in the access data byte 996, used transmitter number number access SENSOR array 998.SENSOR array 998 each member are contained the directive of a suitable data byte.Each data byte 996 contains 8 binary digits of sensing data information.The binary digit that contains given valve valve data information by its data byte 996 the address and representative data byte 996 in the movement value of binary digit relative position, be accessible.

H. window interface

Control software can comprise Window-type interface and working space.Usually, window interface is a rectangular graph user interface (GVI), its provide one or more on screen window displayed.The assistant window object as desired can all size and form (for example brick shape or trellis) demonstration.At the window top is an option wall scroll shape that a large amount of user's command selection are arranged, and each bar shaped can be called auxiliary inferior menu and Software tool is that application purpose is used.Window also comprises the dead zone that is used to show and handle screen object.This dead zone person of being to use and the working space or the viewport that are stored in the data object exchange message in the control computer system memory.

Window interface comprises screen cursor and is used for selecting and or calls the directive of institute sensitization interest screen object.The motor message of response user's indicating unit such as mouse, cursor drift about (promptly moving freely) to required screen location on screen.During cursor movement or afterwards, the user can provide customer incident signal (for example mouse key button " location " and " braking ") selecting and manipulating objects, as known in the art.For example, window can be closed, sizing again, or by rolling up screen assembly in " location " (selections).The equivalence of key stroke comprises that keyboard quickens or " hot key " is provided and is used for finishing these and other user's operation by keyboard.Therefore, window interface provide with control computer alternative more direct by way of.

Below the window interface information or event-driven system structure.This model can provide best description by the mode that its operation and tradition are used or the operation contrast of sequential architectural, as the institute of the command interface among Figure 25 example.In this mode, the handiness of the event driven system that reader can help increasing and complicacy.

The mode program comprises a series of in beginning, the discrete operations module or the mould that define when middle process and end.Therefore program is according to the working order that is completely fixed, and each step must be finished before carrying out next step.

The relatively easy design of mode program and when carrying out, usually and be not easy to use.Design guarantees that certainly all required information enter, and just enter in the cost that makes the user with the programmed model operation.Particularly, because program has been set up the pre-mould of arranging of a cover, the user does not at first finish the pre-mould that needs just can not enter another by a mould.Do not allow the user this to be had in proper order any deviation.The ineffective activity of this mode program is unfavorable for handling realistic task.

On the other hand, the event-driven system structure is avoided preselected order, optimizes (opting) and replaces " event loop ".Event loop is the centralization mechanism system of handling about user and system event information.It comprises event queuing and process, is used for retrieving and sending information to each group of windows.

Information is how operating system manages to make multiple application and hardware synchronization, and as mouse location with knock keyboard, the information of MS-window is converted into the window events handling procedure.From program, an information just comprises the numeric structure of special incident information.Message structure can contain the information identification as special incident permanent marks.For example, can comprise about window from the information of window object producing, close, move and become the information of size.Auxiliary event data can be used as information parameter and obtains; The definite explanation of given parameter changes according to the event type of each representative.

Provide process with retrieval from the information of system queuing and will pacify send to suitable applications, then application can begin to handle the information of any arrival.Each window belongs to a special window type, and it has defined the total feature of some this type windows.What interrelate with each type is the window function with all information of delivering to this class window.Provide and use queuing, wherein Windows can place the information that belongs to application-specific.When input has been received in this application, its read-only information of waiting for.If if do not find or exist with higher priority, other application message, Windows is by other application controls.

The general process of the concurrent carry information of retrieval in event system is as Mi-crosoft ^RWindows ^TM, be well known in the art, referring to for example Petzold, C., Programming Windows, second edition, Microsoft Press, 1990 and Custer, H., Inside Windows NT, Microsoft Press, 1993. auxiliary information can be found among the Mi-crosoft Window Software Development that Redmond, WA buy by Microsoft Corp..Above the disclosure of each document that is drawn is incorporated by reference for all purposes.

The GUI that Figure 33 explanation realizes on control computer.This GUI includes the rectangular window 1301 of working space 1303.At the window top is to select single line 1305, and it has user's command selection 1306 and 1313.Each these command selection comprises the inferior menu that contains control synthesizer operating command.These command selection provide automatically or have finished by hand the synthetic handiness by call suitable order with mouse to the user.

Good environment to the user is noticed in the GUI design, has therefore reduced the effort of time variable control synthesizer.For example a dialog object or a cover dialog object and each order interrelate.When calling an order, suitable dialogue object is presented at working space, and man-machine interaction ground reminds the user to import required information.A Help order 1313 provides intelligence and helps the user to pass through this program.Use the dialogue object, the user need not use written order can operate synthesizer.

Figure 34 has illustrated the inferior menu 1320 that interrelates with macro instruction (Macros) selection.The user can by respectively on inferior

menu exercise question

1321 or 1322 mouse location call Learn or Run order.In the Windows environment, macro instruction is the object that comprises a cover discrete command.Order as the discussion relevant with Figure 16-24 control synthesizer.Use Learn order 1321, user's definable one macro instruction is finished specific function.Inferior menu also can identifying call can be ordered the key stroke equivalence of design.For example " Alt+L " is used for carrying out the Learn order.

Macro instruction is " Learned " in a single day, macro instruction and other predefined macro instruction that Run order 1322 can selected steering routine design learning be crossed.Figure 35 illustration when calling the dialog object that Run when order shows.Dialog object comprises 1325 (linking frames), and it has listed the macro instruction that can get.In order to select a macro instruction, the name of user's 1324 input macro instructions in Selet Macro space.Perhaps the user goes up volume by mouse location and braking, required macro instruction file in selecting space 1325.Then the user in the space 1326 the input macro instructions with the number of times that is repeated.Last run macro instruction, the user locatees mouse on RunMacro button 1327 or RunSMacro button 1328.The RunSMacro order makes systematic order finish the macro instruction function, i.e. next reactor.When selecting Cancel to select 1329, withdraw from dialog object 1326.Help selects 1330 to provide intelligence according to dialog object.

Figure 36 explanation Groups on selecting the single line bar selects the inferior menu 1335 of 1310 demonstrations when selected.Inferior menu 1330 comprises Define order 1331, and it is used to define the valve that a cover is connected with one group of particular reactor.In case after the definition, the valve group just is used as a group objects and is stored in the holder.Computer storage can contain a lot of group objects, each object definition one special valve group.

Inferior menu 1330 also comprises Open/Pulse order 1332 with predetermined time cycle filling and drainage selecting reactor.When calling, show and the relevant dialogue object box of Open/Pules order.This dialogue object box is similar among Figure 35 illustrated a bit, contains a linking frame, and it lists the got group objects of therefrom selecting.The user selects the selector valve required time cycle in the design space that required group and input pulse provide by the dialogue object.In order to load reactor, the user locatees on the Open button and shows that valve will open.Then the user begins the filling process by location Pulse or Spules (order is loaded reactor) button.Be the emptying reactor, the user locatees Close button and Pules or SPules button.

Use Fill/Drain order 1333, the user can load or emptying receptacles automatically with transmitter.When selecting the Fill/Drain order, shown and the similar dialog object of Open/Pules order.The user selects one group of valve, and location AutoFill or SAutoFill button are finished load function or location AutoDrain or SAutoDrain button emptying reactor.In some instances, can provide Time Delay to select, after triggering transmitter, postpone the closed or unlatching of valve.It is particularly useful under the situation of desired content that this function reactor when transmitter is touched does not also have to reach with limitting.Select by the delay that establishes the specific delays cycle, reactor can suitably be loaded.As can be seen, relevant with Group menu order application response device in the selection combination is synthetic only provides handiness to the user.

Figure 37 has illustrated and has contained the inferior menu 1340 that is suitable for changing the selection that menu selects.The order 1341 that produces has defined the variable that can carry out in macro instruction.Variable can be typically, for example in value, as the time, can use under a situation that is blended into another variation.Replace producing the macro instruction of each time value, a variable can be defined as the corresponding time simply.Use the order 1342 of setting up before each synthesis cycle, variable can be created as desirable value.

Figure 38 explanation is selected relevant inferior menu 1345 with Diagnostics.Inferior menu 1345 comprises Valves 1346, Sensor 1347 and Mix 1348 orders, and these orders pass to user prompt control synthesizer and deposit the information of connection about the diagnostic purpose synthesizer.When selecting Mix to order 1348, start vortex motor mixing reactor, carry out the user-defined time cycle.

With reference to Figure 39, when selecting the Valves order, show Valve Diagnostic dialog object 1390.The user by selecting the inlet of required valve correspondence, can control the operation of any valve in the synthesizer by dialog object 1390.For example the valve 100 in the storehouse 1 can be opened by positioning cursor on frame 1391.For convenience, all valves can be closed by selecting Close All order 1397.One Can-cel orders 1398 close box objects 1390.

Figure 40 explanation is corresponding to the Sensor diagnostics dialog box object 1391 of transmitter order.As described, dialog object 1391 contains a frame 1396 relevant with each transmitter.Frame 1396 is notified user's respective sensor state.For example, if in the frame be " X ", respective sensor is connected when long.On the contrary, the bright transmitter of empty frame table cuts out.

Figure 41 explanation shows inferior menu 1350 when selecting File to select.Newer command 1351 produces one and newly syntheticly sets up document control and synthesize, and Modify order 1352 makes the user select editor with being pre-stored in composite document.In case synthetic set up file and finish, the user calls Save 1354 or Save As order 1355 file is existed in the holder.Print order 1357 is printed and is selected composite document.Print Setup orders 1358 configure printer to required specification, so print file come out with specification attractive in appearance.Call Exit order 1359 and withdraw from the File selection.

In some instances, as each synthetic before, maybe when one group of new valve of selection, the user may wish to dispose this system by calling Load cfg file order 1356.The file that system loading is suitable informs which is the suitable valve that will use.In fact, cfg file conversion or with on valve and each selecting reactor " contact ".

Figure 42-44 explanation is used in and produces and modify a synthetic dialogue object of setting up in the file.With reference to Figure 42, Set Assozciatedi dialog box 1360 allows the user select required reactor by the suitable frame that inspection is contained in the space 1361.For convenience, provide Check All button 1362 and Uncheck All button 1362 easily to select or do not select all reactors.Check Bank button 1364a-1364d allows the user select all to belong to the reactor of particular bin.When selecting the Cancel button, withdraw from synthetic creation facilities program (CFP).As previously explained, Help button 1369 information of user by this program of offering help.Set up process for continuing to synthesize, the OK button 1365 of user's locating closing Set Associate dialog object also shows Syn-thesis Setup dialog object.

Figure 43 illustrates Synthesis Setup dialog object 1370, defines synthetic Start Macro 1371 with it, Loop Macro 1372 and End Macro1372.Start Macro and End Macro for example, comprise the order of washing reaction device before or after each synthesis step.Loop Macro contains and carries out the synthetic order.These orders comprise with amino acid structure unit and oligonucleotide injecting reactor, merge reactor content and mix their generation bead suspension in the parent container.Bead suspension is distributed to selected reactor.Inject, merge and synthesis cycle of allocation step formation.Number according to input in Number Synthesis Steps parameter 1374 repeats synthesis cycle.In case defined macro instruction, the user has deposited Synthesis Setup dialog object content and has continued to set up process by location OK button 1375.The user still can locate Cancel button 1376 and stop this process.

Figure 44 has illustrated Amino ﹠amp; Oligo Setup dialog object 1380.As seen, frame contains the inlet relevant with each reactor 1381.The user is at inlet amino acid needed symbol of input and oligonucleotide sign indicating number.For example, reactor 1 (RVo1) inlet contains " V ATGCCGA ", will make synthesizer inject amino acid V and oligonucleotide ATGCCGA in reactor 1.After importing suitable sign indicating number, the user locatees the process that OK button 1382 is finished foundation.Provide Cancel button 1383 to withdraw from this program.Can see, set up synthetic storehouse easily with synthetic creation facilities program (CFP).

In case finish synthetic foundation, the user can begin building-up process.With reference to Figure 45, the user at first selects Synthesis to select 1308, is that Go order 1391 begins this method then.Perhaps, the user can set up a single macro instruction and finish the function that stores in the composite document.

With reference to Figure 46, status screen 1400 term of execution that GUI being presented at synthetic or macro instruction.As shown, status screen is divided into two isolating districts 1401 and 1402.First district 1401 shows and synthetic relevant information, for example lists Start Macro, Loop Macro, and the name of End Macro, Loop Number program line 1408 is notified the user the synthetic middle round-robin number of times that keeps in addition.

Second district 1402 shows the Macro filename of now carrying out.As mentioned above, a macro instruction can be nested with another macro instruction to finish high-level function.The design point screen is listed the 1409a-1409j macro instruction that is called as 10 nested levels.Command program line 1410 can be provided, Timing program line 1411, Message program line 1412, and RV ' S program line 1413 show secondary status or configuration information.For example, the Command program line can show which macro instruction order carries out, when using the Message program line, and the written information of the pre-program of explicit user.The Timing program line shows opening of valves or the closed time that keeps.RV ' S program line informs which reactor the user uses.Time to Complete lines 1414 are informed the synthetic per-cent of finishing preceding remaining time of user.

Whenever, the user can be by the function key of pre-design term of execution of synthetic or macros, and promptly F12 connects " User Abort " state.User Abort state suspends the user or carries out the macro instruction that does not define as a synthetic part, and need not withdraw from synthesis program.When calling, system temporarily suspends execution, and shows UserAbort dialog object 1420, as Figure 47 explanation.At the moment, the user can insert and carry out any macro instruction.Can be by in Select Macro frame 1421, knocking in required macro instruction title and on Run Macro button 1422, locating mouse and carry out.

User Abort dialog object also provides an Ignore button 1424, and it makes the user continue to use synthesis program, just looks like that this system never entered UserAbort mode.Jump to select 1423 order set the current command in the macro instruction that continues to jump for the first time before synthetic for one.Retry button 1425 makes system from the beginning carry out current macro instruction.It is synthetic that Abort button 1426 allows the user withdraw from.

Figure 48 explanation and the relevant inferior menu 1430 of Edit order.The Edit order is easy to by selecting to be listed in the suitable title access on the inferior menu and changing various objects the user.When selecting Associate to order 1431, be presented at the dialog object of classifying Set Associate object as that stores in the holder.By selecting required object, show Set Associate dialog object.This moment, the user can use the content of mouse editing dialog frame object.After finishing, the user locatees the OK button and has deposited revised file.Similarly, the user can be by selecting required object editing Group, Macro, Vari-able and Code object.Then alternative is placed in the editing machine, allows the user's modification file.

As above mentioned, transmitter can touch accidentally owing to the existence of drop, and this is a problem, and particularly the solution that uses in synthesis cycle contains the high density bubble.For avoiding or slow down the accidental triggering of transmitter, its susceptibility can be designed program.

Transmitter is designed program with Machine Config order 1456.This order shows several inlets, Fill%, Drain%, the Config dialogue object box of Time Out% and PtsAve.Fill% regulation transmitter keeps connecting measures the percentage ratio when reactor is filled with.For example, if enter 80%, transmitter is bound to connect 80% of its reading.Drain% regulation transmitter keeps cutting out the percentage ratio of measuring reactor emptying when.As an embodiment, if enter 20%, transmitter must be to cut out 80% of its reading.Time out is defined in transmitter and connects or close the time cycle that prolongs before.PtsAve regulation is used for counting or reading of time percentage ratio that determination sensor connects or close.The user also can stipulate to want the transmitter or the sensor groups of reading.Afterwards, the user locatees the OK button, and it is according to the parameter automatic configuration system.

Figure 49 is the event-driven system flow sheet of explanation as the described control software of Figure 33-48.As shown in the figure, help connection between each object program piece as the GUI heart from I/OObject program block of synthesizer.When starting this control software, a program block 1460 is loaded into configuration file on the I/O Object program block.These files are used to dispose Variable Object program block 1456, Lookup Table program block 1457, Macro Objects program block 1458, Group Objects program block 1459, Valve Object Array program block 1461 and Sensor Object Array program block 1462.

Lookup Table Objects program block comprises a file that is used for corresponding its respective reaction device of instruction valve and transmitter, like this to reduce each valve of manual record or the transmitter address corresponding to particular reactor.The variable of Variable ObjectS program block area definition, and the group objects of definition exists in the Group Objects program block.

Valve Object Array program block has the array of each valve in the recognition system.For closing or open a valve, the signal of a control of I/O Oject program block scanning Valve Ob-ject Array program block output particular valve.For controlling one group of valve, the I/O program block scans Valve ObjectArray with Group Objects program block, allows it send the signal of suitable control valve.

Sensor Object Array program block has the array of each transmitter in the recognition system.The I/O program block can be read a transmitter by scanning Sensor Object Array program block, up to finding a coupling that makes it read a particular sensor.In order to read one group of transmitter, I/O and Group Objects program block scanning Sensor Object Array program block are measured the right sensors that will read.

GUI also comprises Dialog Box Objects program block 1453 and Synthesis Object program block 1454.Dialog Box Objects program block contains the dialog object that shows when calling some order.Synthesis Object program block has the current synthetic file of setting up.Different synthetic for finishing, GUI will requiredly synthesize and set up file and read in Synthesis Object program block by storer.On the contrary, the content of Synthesis Ob-ject program block is written into holder and deposits the synthetic file of setting up in.

Main Window Object (MWO) program block 1452 is connected with the View Object program block 1455 that shows GUI ' main window (Fig, 33) on screen.A Message of response user command is sent to MWO.Analyze this information and provide the suitable order that will carry out.For example, if receive the Macro order, MWO instruction I/O object is from Dialog Box Objects program block retrieval macro instruction dialog object, and from Macro Objects program block retrieval macro instruction catalogue, View Object program block is received from the information of I/O program block and with it and is presented on the GUI.

In one embodiment, if computer generation obstacle, the I/O program block contains a Watchdog by way of closing synthesizer.For example, I/O object program piece can be designed program and be given pulse of synthesizer per 2 seconds.If because some reason synthesizer do not receive these pulses, can suppose that then control computer breaks down and will close.

Embodiment

Following embodiment is described in more detail the present invention but does not limit the present invention.Embodiment 1: the preparation in storehouse and screening

On the present embodiment explanation resin balls peptide synthetic product capable of being combined how by the name oligonucleoside join the contact of recognizer mark with synthetic in the common bead that exists of each amino acid coupling step and given clearly.Each mark transhipment by the link coupled amino acid monomer, can be inferred total sequence of peptide on the bead by the mark of reading on the bead in the synthetic particular step.The bead of collecting can screen, and combines with fluorescently-labeled anti-peptide antibody with fluorescence-activated cell sorter (FACS).Those antibody can separate with FACS with its bead of combining closely, and can be passed through pcr amplification by the oligonucleotide identifier that the bead of sorting contacts with each.The determined identity that has and have high-affinity antibody bonded peptide sequence of DNA amplification sequence.By in conjunction with Gao Rong, oligonucleotide is encoded to the information storage on basis, amplification method is learned, with sorting based on fluorescence, the invention provides a kind of method of determining by each molecular nature in natural or the huge library of molecules of the chemical macromolecule chain section synthetic of non-natural, and the method for effectively separating each bead with biological acceptor high-affinity part.

In this embodiment, single stranded oligonucleotide be used to the to encode peptide capable of being combined that uses L and D-amino acid structural unit and 10 μ m diameter polystyrene spheres is synthetic.Oligonucleotide tags has high information and includes, and is suitable for very high sensitivity detection and decoding, and in the methods of the invention, is stable to being used for peptide synthetic reagent.The parallel assembling with oligonucleotide of peptide is alternately synthetic, and each bead has the replica of many single peptide sequences and unique oligonucleotide identification marking like this.That oligonucleotide is shared is common 5 '-and 3 '-the PCR priming site, so bead can be used as the template of PCR.The synthetic storehouse of coding contains about 8.2 * 10 ⁵Individual seven peptides and screening are to be attached to anti-dynorphin B monoclonal antibody D32.39 (referring to Barrett ﹠amp; Goldstein, 1985, Neuropeptides 6:113-120, incorporated by reference here), with each bead of fluorescence-activated cell sorter (FACS) selection mortise antibody.After dividing the upward oligonucleotide pcr amplification effect of choosing shuttles, the determined sequence of DNA is to measure the characteristic of peptide part.

A. reagent and general method

The single 10 μ m diameter bead raw materials that disperse that are used in this work are buied by Pharmacia, and commercial synthetic is with 1, the macropore styrene-divinylbenzene copolymer of 12-diamino dodecane linking agent functionalization.This bead is the Phar-macia Monobeads of Pharmacia ' s Gene Assembler Support linking agent derivatize of no use ^TMReferring to Ugelstad and Mork, 1980, Adv.ColloidInterface Sci., 13:101-140, incorporated by reference here.

All protection amino acid are obtained by Bachem Bioscience Inc..PCR and sequencing primer are synthetic with Applid Biosystems model 394 nucleotide oligonucleotide synthesizers.The reliable sample of some peptides is with Applied Blosystems model 431A peptide synthesizer synthetic, uses Fmoc-protection amino acid, and HBTU/HOBt is chemical activation on the spot, and with 40: 1: 1TFA/ water/dithioglycol goes protection.These peptides pass through at Rainin C ₁₈HPLC on the reversed-phase column (＞95% purity) carries out purifying, and water/acetonitrile/0.1%TFA is an eluent, confirms structure with mass spectrum.

B.69 base oligonucleotide and opioid peptides dynorphin B is parallel synthetic

The C-terminal seven amino acid fragment H-Arg-Gin-Phe-Lys-Val-Val-Thr-NH of opioid peptides dynorphin B ₂(RQFKVVT) (SEQ IDNO:2) and 69 chain link oligonucleotide (ST08) are parallel synthetic on 10 μ m diameter beads, and the sequence of ST08 is 5 '-ATC CAA TCT CTC CAC (ATC TCTATA CTA TCA) TCA CC[TA TC CT AT TT TIAC] CTC ACTCAC TTC CAT TCC AC-3 ' (SEQ ID NO:20)

This sequence line part is corresponding to the PCR primer sites, and district in the bracket and the primer homology that is used for sequencing template.14 base sequences are represented the template coding region in the bracket.

At first use succinimide 4-O-DMT-oxidation butyric ester (molecular probe) and N-Fmoc-2,4-dimethoxy-4 '-(carboxymethyl oxygen)-benzhydryl amine (being acid cleavage Knorr carboxylic acid amides linking agent) or N-Fmoc-Thr ( ^tBu)-the mixture process bead of the 1-oxidation benzotriazole ester of OH (be used for non-cracking experiment).Spectrophotometric records the amino ratio with DMT-protection hydroxyl residue of the protection of Fmoc-on the bead and is approximately 20: 1.3 of the following nucleosides of use '-methyl-N, N-diisopropylaminoethyl phosphoric acid ester makes bead carry out 20 circulation: N of oligonucleotide synthetic on automatic DNA synthesizer DNA ⁶-Bz-5 '-O-DMT-(the 7-denitrification is assorted)-2 '-Desoxyadenosine (Berry and Associates, Ann Arbor, Michi-gan), N ⁴-Bz-5 '-O-DMT-2 '-Deoxyribose cytidine and born of the same parents 5 '-O-DMT-thymus pyrimidine deoxidation nuclear) glycosides (Glen Research).

Then bead is taken out from synthesizer and showed in 5 minutes and remove the Fmoc protecting group with the DMF solution-treated of 10% piperidines.In the coupling behind first amino-acid residue (N-Fmoc-Thr ( ^tBu)-OH), with the DMF solution-treated bead of acetic anhydride and 1-Methylimidazole to cover unreacted amine.All peptide linked reactions are carried out 20 minutes, and contain 0.11M Fmoc-amino acid, 0.1M HBTU, the DMFF solution of 0.1M HOBt and 0.3MDIEA.Allow bead carry out the circulation (carry out detritylation with TCA, the catalytic phosphorous acid base of tetrazolium turns usefulness into, covers with acetic anhydride, with the iodine oxidation in the acetonitrile/water) of Nucleotide addition on twice synthesizer then.Repeat the sequential steps of amino acid coupling and dinucleotides addition, up to finishing synthetic that peptide sequence RQFKVVT (SEQ ID NO:2) and oligonucleotide coding region make up.After carrying out 35 times other oligonucleotide synthesis cycle; use piperidines/DMF (1: 9 successively; 8 minutes); thiophenol/triethylamine/diox (1: 2: 2; 4 hours), ethylene diamine/ethanol (1: 1, handled 5 hours down for 55 ℃); handle so that peptide and two chains of oligonucleotide are gone protection fully with TFA/ water (20: 1,1 hour).In the experiment of using the acid cleavage linking agent, concentrate TFA protective reaction supernatant liquor under the vacuum, isolating then thick peptide product is analyzed with HPLC.

C. the structure of code database

The parallel synthetic chemistry of ruling above has been used in the structure in this storehouse.By with N-Fmoc-Thr ( ^tBu)-mixture of OBt and succinimide 4-O-DMT oxobutanoic acid esters is coupled on all beads, as mentioned above, makes that the synthetic site of peptide is different from the synthetic site of DNA in this experiment.The oligonucleotide tags sequence is only in the coding region in the storehouse different with ST08.3 of oligonucleotide ST08 '-conserved regions is at first at 35mg (～1.75 * 10 ⁸Individual bead) synthetic on total ball amount.Remove the Fmoc protecting group, the bead quality is divided into seven equal portions.Each equal portions coupling-individual seven different 2-N-Fmoc-protection amino acid (Side chain protective group is represented in bracket): Ary (NG-Pmc), Gln (Trt), Phe, Lys ( ^tBoc), Val, D-Val and Thr ( ^tBoc). allow each part carry out twice automatic oligonucleotide then and synthesize.Each sequence of augmenting dinucleotides of unique definite each different aminoacids residue is TA, TC, CT, AT, TI, CA and AC.Then bead is merged, thorough mixing carries out Fmoc with whole beads and goes protection.

Bead distributes, the peptide coupling, and the oligonucleotide dimer is synthetic, and bead recombination, Fmoc are removed this circulation and are repeated altogether seven times.Last Fmoc protecting group is not removed.Perhaps, allow a large amount of beads that merge carry out the oligonucleotide synthesis cycle 35 times.Then as mentioned above, protection is gone in the storehouse fully.

D. dye and facs analysis in the storehouse

With the part (typically 0.5-2mg bead) in storehouse be suspended in the buffer reagent of blockading (PBS, 1%BSA, 0.05%Tween-20) in and at room temperature be incubated 1 hour.Precipitate into sheet and be resuspended in mAbD32 by centrifugal bead, in 39 solution (10mg/mL blockade buffer reagent).Suspension is incubated 30 minutes on ice bath, be centrifuged into flaky precipitate, and washs with the buffer reagent of blockading.On ice bath, bead is suspended in sheep anti-mouse antibody (molecular probe) solution that phycoerythrin puts together 20 minutes then.Bead washs in the buffer reagent of blockading and dilutes in PBS, delivers to the fluorescence activated cell branch and washes (FACS) (Becton Dickinson FACStar Plus).The fluorescence identification that needs by their with mABD32,39 bonded balls.From the most obvious painted storehouse 0.17% and have both each beads of minimum fluorescence volume district (about 98%) and be classified and enter PCR miscrofuge bottle.D32.39 is blockaded by the pre-incubation of mAb and solvable peptide Ac-RQFKVVT-OH (SEQ IDNO:2) D XTU IPE YA O 10M to the characteristic combination of bead.

E. chou matched moulds plate PCR

Has a 0.2mM dATP what the producer provided, dCTP and dGTP, 0.8mM dVTP, every kind of primer of 2mM, buffering system (the 50mM KCl of 3 Tag of unit polysaccharases (Promega) and 1 unit uracil-DNA glycosidase (Gibco BRL), 10mM Tris-HCl, pH9.o, 0.1%Triton X-100,2mM MgCl ₂) carry out pcr amplification in (cumulative volume is 70L).Primer sequence 5 '-ATC CAA TCT CTC CAC-3 ' (SP13) (SEQ ID NO:21) and 5 '-(vitamin H)-GTG-GAA-TGG-AAG-TGA-3 ' (SP14) (SEQ ID NO:22) respectively with ST08 template homology or complementation.PCR reaction by 45 95 ℃ of sex change 30 seconds, 50 ℃ of primer annealings 1 minute, constitute in the circulation in 1 minute of 72 ℃ of extensions.React with the electrophoretic analysis in 20% acrylamide or the 2% low melting-point agarose gel.

F. measure PCR product sequence

(Dynal Inc.) separates biotinylated PCR product from each reaction with the magnetic bead of streptavidin parcel.Abiotic elementization chain alkalescence wash-out after scouring, each bead sample order-checking mixture process.Explanation according to the producer, use primer 5 '-(SP15) (SEQ IDNO:23) and Bst polysaccharase (Bio-Rad) carry out dideoxy sequencing, different deoxidations that has been to use 1: 100 ratio and dideoxy nucleotide triguaiacyl phosphate (Pharmacia) to ATC TCT ATA CTA TCA-3 '.

G. the mensuration of peptide binding affinity

Various peptides are determined in a competition combination experiment to the binding affinity of monoclonal antibody D32.39.Trace peptide (LRRASL GGGRRGFKVVT (the SEQ ID NO:24 that contains the known epi-position of the D32.39 that merges with CAMP dependent kinases consensus substrate sequence; 50pM) used [g- ³³P] the paramount specific activity of ATP radio-labeled (referring to Li etal., 1989, Proc.Natl, Acad, Sci.USA 86:558-562, incorporated by reference here), and mix (10 μ M-1pM) with various concentration peptide to be measured.Peptide mixt is joined in the polystyrene well with D32.39 (0.1Gmg/mL) coating, and sample washs well 4 ℃ of insulations 2 hours with PBS, and the radioactivity relevant with each well and be used for making the competition binding curve counts.Under condition determination, IC ₅₀Should be near the dependent constant (Kd) of peptide.Embodiment 2: the synthetic and stability study of thiazolidino-ketone (thiazolidinones)

Following embodiment relates to the inventive method synthetizing thiazolium alkane and ketone.This synthesizes at U.S. number of patent application No.08/256090, applies for illustrating in greater detail in the patent on June 23rd, 1994 of Shen, and this paper is that all purposes are incorporated by reference.

A. prepare double-tagging thiazolidino-ketone

H ₂N-S-TentaGel (500mg), a kind of polystyrene resin (Rapp Pdymere, Tubingen, Germany, 1g, 0.30mmol/g loading) that is purchased uses Fmoc-Gly-OH at the enterprising row labels (2-of alpha-carbon ¹³C, 99%, by Cambridge Isotope Laboratories Inc., And Over, MA buys).Resin Ac ₂O covers, and goes protection with piperidines, with its OBt-activatory ester coupling Fmol-light linking agent.Resin is capped again, goes protection, and with the anhydride reaction of unlabelled Fmoc-Gly-OH.Cover and go protection generation unhindered amina resin again.With the PhCHO of 0.75M mark on carbon back (carbonyl- ¹³C, 99%; Available from Cambridge Isotope Laboratorios, Inc., Andover, MA) the ACN solution that contains 3 molecules with 2.0 Thiovanic acids reacted 2 hours at 70 ℃, generated double-tagging thiazolidino-ketone resin.With resin thorough washing (3 * 5mlCH ₂Cl ₂, 3 * 5ml DMF, 3 * 5ml CH ₂Cl ₂, 3 * 5ml MeOH, 3 * 5mlCH ₂Cl ₂, 3 * 5ml Et ₂O) dry and under vacuum.

The B.TFA stability study

Part resin (20mg) is used 95%TFA 15%H ₂O handled 1 hour, used CH then ₂Cl ₂, MeOH and Et ₂The O washing.Gel- ¹³CNMR analyzes resin and shows the loss that does not have thiazolidino-ketone, as confirming by the relative integration of two mark carbon.See bar 1, Figure 30.Any destruction of wishing light linking agent or thiazolidino-ketone can cause the minimizing of the integration on the carbon of benzyl position.This experiment shows that it is stable that the light linking agent of thiazolidone is handled TFA.

C.DNA synthesizing stable Journal of Sex Research

A part of resin (20mg) is loaded on the synthetic post of standard DNA, and uses A with the formation (Phosphoramidites) of its phosphoramidate, C and T nucleosides carry out 40 circulations of DNA synthetic.Each circulation back iodine oxidation.Begin to use 2%TFA/CH in each circulation ₂Cl ₂Process resin is removed " model " two pairs of methoxy trityls (DMT).By taking out resin in the post, with the DMF washing, usefulness silica gel- ¹³The CNMR spectrum analysis.See Figure 30, bar A.The gained collection of illustrative plates shows the destruction that almost is provided with or is provided with light linking agent or thiazolidino-ketone molecule.

Part resin (2mg) has also carried out photodissociation in three hours in pH7.4 PBS damping fluid, analyze the thiazolidinedione that discharges with HPLC.See Figure 31 and Figure 32, bar A.Data show that thiazolidino-ketone discharges under high purity, and handle the neither considerable change of light linking agent and thiazolidinedione with the standard DNA synthetic agent.Embodiment 3: capable of being combined synthetic

Can carry out the capable of being combined synthetic of YGGFL with synthesizer.Synthesize in reactor 1-6 and carry out, 7-9 is unconnected.Six seed amino acids that add in bead are L, E, G, Y, A and F.YGGFL and other peptide are stipulated together.Bead is added in the parent container (29.5mg) and is suspended among the DMF.Synthesis cycle comprises distribution more at every turn, and the peptide coupling covers, and amine goes protection, goes to protect the collection of back FMOC, uses DMF drip washing, reaches these several steps of recombinant in the parent container.

After synthetic, mark Hettz antibody is introduced the blended bead.Hertz antibody is most of to be combined with YGGFL.The existence of facs analysis proof YGGFL proves that this regulation combination is a synthetic in synthesizer.Originally experimental results show that the different collection of peptide, comprise the YGGFL chain, can be by synthesizer regulation and synthetic.

Although the present invention clearly can carry out some variations and modification for the purpose that the is aware and understand mode with specification sheets and embodiment is illustrated in detail in claims scope of augmenting.Embodiment 4: use Instrument measuring bead of the present invention to distribute

For whether the mixing bead of measuring in the parent container is distributed in the reactor equably, with some bead biotinylations.Manually the bioid ball is placed in the reactor.Abiotic elementization ball manually is placed in other 8 reactors.Synthesizer is delivered to the parent container with the ball in 9 reactors.From the parent container, get a sample, allow combine with vitamin H on the biotinylation bead with the glimmering streptavidin that fills.About 9.1% bead is by biotinylation in fluorescence-activated cell sorter (FACS) the analytical proof parent container.Then bead is reallocated in 9 reactors, the biotinylation bead is measured with the facs analysis instrument the per-cent of total bead in each container.Table 5 shows in each reactor and to mix the ratio that bead has biotinylation bead approximately identical with the parent container and total bead.

Table 5

	Fluorescence ball percentage composition
	Fluorescence ball percentage composition	The parent reaction	?????9.1
		The parent reaction	?????9.1
		??????1	?????9.7
??????2	?????9.7	??????1	?????9.7
??????2	?????9.7	??????3	?????9.4
??????4	?????8.7	??????3	?????9.4
??????4	?????8.7	??????5	?????9.4
??????6	?????8.9	??????5	?????9.4
??????6	?????8.9	??????7	?????9.1
??????8	?????8.9	??????7	?????9.1
??????8	?????8.9	??????9	?????9.3
Mean value	?????9.2	??????9	?????9.3
Mean value	?????9.2	Standard set-up	?????0.32

Should be understood that only not conduct restriction of foregoing description for explanation.By above-mentioned explanation, a lot of specific exampless are bright to those skilled in the art conspicuous, so the scope of the invention should not be subjected to the limitation of above-mentioned explanation, and should be with reference to claims and the four corner that is equivalent to claims right.

Sequence table (1) integrated information:

(I) applicant: AFFYMAX TECHNOLOGIES N.V.

(II) denomination of invention: Synthesizing and Screening Molecular Di-versity

(III) sequence number: 24

(IV) contact address:

(A) address: Townsend and Townsend Khourie and Crew

(B) street: One Market Plaza, Steuart Tower, Suite2000

(C) city: San Francisco

(D) state: California

(E) country: USA

(F) postcode: 94105

(V) computer-reader form

(A) medium type: Floppy dish

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release # 1.0, Version # 1.25

(VI) flow process application data:

(A) application number:

(B) applying date:

(C) classification:

(VII) priority data

(A) application number: US 08/146,886

(B) applying date: 1993.11.02

(VII) priority data

(A) application number: US 08/149,675

(B) applying date: 1993.11.02

(VIII) proxy/business quarters's information

(A) title: Norviel, Vernon A.

(B) registration number: 32,483

(C) reference/summary number: 16528J-000740PC

(IX) telecommunication information:

(A) phone: 415-326-2400

(B) information of fax: 415-326-2422 (2) SEQ ID NO:1:

(I) sequence signature

(A) length: 13 amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:1:

Tye?Gly?Gly?Phe?Leu?Arg?Arg?Gln?Phe?Lys?Val?Val?Thr

The information of 15 10 (2) SEQ ID NO:2:

(I) sequence signature

(A) length: 7 amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:2:

Arg?Gln?Phe?Lys?Val?Val?The

The information of 15 (2) SEQ ID NO:3:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:3:

Thr?Phe?Arg?Gln?Phe?Lys?Val?Thr

The information of 15 (2) SEQ ID NO:4:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:4:

Thr?Thr?Arg?Arg?Phe?Arg?Val?Thr

The information of 15 (2) SEQ ID NO:5:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:5:

Thr?Val?Arg?Gln?Phe?Lys?Thr?Thr

The information of 15 (2) SEQ ID NO:6:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:6:

Gln?Val?Arg?Gln?Phe?Lys?Thr?Thr

The information of 15 (2) SEQ ID NO:7:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:7:

Arg?Gln?Phe?Arg?Thr?Val?Gln?Thr

The information of 15 (2) SEQ ID NO:8:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:8:

Lys?Gn?Phe?Lys?Val?Thr?Lys?Thr

The information of 15 (2) SEQ ID NO:9:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:9:

Gln?Gln?Phe?Lys?Val?Val?Gln?Thr

The information of 15 (2) SEQ ID NO:10:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:10:

Lys?Gln?Phe?Lys?Val?Thr?Gln?Thr

The information of 15 (2) SEQ ID NO:11:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:11:

Thr?Gln?Phe?Lys?Val?Thr?Lys?Thr

The information of 15 (2) SEQ ID NO:12:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:12:

Thr?Phe?Arg?Val?Phe?Arg?Val?Thr

The information of 15 (2) SEQ ID NO:13:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:13:

Phe?Arg?Arg?Gln?Phe?Arg?Val?Thr

The information of 15 (2) SEQ ID NO:14:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:14:

Arg?Gln?PHe?Lys?Gln?Val?Gln?Thr

The information of 15 (2) SEQ ID NO:15:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:15:

Gln?Thr?Val?Thr?Val?Lys?Lys?Thr

The information of 15 (2) SEQ ID NO:16:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:16:

Gln?Gln?Val?Gln?Arg?Gln?Thr?Thr

The information of 15 (2) SEQ ID NO:17:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:17:

Lys?Thr?gln?Val?Val?Gln?Phe?Thr

The information of 15 (2) SEQ ID NO:18:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:18:

Gln?Val?Thr?Gln?Val?Arg?Val?Thr

The information of 15 (2) SEQ ID NO:19:

(I) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:19:

Phe?Val?Val?Thr?Val?Arg?Val?Thr

The information of 15 (2) SEQ ID NO:20:

(I) sequence signature

(A) length: 69 base pairs

(B) type: nucleic acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: DNA (oligonucleotide)

(XI) sequence description: SEQ ID NO:20:

The information of ATCCAATCTC TCCACATCTC TATACTATCA TCACC-TATCC TATTITTACC TCACTCACIT CCATTCCAC (2) SEQ ID NO:21:

(I) sequence signature

(A) length: 15 base pairs

(B) type: nucleic acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: DNA (oligonucleotide)

(XI) sequence description: SEQ ID NO:21:

The information of ATCCAATCTC TCCAC (2) SEQ ID NO:22:

(I) sequence signature

(A) length: 15 base pairs

(B) type: nucleic acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: DNA (oligonucleotide)

(XI) sequence description: SEQ ID NO:22:

The information of GTGGAATGGA AGTGA (2) SEQ ID NO:23:

(I) sequence signature

(A) length: 15 base pairs

(B) type: nucleic acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: DNA (primer)

(XI) sequence description: SEQ ID NO:23:

The information of ATCTCTATAC TATCA (2) SEQ ID NO:24:

(I) sequence signature

(A) length: 17 base pairs

(B) type: amino acid

(C) number of share of stock: strand

(D) topological framework: linearity

(II) molecule type: peptide

(XI) sequence description: SEQ ID NO:24:

Leu?Arg?Arg?Ala?Ser?Leu?Gly?Gly?Gly

1???????????????5

Arg?Arg?Gln?Phe?Lys?Val?Val?Thr

10??????????????????15

Claims

1. the method for multiple molecule on synthetic a large amount of substrates, form by following steps:

Described substrate is distributed in a plurality of reactors;

With different reagent in each described reactor with the first part of described various molecules and the described substrate coupling in the described reactor;

With described substrate by flow line deliver to mixing tank again in and mix described substrate;

Described substrate is distributed to the described reactor from described mixing tank by described flow line;

The described first part coupling of the second section of described various molecules and described molecule is generated various molecules on the described substrate.

2. the process of claim 1 wherein described distribution step again.Comprise by the common conduit described substrate of at least a portion that distributes again.

3. the instrument of parallel linked reaction on the solid phase carrier comprises:

The parent container;

The reactor storehouse that at least one links to each other with the parent container, the reactor that massive parallel carries out linked reaction is contained in described reaction storehouse;

Between described parent container and reactor a large amount of flow lines are arranged, described flow line forms the stream between described reactor and described parent container;

Reagent is delivered to the delivery system in described reactor storehouse; With

Between described parent container and described at least one reactor storehouse, coordinate to shift the programmable calculator of described solid phase carrier.

4. the instrument of claim 3 further comprises a shared pipeline, and described common conduit is connected with at least one described a large amount of flow line.

5. the instrument of claim 3, wherein said delivery system comprise further reagent are transported to the device of instrument second section by the first part of instrument that described first part has the pressure higher than second section.

6. the instrument of claim 3, wherein said reactor storehouse further comprises the device of the described a large amount of reactor content of parallel stirring.

7. the instrument of claim 3, wherein said reactor storehouse further comprises the valvegear of response from described programmable calculator command signal, select in described a large amount of reactors in order to first pattern described reagent is transported to, and select in described a large amount of sequential reactors in order to reagent is delivered to second mode sequence with parallel mode.

8. the instrument of claim 3, wherein said parent container, the described device storehouse of answering is with described delivery system airtight separating atmospheric in described parallel coupling building-up reactions.

9. the instrument of claim 4 further is included in the valve of the described solid phase carrier of movement between described parent container and the described a large amount of reactor, and described valve is unique valve that described carrier passes through during movement between described parent container and described a large amount of reactor.

10. the instrument of claim 9, wherein said reactor storehouse further comprises the valvegear of response from described programmable calculator command signal, select in described a large amount of reactors in order to first pattern described reagent is transported to, and select in described a large amount of sequential reactors in order to reagent is delivered to second mode sequence with parallel mode.

11. a parallel method of carrying out linked reaction on bead, described method comprises:

Described bead suspension is transferred in a large amount of reactors by the parent container;

In described a large amount of reactors, on described bead, carry out described linked reaction;

Described bead is transferred to described parent container by a large amount of reactors; And mix described bead.

12. the method for claim 11, wherein said step repeats the preliminary assay number of times.

13. further comprising by common conduit, the method for claim 11, wherein said transfer step shift the described bead step of at least a portion.

14. the fairing preface block that is used for fluorescence detector that liquid exists in detecting translucent tube basically, described fairing preface block comprises:

The setting of described photodetector receptor part is arrived in control by described tube hub from the light beam of described photodetector sender; With

Inhibition by the emission of described photodetector sender except that the described described detector receptor of arrival part of filling the bundle.

15. the fairing preface block of claim 14, wherein said control setting comprises a pinhole openings by fairing preface block.

16. the fairing preface block of claim 15, wherein said fluorescence detector has two plugs, and sender is connected on first plug, and receptor is connected on second plug, and described optics generic sequence module further comprises:

The setting of the fairing preface block between described two plugs is frictionally engaged; With

Place described pipe and make the setting of described pinhole openings longitudinal axes and described pipe radial axle with an angle of 90 degrees meet.

17. the fairing preface block of claim 14, wherein said control are provided with the device of the described pipe in location between the receptor that further is included in sender and described fluorescence detector.

18. the delivery system of delivery of therapeutic agents comprises:

2-port valve with first port and second port;

3-port valve with a first channel and one the 3rd port;

The setting of response programmable calculator is used for selectively fair described the 3rd port and described first channel UNICOM;

Respond the setting of described programmable calculator, in order to selectively to allow described first port and the described second port UNICOM; With

Described first port and described first channel be tightly connected and form the setting of pipeline.

19. the system of claim 14, wherein said the 3rd port and first pipe coupling that is loaded with reagent, and another described first port and described second port be loaded with second pipe coupling of argon gas, wherein said first pipe carries described reagent to described pipeline or flow out described pipeline.

20. a synthesis device capable of being combined that carries out linked reaction on bead comprises:

At least one reactor storehouse, described reactor storehouse comprise a large amount of reactors and a large amount of monomer reservoir, and each described reservoir is associated with a described reactor; With

A shared reagent reservoir, described shared reagent reservoir are connected by common conduit with described at least one reactor storehouse shared reagent are transported in described a large amount of reactor.

21. the method for a complex sign library of molecules, wherein each differing molecular and a solid phase carrier are covalently bound in the storehouse, and with one or more different marker marks, wherein each described one or more different marker contains variation hydrocarbon district and a part hook, each marker also is that covalency is connected with described solid phase carrier, and described method comprises: (a) distribute carrier with random fashion between a large amount of reactors; (b) carrier in each reactor is exposed to the first chemical structure unit; (c) merge carrier; (d) random assignment carrier between a large amount of reactors; (e) carrier in each reactor is exposed to a chemical structure unit; (f) repeating step (a) to (e) is at least once to 20 times.

22. the method for claim 21, wherein said solid phase carrier are joints.

23. the method for claim 21, wherein said solid phase carrier is Monobead ^TM

24. the method for claim 21, wherein said molecule is connected with described solid phase carrier by a joint.

25. the method for claim 24, its center tap is a cleavable.

26. the method for claim 21, wherein each described one or more different marker contains:

The cleavable joint that each described one or more different marker is connected with described solid phase carrier;

A molecular hook; With

A variable-length hydrocarbon chain that connects described joint and described molecular hook.

27. the method for claim 21, wherein said each one or more different marker have following formula: wherein n is 1 to 10 integer, and X is the cleavable joint, and R is a part hook.

28. the method for claim 27, wherein X is the photodestruciton joint.

29. the method for claim 21, wherein said molecular hook is selected from vitamin H, but the group that activating group and high association peptide are formed.

30. the cracking from the described solid phase carrier of a method of screening claim 25 tagged molecule storehouse, wherein said molecule, and with acceptor incubation under the condition that helps part is attached on the described acceptor.

31. the method for claim 30, wherein said molecule is a peptide, described solid phase carrier is that diameter is about 50 beads to about 500 μ m, and described cleavable joint is the mixture of a cleavable joint, and has only the described molecule of the part cracking before described incubation step on the described bead.

32. synthetic method that contains a large amount of different members synthesis peptide libraries; each member comprises by the amino acid monomer sequence synthetic peptide that is connected with bead; described bead also connects one or more oligonucleotide identification tag; discern the monomer sequence of described peptide; wherein said amino acid monomer is removed the Fmoc protecting group with the Fmoc protection and with piperidines, and innovative approach comprises with 5-15% piperidines processing 5-60 minute or with the processing of 15-30% piperidines effectively removed Fmoc in 1 to 30 minute.

33. the innovative approach of claim 32, wherein said bead have about 10 μ m diameters, and are made up of lauryl amine joint deutero-macropore styrene-divinylbenzene copolymer.

34. the innovative approach of claim 32, wherein said bead is Monobeadu ^TM

35. the innovative approach of claim 32, wherein said amino acid monomer have side chain ( ^tBu) Side chain protective group, with TFA remove described ( ^tBu) Side chain protective group, and described oligonucleotide tags comprise the 7-denitrification assorted-2 '-Desoxyadenosine.

36. a test right requires to exist in 26 methods method of one or more different markers, this method comprises:

The one or more different markers of cracking from the described solid phase carrier;

Fixing one or more different markers;

Coupling one can be increased on described one or more different markers, and detectable group is to described molecular hook;

Increasing, this can increase, but detection moiety; And

But detect the existence of the described detection moiety that increases, but wherein exist the detection moiety explanation of to increase to have one or more different markers.

37. the method for claim 35, the wherein said amplification, but detection moiety contain can with described molecular hook bonded oligonucleotide sequence.

38. a mensuration is connected to the method for the composition sequence of the molecule of solid phase carrier in the claim 21 tagged molecule storehouse, this method comprises that each measures the existence of described one or more different marker that is connected with described solid phase carrier, the existence of described one or more different markers or do not have to show whether specific synthesis step in the described molecule synthesis order has taken place.

39. the method for claim 38, described each detection of wherein said one or more different markers comprises:

According to its structure, the described one or more different markers of physical sepn, used unpack format is from described one or more different markers;

Fixing described one or more different markers are to keep described unpack format;

Handle described marker with oligonucleotide sequence, used oligonucleotide sequence is selectively in conjunction with described one or more different markers;

Described oligonucleotide sequence increases;

Detect the existence of described oligonucleotide sequence, the existence of wherein said oligonucleotide sequence with do not have the existence of expressing described one or more different markers and do not exist;

By discern a described existence with a plurality of different markers at the relative position of one or more different markers described in the described unpack format; With

With the existence of described one or more different markers with do not exist with described molecule synthesis order in the connecting of specific synthesis step.