CN1134156A - Synthesizing and screening molecular diversity - Google Patents

Synthesizing and screening molecular diversity Download PDF

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Publication number
CN1134156A
CN1134156A CN 94193984 CN94193984A CN1134156A CN 1134156 A CN1134156 A CN 1134156A CN 94193984 CN94193984 CN 94193984 CN 94193984 A CN94193984 A CN 94193984A CN 1134156 A CN1134156 A CN 1134156A
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reactor
valve
library
synthesis
method
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CN 94193984
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Chinese (zh)
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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
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阿菲马克斯技术公司
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Priority to US08/149,675 priority Critical patent/US5503805A/en
Priority to US08/146,886 priority patent/US5639603A/en
Application filed by 阿菲马克斯技术公司 filed Critical 阿菲马克斯技术公司
Publication of CN1134156A publication Critical patent/CN1134156A/en

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Abstract

有效地在底物上合成不同分子产物的设备和方法。 The method and apparatus of various products efficiently synthesized molecules on a substrate. 一透明容器(200)含有底物的悬浮体。 A transparent container (200) containing a suspension substrate. 将底物用氩气加压并转移至发生单体反应的一个或多个反应容器库的多个反应容器中(201-209)。 The substrate was pressurized with argon and the reaction vessel was transferred to a plurality of reaction vessels or more monomers libraries reaction occurs (201-209). 选择性地,底物可用一标记单体标记。 Alternatively, the substrate can be used a monomer labeled marker. 在单体加成反应期间,用旋涡马达旋流反应容器(201-209)中的内含物以促进合成。 During the monomer addition the reaction, the reaction vessel (201-209) with a vortex swirl the contents of the motor to facilitate synthesis. 在期望的单体和/或标记单体加成反应之后,将悬浮体用氩气加压,转移回到透明容器(200)中混合。 After the desired monomer and / or addition reaction of the monomer tag, the suspension was pressurized with argon, transferred back to a transparent container (200) were mixed. 此后,可将悬浮浮体用氩气加压,并被再分配在反应容器中(201-209)用于进一步的合成。 Thereafter, the suspension can float with argon and pressurized reaction vessel and redistribution (201-209) for further synthesis.

Description

合成和筛选多种分子 A variety of molecular synthesis and screening

本申请是1993年11月2日申请的美国专利系列号为08/146,886和08/149,675的部分继续申请,每一篇均被本文引作参考。 This application is November 2, 1993, US Patent Application Serial No. part 08 / 146,886 and 08 / 149,675 a continuation, each one are incorporated herein by reference.

本专利文件公开的部分包含受版权保护的内容。 Section of this patent disclosure document containing content subject to copyright protection. 版权拥有者不反对任何人的专利文件或专利公开的传真复制,因为它出现在专利和商标局的专利文件或记录中,但在其它方面无论如何保护所有版权权利。 The copyright owner has no objection to any person facsimile reproduction of the patent document or the patent disclosure, as it appears in the patent file or records of the Patent and Trademark Office, but otherwise all copyright rights protection anyway.

本发明一般涉及合成大量不同分子和从中鉴定和分离具有用和所需活性的化合物的方法和设备。 The present invention generally relates to the synthesis of a large number of different molecules and isolated and identified from compounds having the desired activity with the apparatus and methods. 本发明还涉及将识别标记掺入这些化合物中以便于具有所需特性的化合物的识别。 The present invention further relates to the identification mark so as to be incorporated into these compounds to identify compounds having a desired property.

大分子受体的配体可通过筛选经分子生物学或合成化学技术制备的大量不同的肽来鉴定。 Macromolecular ligand receptor can be identified by a number of different screening peptides produced by biological or chemical synthesis of molecular techniques. 重组肽库已通过将简并寡核苷酸插入到编码丝状噬菌体壳蛋白和DNA-结合蛋白LacI.的基团中而被建立。 By recombinant peptide library has been inserted into the degenerate oligonucleotides encoding a filamentous phage coat protein and the LacI DNA- binding proteins. The group is established. 见于Cwirla et al.,1990,Proc.Natl.Acad.Sci.USA.87:6378-6382;Scott & 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;和PCT公开号WO 91/17271,WO 91/19818,WO 93/08278,每一篇均被本文引作参考。 Found in 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; and PCT Publication No. WO 91/17271, WO 91/19818, WO 93/08278, each incorporated herein are a. reference. 这些随机库可包含大于109的不同肽,其中每一种肽融合到与编码它的遗传物理性连接的大的蛋白质序列中。 These random library can contain greater than 109 different peptides, wherein each peptide sequence fused to a large protein encoding it is connected to the physical genetic. 这些库与受体的相互作用,通过几次亲和纯化,选择性曝光或显示在E.Coli和DNA单克隆中被扩增以定序揭示负责与受体结合的肽的同一性的载体而被有效地筛选。 The interaction of the library with the receptor, and affinity purified by several times, exposing selectively amplified in E.Coli or display and DNA sequencing to reveal the monoclonal identity to the carrier responsible for receptor binding peptide and It is effectively screened. 还可见于PCT公开号WO 91/05058和WO 92/02536。 It may also be found in PCT Publication No. WO 91/05058 and WO 92/02536.

建立肽或其它分子库的化学方法不限于使用仅20种基团编码的氨基酸的合成。 Peptide or other molecule libraries established chemical synthesis method is not limited to using only 20 kinds of amino acids encoded group. 通过扩增结构单元组使其包括非天然氨基酸和其它分子结构单元,大大地增加了可达到的序列和结构的差异。 By amplification of the structural unit group to include unnatural amino acids and other molecular constituent unit, greatly increases the differences in sequence and structure that can be achieved. 在一些描述作为产生合成分子库的方法中,这些反应产物被立体地分离,各库成员的同一性通过合成的性质而被明确地确定,这些合成见于Geysen et al.,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. & Wheelan,J.(Wiley,New York)PP.134-146;Fodor et al.,1991,Science 251:767-773;美国专利号5,143,854和PCT专利公开号WO84/03564;86/00991;86/06478;90/15070;和92/10092,每一篇均被本文引作参考。 In some methods described as generating the library of synthetic molecules, these reaction products are separated three-dimensionally, the identity of each library member is clearly determined by the nature of the synthesis, the synthesis found in Geysen et al., 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 No. 5,143,854 and PCT Patent Publication No. WO84 / 03564; 86/00991; 86/06478; 90/15070; and 92/10092, each of one are incorporated herein by reference.

通过“茶袋”(tea-bag)多肽合成法已制备出具有大于3千万溶性肽的库。 By "teabag" (tea-bag) polypeptide synthesis having been produced library of greater than 30 million soluble peptide. 见于Houghten,1985,Proc.Natl.Acad.Sci.USA 82:5131-5135和美国专利4,631,211,每一篇被本文引作参考。 Found Houghten, 1985, Proc.Natl.Acad.Sci.USA 82: 5131-5135 and U.S. Patent No. 4,631,211, each one being incorporated herein by reference. 每一个库被合成和筛选为简并肽混合物,其中此序列中的各氨基酸被清楚地定义。 Each library is synthesized and screened degenerate peptide mixtures, wherein each amino acid in this sequence is clearly defined. 迭代法筛选(如在竞争结合分析中)和再合成被用于分馏这些混合物以及确定库中最具活性的肽。 Iterative Screening (e.g. in a competitive binding assay) is used for resynthesis and fractionating such mixtures and to determine the most active peptide library. 见Houghten et al.,1991,Nature 354:84-86;Pinilla et al.,1992,Peptide Research 5:351-358;Blake,J.& Litzi-Davis,1992,Bioconjugate Chem.3:510-513;和PCT专利公开号WO92/09300,每一篇均被本文引作参考。 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 ; and PCT Patent Publication No. WO92 / 09300, each incorporated herein by reference are a.

运用FurKa等的裂解合成协议,见于第十四届国际生物化学大会(1988年)摘要,Prague,Czech.5:47(还见于FurKaet al.,1991,肽蛋白质研究国际杂志37:487-493;和Sebestyen et al.,1993,Bioorg.Med.Chem.Lett.3:413-4187,Lam和他的同事们制备出了包含106个与100-200μm直径的树脂小球相连的肽的库。见Lam et al.,1991,Nature 354:82-84;Lam et al.,1993,Bioorg.Med.Chem.Lett.3:419-424;和PCT专利公开号WO92/00091中,每一篇均被本文引作参考。这些小球通过与一标记的受体一起温育而被筛选:与受体结合的小球通过肉眼观察被确定并在显微操纵器的帮助下被选择出来。每一个小球包含50-200Pmol可通过埃德曼降解或质谱分析直接确定的单肽序列。原则上,可用这种方法通过降低这些珠的大小来产生具有极大不同的库。肽序列测定技术的灵敏度限于1Pmole,然而对直接肽序列测定分析的范围有明显 Such as the use of FurKa cleavage synthesis protocol, found in the fourteenth session of the International Conference of Biochemistry (1988) summary, Prague, Czech.5: 47 (also seen in FurKaet al, 1991, International Journal of Peptide Protein Research 37: 487-493; ., 1993, Bioorg.Med.Chem.Lett.3 and Sebestyen et al: 413-4187, Lam and his colleagues have prepared a peptide library containing 106 100-200μm diameter of the resin beads is connected to the common. . Lam et al, 1991, Nature 354: 82-84; Lam et al, 1993, Bioorg.Med.Chem.Lett.3:. 419-424; and PCT Patent Publication No. WO92 / 00091, each are a incorporated herein by reference the pellets are screened by incubation with a labeled receptor: receptor bound to the beads was determined by visual observation and are selected with the aid of each of the small micromanipulator 50-200Pmol ball comprising a single peptide sequence may be determined directly by Edman degradation or mass spectrometry. in principle, this method can be used to generate libraries having different maximum by reducing the size of these beads. Determination of the sensitivity of peptide sequences is limited art 1Pmole, although the scope of the direct measurement of peptide sequence analysis significantly 限制。然而,当库的结构单元组被扩展至包括D-或其它非天然氨基酸或其它化学结构单元时,没有分析方法提供直接和明显的序列分析。 Limit. However, when the structural unit repertoire is extended to include D- or when other unnatural amino acids or other chemical structural units, and there is no apparent analytical methods provide direct sequence analysis.

一批化学合成分子和天然产物(如微生物发酵液体培养基)的高效率筛选在新的药理试剂的引线化合物的研究中传统地起着关键的作用。 A number of chemically synthesized molecules and natural products (e.g., fermentation broth) is efficiently screened conventionally plays a key role in the study of the lead compound in a new pharmacological agents. 对制造和估算分子差异的组合化学以及相关技术的兴趣的显著高涨意味着在药物发现paradigm的发展中一个重要的里程碑。 Interest in combinatorial chemistry and molecular manufacturing differences in estimates and related technology means a significant rise development paradigm of discovery is an important milestone in the drug. 见于被本文引用的Pavia etal.,1993 Bioorg.Med.Chem.Lett.3:387-396。 Pavia etal found in the referenced article, 1993 Bioorg.Med.Chem.Lett.3:. 387-396. 今天,肽化学已成为开发组合方法在配体识别中运用的主要工具。 Today, peptide chemistry has become the main tool developers use a combination of methods in ligand recognition. 见于被本文引用的Jung & Beck-Sickinger,1992,Angew.ChemInt.Ed.Engl.31:367-383。 Found Jung & amp cited herein; Beck-Sickinger, 1992, Angew.ChemInt.Ed.Engl.31: 367-383. 这归结于大量结构不同的氨基酸单体的有效性,相对种属的高产率固相偶合化学以及与制造重组肽库的生物学方法的协同作用。 This is due to the effectiveness of a large number of different configurations of the amino acid monomers, relatively high yields of species and the solid phase coupling chemistry and biology synergy producing a recombinant peptide library. 而且,许多低分子量的肽的潜在和特殊的生物学活性使这些分子成为治疗药物发现的有吸引力的起点。 Moreover, the potential and specific biological activity of many peptides of low molecular weight molecules making them the starting point for the treatment of drug discovery attractive. 见于Hirschmann,1991,Angew.Chem Int.Ed.Engl.30:1278-1301和Wiley & Rich,1993,Med.Res.Rev.13:327-384,每一篇均被本文引作参考。 Found Hirschmann, 1991, Angew.Chem Int.Ed.Engl.30: 1278-1301 and Wiley & amp; Rich, 1993, Med.Res.Rev.13: 327-384, each incorporated herein by reference are a. 不利的药物动力学特性如差的口服生物利用率和在体内被迅速解除,制约了肽化合物作为药物的更广泛的开发。 Unfavorable pharmacokinetic properties such as poor oral bioavailability and are rapidly released in vivo, as a medicament restricted peptide compound broader development. 最近这种认识激起了研究人员将肽化学之外的组合有机合成概念扩展到制造已知的药品如苯并二氮杂(见本文引用的Bunin & Ellman,1992,J.Amer.Chem.Soc.114:10997-10998)和聚合分子如寡N-取代的甘氨酸(“肽”)和寡氨基甲酸酯。 This recognition has recently aroused researchers combined organic synthesis concept extends to outside of peptide chemistry known in the manufacture of drugs such as benzodiazepines Bunin & amp occlusion (see cited herein; Ellman, 1992, J.Amer.Chem .Soc.114: 10997-10998) and polymeric molecules such as N- substituted glycine oligonucleotides ( "peptide") and oligo carbamates. 见于Simon et al.,1992,Proc.Natl.Acad.Sci.USA89:9367-9371;ZucKermann et al.,1992,J.Amer.Chem.Soc.114:10646--10647:和Cho et al..1993.Science261.1303-1305,每一篇均被本文引作参考。 Found in 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 one are incorporated herein by reference.

虽然分子的大型库在识别有用化合物或提高引线化合物的性能可具有大的值,但筛选这些库,特别是大型库的困难制约了这些库在降低如药物发现和开发的成本中应取得的效果。 Although large libraries of molecules in identifying useful compounds, or improve the performance of the lead compounds may have a large value, but screening these libraries, in particular the difficulties of large libraries restrict the effect of these libraries in cost reduction such as drug discovery and development should be made . 结果,对制造和筛选分子库的方法的开发成为热衷研究的对象,其中库中的每一个分子用一个专门的标志符来标记以便于化合物的鉴定。 As a result, the development of a method for producing and screening libraries of molecules of the subject of keen study, in which each of the molecules in the library with a special flag to mark in order to identify the compound. (见本文引用的PCT专利公开号WO93/06121;还见美国专利申请系列号946,239,1992年9月16日提出和762,522,1991年9月18日提出,supra)。 (See article cited PCT Patent Publication No. WO93 / 06121; see also United States Patent Application Serial No. 946,239, September 16, 1992 and made 762,522, September 18, 1991 proposed, supra). 在此方法中,化学合成方法典型地为树脂小球上的组合合成的产物通过与合成中每一偶合或其它产物产生反应步骤一致将标志符标记与这些小球连接而被清楚地确定。 In this method, chemical synthesis typically produces a combination of the reaction step on the resin beads synthesized by synthesizing each of the coupling product or other product is clearly consistent with the determined identifier tag is connected with these pellets. 每一标记规定了感兴趣的反应步骤中发生的,例如哪一个氨基酸单体在肽合成方法中的某一特定的步骤中被偶合。 Each labeled step of reacting a predetermined interest in, for example, a particular coupling step in which a single amino acid in the peptide synthesis method. 在任意珠上的一个化合物的结构和鉴定,例如肽的序列可通过阅读小球上的标记组来推出。 Structure and Identification of a compound on any beads, for example, a peptide sequence may be introduced by the reading mark group pellets. 理想地,这些标记具有高信息含量,适合于非常高的灵敏度检测和译码以及对合成中采用的试剂稳定。 Ideally, these markers have a high information content, suitable for very high sensitivity detection and decoding, and the stability of the reagents employed in the synthesis. 寡核苷酸编码的化学合成的概念还由Brenner和Lerner提出过。 Concept of chemically synthesized oligonucleotides encoding is further proposed by the Brenner and Lerner. 见于本文所引用的1992,Proc.Natl.Acad.USA 89:5181-5183。 Found in the cited herein 1992, Proc.Natl.Acad.USA 89: 5181-5183.

采用编码方法表明首先从正交分化的二胺衍接物开始,平行组合合成法可用来产生一包括“粘合的”线和“编码”线的可溶性嵌合肽的库。 Using the coding method shows that starting with the diamine derivative differentiation Receiving orthogonal and parallel synthesis methods, may be used to generate a library including "bonded" line and a "coding" line of soluble chimeric peptides. 见于本文引作参考的Kerr et al.,1993,J.Amer.Chem.Soc.115:2529-2531。 Found herein incorporated by reference Kerr et al, 1993, J.Amer.Chem.Soc.115:. 2529-2531. 通过建立“编码”线上的四个L-氨基酸组成的氨基酸编码记录天然或天然氨基单体与粘合线的偶合。 Amino acids encoded by the establishment of "coding" line recording four amino acids L- coupling with native or naturally-amino monomers bond line. 通过受体亲和纯化从等摩尔的肽混合物中选择化合物,并通过HPLC进行分辨。 Compound selected from an equimolar peptide mixture and purified by affinity receptors, and resolved by HPLC. 通过爱德曼降解揭示粘合线的结构确定各纯化分子的编码线的序列。 Edelman Degradation by bond line structure determination revealed a sequence encoding the purified molecules each line. 类似的肽编码方法最近也由NiKolaiev等报道在1993年的肽研究6:161-170中。 Similar peptide coding method also study 6 in 1993 from the NiKolaiev peptide recently reported: 161-170 in.

对爱德曼方法的灵敏度和效率的限定将最终限制分析具有限差异的库的编码方法的这方面的范围。 Edelman sensitivity and efficiency of the method is defined to limit the scope of this aspect of the final analysis coding method having a current difference of the library. 寡核苷酸的利用提供了较大的希望,但仍需合成寡核苷酸标记分子库的改进方法。 Oligonucleotide provides a larger desired use, but still need improvement for nucleotides labeled oligonucleotide molecule library synthesis. 而且,仍需要合成和筛选非常大量的被标记分子库的替换方法。 Moreover, still we need to synthesize and screen a very large number of alternative methods are labeled molecule library.

期望在大量固体支持物如小球上合成一批不同的分子时,可出现另外的问题。 When the desired solid support such as a large number of pellets from a batch of different molecules, additional problems may occur. 使用带有在其上合成的不同的分子产物的小球的例子公开在被本文引作参考的例如下面的申请中,它们是1992年4月29日提出的美国申请系列号为07/876,792;1991年9月18日提出的申请系列号为07/762,522和1992年9月16日提出的申请系列号为07/946,239中。 With the use of pellets in which the synthetic product of different molecules, for example, an example is disclosed in the following application is incorporated by reference herein, which is April 29, 1992 proposed by U.S. Application Serial No. 07 / 876,792; September 18, 1991 submitted application Serial No. 07 / 762,522 and 16 September 1992 submitted application Serial No. 07 / 946,239.

虽然获得了相当大的成功,上述技术仍遇到了一定的限制。 Although gained considerable success, the above techniques are still experiencing a certain limit. 例如,不同产物的合成在小球上开始时,对这些小球的许多人工控制变为必需。 For example, the synthesis of different products on the ball start, many of these manual control pellets becomes necessary. 例如在1992年4月29日提出的被本文引作参考的美国申请系列号07/876,792中,必须在载体中悬挂一批小球将小球分开,完成分开组的小球上的单体添加反应,有时再分开和有选择性重组如此合成的小球混合这些重组的小球,并且重复此过程。 For example, in April 29, 1992 presented herein incorporated by reference US Application Serial No. 07 / 876,792, to be hung in a number of small ball carrier to separate the ball, completion of the monomer on the separate group of small ball added the reaction, sometimes separated again and selective recombinant beads thus synthesized recombinant mixing these pellets, the process is repeated. 当包括大量单体和反应包括许多单体添加步骤时,人工操作变得极为乏味。 When included a large number of monomers and monomer addition reaction involves many steps, it becomes extremely tedious manual operation. 除此,“计量”合成的许多产物成为一使人胆怯的任务。 In addition, many products of "metering" synthetic become a people timid task.

从上可见,合成和筛选非常大量的标记的分子库的改进方法和设备为人们期望。 It is seen from the above, synthesis and screening of very large numbers of labeled improved method and apparatus for the expected molecular libraries. 本发明满足了这些以及其它的需要。 The present invention satisfies these and other needs.

本发明提供了制备和筛选分子库的改进方法,其中库中各分子用特定的,容易译码的标定符标记。 The present invention provides an improved process for preparing and screening libraries of molecules wherein each molecule in the library with a labeled specific calibration symbol easily decoded. 还提供了一种快速有效地合成不同分子产物的设备和方法。 Also provides an apparatus and process for the synthesis of different molecules product quickly and efficiently.

在一个实施例中,本发明提供了将组合化学方法的产物标记到结构被编码的合成化学库上的方法和试剂。 In one embodiment, the present invention provides a combination product of a chemical process to methods and reagents marking structure is encoded on a synthetic chemical libraries. 在一个重要的实施例中,本发明提供了一种通过交替和相容的合成方法在细微的小球上完成肽和寡核苷酸的合成的方法。 In one important embodiment, the present invention provides a method of synthesis and synthetic methods compatible by alternately completion peptides and oligonucleotides in the fine beads. 由这种组合合成方法制备的大量寡核苷酸编码的合成肽库由许多小球组成,每个库中包含简单肽(具确定顺序)和序列人为明显地编码关联肽的结构的单链DNA标记的许多拷贝。 A large number of synthetic oligonucleotides encoding the peptide library prepared from such compositions by the synthetic methods of many small balls, each library contains a simple peptide (having the sequence determination) and the sequence encoding the artificial single-stranded DNA significantly associated peptide structure many copy mark. 可有效地提出有关库与通过流动血细胞计数得到的荧光标记的生物受体和通过利用FACS设备分类单个小球的能而选择出来的各小球之间的相互作用的问题。 Effective to questions related to the interaction between the library by flow cytometry with a fluorescent marker obtained by using a biological receptor and FACS Classified single pellet can be selected out of each pellet. 在分类的小球上的DNA标记通过PCR技术放大和测序来确定被编码的肽配体的结构。 DNA markers on the classification of the pellets to determine the structure of the peptide ligand is encoded by PCR amplification and sequencing techniques. 这些库可用来,例如寻找受体的高亲和性(毫微克分子)配体如抗-肽单克隆。 These libraries can be used, for example, to find high-affinity receptors (nanomoles) ligands such as anti - peptide monoclonal.

本发明的合成分子库可通过在大量固体载体的每一个上合成化合物来制备,该化合物由于不同的固体载体而不同。 Synthesis of molecule libraries according to the present invention can be prepared by each of the synthesized compound, which due to different solid supports and solid support in a different lot. 用包括下列步骤的方法制备化合物:(a)在多种反应容器中以随机方式均分载体;(b)将每一反应容器中的载体暴露于第一化学结构单元中;(c)将载体汇集起来;(d)在许多反应容器中以随机方式均分载体;(e)将每一容器中的载体暴露于化学结构单元中;和(f)至少重复步骤(a)至(e)1至20次。 Compound prepared by the method comprising the steps of: (a) a reaction vessel in a more random manner average vector; (b) each of the reaction vessel exposed to the first carrier in the chemical structural unit; (c) The carrier pooled; (d) in the reaction vessel to a number average random vector; (e) each container is exposed to the chemical structure of the support units; and (f) repeating at least steps (a) to (e) 1 to 20 times. 典型地,大体上等量的固体载体将被均分至每一反应容器。 Typically, substantially the same amount of solid carrier will be divided equally to each of the reaction vessel. 在本方法的一个实施例中,化学结构单元从氨基酸组中选择,得到的化合物为肽低聚物。 In one embodiment of the method, the chemical structure of an amino acid unit selected from the group, the compound obtained is a peptide oligomer.

更具体地,本发明涉及对与这些方法相关的偶合化学一些改进。 More particularly, the present invention relates to some improvements associated with these coupling chemistry methods. 其中之一的改进涉及用于从这些合成中的小球,接头或生长肽链的α-氨基中去除Fmoc保护基的化学。 One of them relates to improved chemical removal of the Fmoc protecting group from the α- amino group in the synthesis of these pellets, or linker peptide chain growing. 优选地,该去除作用通过用5%一15%,优选为10%的哌啶处理5至60分钟,优选为5至10分钟的而变得有效,虽然可采用其它条件如15%至30%的哌啶处理5至30分钟。 Preferably, the action is removed by treatment with a 5% to 15%, preferably 10% piperidine for 5 to 60 minutes, preferably 5 to 10 minutes becomes active, although other conditions may be employed, such as 15-30% piperidine for 5 to 30 minutes. 其它改进涉及肽偶合反应的活性化学,因为当某些自动化设备被用于完成寡核苷酸标记的肽库的合成时,本发明提供了一减少试剂供应瓶的HoBt/HBTU的简单混合物。 Other improvements relate to active chemical peptide coupling reaction, as when the synthetic peptide library is used to perform some automated equipment labeled oligonucleotide, the present invention provides a simple mixture of a reducing agent supply bottle HoBt / HBTU in.

在其它方面,本发明涉及合成标记的分子库的方法,其中库中的每一分子与固体载体共价连接并用一个或多个不同化学惰性烃标记物标记。 In other aspects, the present invention relates to a method of synthesizing libraries of molecules labeled solid support wherein each molecule in the library are covalently connected labeled with one or more different chemically inert hydrocarbon markers. 其中所述标记物包括可变烃区域和分子钩。 Wherein the marker comprises a variable region and a hydrocarbon molecular hook. 优选地,该标记物包括将该标记物与固体载体相连的可裂解的接头,分子钩和将分子钩与可裂解接头相连的具可变长度的烃链。 Preferably, the marker comprises the variable cleavable linker attached to a solid support with a marker molecule and a molecular hook with a hook having a cleavable linker attached to the hydrocarbon chain length. 更优选为其中所述标记物包括下式的那些例子: More preferably wherein the marker examples include those of the formula: 其中n为1至10或更大,x为可裂解的接头,R为分子钩。 Wherein n is 1 to 10 or more, x is a cleavable linker, R is a molecular hook. 这些分子钩优选地选自生物素,高缔合肽对的补体和被保护的能被活化的基团,如能被光活化的基团。 The molecular hook is preferably selected from biotin, complement-associated peptide highly protected and can be activated groups, such as groups that can be photoactivated. 优选的可裂解的接头可被光裂解的接头。 Preferred cleavable linker may be a photocleavable linker.

还提供了检测这些化学惰性烃标记物存在的方法。 Also it provides methods for detecting these markers chemically inert hydrocarbon present. 该方法包括从固体载体中裂解出一个或多个不同标记物,接着将标记物固定在第二固体载体上。 The method cleaved from the solid support comprising one or a plurality of different markers, then the marker immobilized on a second solid support. 接着将寡核苷酸序列放大,检测它的存在,其中寡核苷酸序列的出现与否是标记物出现与否的标志。 The oligonucleotide sequence is then amplified, its presence is detected, wherein the occurrence or absence of the oligonucleotide sequence is a marker of the presence or absence of signs.

本发明的另一实施方案提供了一种确定了被标记分子库中的固体载体相连的合成分子的合成步骤的顺序的方法,其中库中的分子用惰性烃标签通过化学方法标记。 Another embodiment of the present invention provides a method of determining the order of the synthetic steps of the synthetic molecule is labeled molecule attached to a solid support in the library, wherein the library of tag molecules labeled with an inert hydrocarbon by chemical means. 该方法包括分别检测所述固体载体上的一个或多个不同标记的存在和作为所述分子合成中的出现特殊合成步骤的标志的所述各标记的存在或不存在情况。 The method includes detecting one or each of a plurality of different markers present on the solid support and the presence of the flag as a specific synthetic steps of the synthetic molecule in each occurrence or absence of markers.

另一方面,本发明涉及在小球上合成太小不能在常规流动血细胞计数设备上分离的被编码的合成化学库的方法和设备。 Another aspect, the present invention relates to a method and apparatus for synthesizing chemical library is encoded in the synthesis of small beads can not be separated in a conventional flow cytometer apparatus. 这些小球使得到的库的大小从没有小球的库的109个增至1013,对于有小球的库可达1018大小。 These small balls the size of the library to get the ball from the library no increase of 109 1013, a small library for the ball up to 1018 size. 本发明还涉及筛选这些库的方法。 The present invention further relates to methods of screening these libraries.

本发明还涉及筛选被编码的合成库以确定有用化合物的方法。 The present invention further relates to encoded libraries of synthetic screened to determine useful compounds. 一个重要的方面,本发明提供了与制备,标记,筛选自然产物库以区别和鉴定具有用活性的化合物的方法有关在自然产物筛选领域中的重要改进。 An important aspect, the present invention provides the preparation, marker, screening of natural product libraries to distinguish and identify compounds having activity related to significant improvements in screening methods used in the field of natural products.

另一方面,本发明涉及一种从本发明的分子库快速有效鉴定库中化合物的改进方法。 Another aspect, the present invention relates to an improved method for rapid and effective identification of library molecules of the library from the compound of the present invention. 在该方法中,将显示所需特性(如与受体结合)来自被标记化合物库的寡核苷酸标记连环和克隆以便于简单序列测定反应中的大量标记的序列测定。 In this method, the desired characteristics of the display sequence determination reaction in order to determine a large number of simple marker sequence (e.g., receptor binding) of the compound library is labeled oligonucleotide tag chain and cloning. 如果被标记的化合物是肽,采用了建立在遗传密码基础上的编码图,那么为了对肽进一步地分析可将来自多联体的各标记亚克隆至其它选择和表达系统如在上述背景部分描述的质粒和噬菌体系统。 If the compound is labeled peptide is used to establish a password based on the genetic code of FIG, then in order to further analyze the peptide from each marker may concatemers selected and subcloned into other expression systems as described in the Background section above plasmids and phage systems.

在另一实施方案中,本发明提供了快速有效合成不同分子产物的装置和方法。 In another embodiment, the present invention provides an apparatus and method for quickly and efficiently different molecules synthesized product. 根据本发明的特殊方面,在如小球的底物中合成不同的聚合物。 According to particular aspects of the invention, the substrate such as pellets of different synthetic polymers. 选择性地,这些小球在与分子标记的合成反应中同时被“标记”,仅通过实施例,在小球上的合成分子可包括肽,而分子标记可包括寡核苷酸。 Alternatively, the pellets simultaneously are "marked" with markers in the synthesis reaction, only by way of example, the synthesis of molecules on beads may include peptides, and may include a marker oligonucleotide. 当然,无论何时分子具有与其它有关分子一致的基本“结构单元”,其它分子产物同样可用本文描述的方法合成。 Of course, whenever a molecule having molecular consistent with other relevant basic "building blocks", product of other molecular methods described herein likewise be synthesized. 实施例包括2,3-苯并二嗪,前列腺素和β-转角拟晶。 Examples include 2,3-diazine, prostaglandins, and quasi crystalline β- corner.

根据本发明的一个实施方案,用透明容器混合小球悬浮体。 According to one embodiment of the present invention, the mixed pellet suspension transparent container. 通过普通的多歧管将混合的小球分配于多个独立的反应容器中,在这些反应容器中,将小球暴露于不同的被选择的单体之中,这些单体在小球上反应并优选地通过共价键偶合至其上。 By a conventional multi-manifold mixing pellets dispensed in a plurality of separate reaction vessel, a reaction vessel in which the pellets are exposed to different selected monomers which react on the ball and preferably coupled thereto by a covalent bond. 这些小球可选择性地暴露于同样共价或通其他方法与这些小球偶合的化学“标记物”之中。 These pellets may be selectively exposed to the same or by covalent coupling with these other methods of chemical pellets "marker." 接着通过多歧管将这些小球重新组合至透明容器中并混合。 The tube is then re-combination of these pellets to the transparent container and mixed by a multi-manifold. 接着在多个反应容器中将混合小球悬浮体再分开,继续添加单体,混合小球和重新分配等过程。 Then a reaction vessel mixed pellet suspension then separated, monomers continue to add, mix pellet and re-allocation process in a plurality. 这个方法导致了一批带有在其表面形成的不同组分子的小球或其它底物的形成。 This process results in the formation of a group with a different set of molecules formed on the surface of pellets or other substrates.

根据本发明的一个方面,本发明包括一种在底物上合成不同分子的设备和方法。 According to one aspect of the present invention, the present invention includes an apparatus and method for synthesizing different molecules on a substrate. 底物被从一透明容器中分配至选择的反应容器中。 The substrate is dispensed from a transparent container to a selected reaction vessel. 接着将试剂加入至反应容器中以合成一部分分子。 The reagent is then added to the reaction vessel to synthesize part of the molecule. 接着将底物移至透明容器中混合。 The substrate is then moved to the transparent container and mixed. 为了进一步合成,将底物重新分配至反应容器中。 For further synthesis, the substrate to redistribute to the reaction vessel. 继续这个操作过程直到合成了所需一组的分子。 This operation continues until the desired synthesized molecule a group. 在合成期间,整个合成器与外界环境密封。 During the synthesis, the overall synthesis is sealed from the outside environment.

广义上来说,本发明提供了制备和筛选分子库的设备和方法,其中库中的各分子用特定,容易译码的识别标记物。 Broadly speaking, the present invention provides an apparatus and method for preparing and screening libraries of molecules, wherein each molecule in the library with a specific, easily coded identification markers.

对本发明实质和优点的进一步理解可参考下面的描述和图。 Further understanding of the nature and advantages of the present invention may be reference to the following description and drawings.

图1表示本发明合成器的示意图;图2表示试剂储蓄器的示意图;图3表示用于合成器中的3-汽门阀;图4表示用于合成器中的2-汽门阀;图6A表示具有一支撑多组试剂储蓄器的可旋转圆盘传送带的反应容器的工作面的可替换设备;图7表示较低多歧管阀,注射阀,试剂储器和反应容器之间的相互连接; 1 shows a schematic diagram of the synthesizer of the present invention; FIG. 2 shows a schematic diagram of the reagent reservoir; FIG. 3 shows a steam gate valve 3- synthesizer; FIG. 4 shows a steam gate valve 2- synthesizer; FIG. 6A shows Alternatively the reaction vessel apparatus having a working surface supporting a plurality of sets of the reagent reservoir of a rotatable carousel; FIG. 7 shows the interconnections between the multiple lower manifold valve, the injection valves, reagent reservoirs and the reaction vessel;

图8表示同尽圆搅拌器;图9表示反应容器工作面的顶部;图10A和10B表示-较低的反应容器固定夹;图11A-11D表示根据本发明的一个方面的利用光传感器来检测大致上为半透明的管中的液体的存在的光校直装置;图12表示根据本发明的一个方面的反应容器;图12A表示在图12的反应容器周围的温度控制套;图12B表示图12A中的套和容器的横截面图;图12C表示具有温度控制套的反应容器的可供选择的实施方案;图12D表示图12C中的套和容器的横截面图;图13表示透明容器;图14是控制合成器的电子设备的简图;图15表示控制电路的简图;图16表示被控制计算机采用来排出反应器中所有液体的步骤;图17表示被控制计算机采用来清洗底部歧管的特质步骤;图18表示被控制计算机采用搅拌透明容器中的内容物的步骤;图19A至19D表示被控制计算机采用将小球悬浮体以透明容 8 shows the same circle do a stirrer; FIG. 9 shows a top face of the reaction vessel; FIGS. 10A and 10B show - the lower reaction vessel fixing clip; FIGS. 11A-11D represent detected by the optical sensor according to an aspect of the present invention. substantially correction light translucent liquid present in a straight tube apparatus; FIG. 12 shows a reaction vessel, according to one aspect of the present invention; FIG. 12A showing around the reactor vessel 12 in FIG temperature control sleeve; FIG. 12B is a 12A is a cross sectional view of the sleeve and container; FIG. 12C shows an alternative embodiment having a temperature control jacket of the reaction vessel; 12D showing a cross-sectional view of FIG. 12C sleeve and container; FIG. 13 shows a transparent container; FIG 14 is a diagram of the electronic control apparatus synthesizer; a control circuit diagram of FIG. 15; FIG. 16 shows the control computer is employed to discharge all the liquid in the reactor of step; FIG. 17 shows the control computer is employed to clean the bottom of the manifold characteristics step tube; FIG. 18 shows the steps employed by the control computer transparent stirred vessel contents; 19A to 19D represents the control computer uses the pellet suspension transparent container 再分配至反应容器的步骤;图20表示被控制计算机采用将来自加压运送系统的试剂装入反应容器的步骤;图21A-21D示意说明被控制计算机采用将来自加压运送系统的试剂装入反应容器的步骤;图22表示被控制计算机采用将氨基酸单体加入至反应容器中的步骤;图23A-23C示意说明被控制计算机采用将氨基酸单体加入至反应容器中的步骤;图24表示被控制计算机采用将小球悬浮体从反应容器转移至透明容器中混合的步骤;图25示意说明控制软件的主要组件的数据变动;图26示意说明命令译码结构;图27示意说明用于获得阀门数据的示意图;和图28示意说明用于获得传感器数据的示意图。 Redistribution step to the reaction vessel; FIG. 20 shows the step of using a computer controlled agent delivery system from the pressurized reaction vessel is charged; FIG. 21A-21D is a schematic illustration of the control computer uses the reagent from the pressurized delivery system was charged the step of the reaction vessel; FIG. 22 represents the step of using the control computer amino acid monomers added to the reaction vessel; FIGS. 23A-23C schematically illustrate the use of the control computer is the amino acid monomer is added to the reaction vessel in step; FIG. 24 represents the step of using the control computer pellet suspension were transferred from the reaction vessel to the mixing of a transparent container; FIG. 25 schematically illustrates the data change control software main assembly; FIG. 26 schematically illustrates the structure of the command decoder; FIG. 27 schematically illustrates the valve for obtaining a schematic view of the data; and FIG. 28 schematically illustrates a schematic view for obtaining sensor data.

图29表示在细微小球上合成组合化学库的设备。 29 shows the device assembled on the fine synthetic chemical library beads. 该设备由一真空歧管或与固体载体相连的磁板组成,其中固体载体有一具一系列合成化合物的反应位点的合成表面。 The equipment consists of a vacuum manifold connected to a magnetic plate or a tube composed of a solid support, wherein the solid support surface a synthetic reaction site of a series of synthetic compounds. 分隔板由一系列与所述反应位点对应的反应孔组成,并且在每一次混合步骤之后它被用来划分库的成员。 The partition plate by a series of wells with the reaction site corresponding to the composition and mixing in each step after it has been used to divide the library members. 该设备还可用来辅助标记化学库的合成。 The apparatus may also be used for the synthesis of chemical libraries assisted tag.

图30说明监测噻唑烷稳定性的13CNMR的使用。 Figure 30 illustrates the use of thiazolidine 13CNMR monitoring stability. Panel C表示载体结合的噻唑烷的13CNMR谱,其中噻唑烷在环的2位和对于接头碳基为α位(被标记的位置用“*”表示)上被13C双标记。 Panel C represents the 13CNMR spectrum thiazolidine bound carrier, wherein the bis thiazolidine 13C is marked to the joint and two carbon group is α position (a position labeled with "*") of the ring. Panel B表示在用95%TFA处理1小时后的载体结合的双标记噻唑烷的13CNMR光谱。 Panel B represents 13CNMR spectrum of doubly labeled thiazolidine after 1 hour with 95% TFA support bound. Panel A表示在40个周期的DNA合成之后载体结合的双标记噻唑烷的13CN-MR谱。 Panel A represents the carrier-bound DNA synthesis after 40 cycles of a dual-labeled thiazolidine 13CN-MR spectrum.

图31进一步说明监测噻唑烷稳定性的13CNMR的使用。 FIG 31 is further described using 13CNMR monitoring thiazolidine stability. Panel C表示在环的α位和对于接头羰基为α位上(标记位置用“*”表示)用13原子进行双标记的载体结合的噻唑烷的13CNMR谱。 Panel C represents the α-position and the carbonyl group to the linker thiazolidine doubly labeled with carrier-bound atom 13 (position marked with "*") of the position α 13CNMR spectrum of a ring. Panel B表示在PBS缓冲液中光解90分钟后的载体结合的双标记的噻唑烷的13CNMR谱。 Panel B represents photolysis thiazolidine 13CNMR spectrum of doubly labeled support-bound after 90 minutes in PBS buffer. Panel A表示在PBS缓冲液中光解3小时后的载体结合的双标记的噻唑烷的13CNMR谱。 Panel A represents photolysis thiazolidine 13CNMR spectrum of doubly labeled support bound after 3 hours in PBS buffer.

图32表示通过将载体结合的噻唑烷经过40个周期的DNA合成和在PBS缓冲液中3小时的光解而制备的反应混合物的HPLC追踪。 32 shows prepared by photolysis of 3 hours in PBS buffer after 40 cycles of DNA synthesis by binding thiazolidine carrier HPLC and reaction mixture tracking.

图33说明被提供作为控制计算机上的图示的操作者连系装置(“GUI”)。 FIG 33 is provided as a control operator icon on the computer associated with means ( "GUI") described below. 如图所示,GUI包括带有一工作空间1303的矩形窗口。 As shown, GUI 1303 includes a working space with a rectangular window. 在窗口的顶部是带有使用者命令选择1306-1313的菜单区域1305。 At the top of the window is a menu selection commands with user's region 13051306-1313. 每一命令选择包括控制合成器操作的辅助的下一级菜单。 Each includes a control command selection operation of the auxiliary synthesizer lower menu. 使用者可通过用鼠标器选择适宜的命令选择来操作。 The user can be selected by selecting the appropriate mouse commands.

图34描述了GUI,显示大菜单中的下一级菜单选择。 34 depicts a GUI, displaying a menu selection menu under a large.

图35描述了运行大菜单的对话箱。 Figure 35 depicts the dialogue box running large menu.

图36描述了GUI,显示在组菜单中的下一级菜单选择。 FIG 36 depicts a GUI, displayed in the menu selection in a menu group.

图37描述了GUI,显示在可变菜单中的下一级菜单选择。 FIG 37 depicts a GUI, displayed in the menu selection menu under a variable.

图38描述了GUI,显示在诊断菜单中的下一级菜单选择。 FIG 38 depicts GUI, displayed in the menu selection menu under a diagnosis.

图39描述了阀诊断屏幕。 FIG 39 depicts valve diagnostic screen.

图40描述了传感器诊断屏幕。 FIG 40 describes sensor diagnostic screen.

图41描述了GUI,显示文件菜单中的下一级菜单选择。 FIG 41 describes a GUI, select the File menu in the menu display of the next stage.

图42-44表示启动合成的对话箱,它允许使用者选择用于合成中的反应容器(图42),选择每一个反应容器中的开始,循环和结束宏命令(图43)以及输入氨基酸符号和寡核苷酸密码(图44)。 42-44 dialogue box showing the synthetic promoter, which allows a user to select a reaction vessel (FIG. 42) Synthesis, selecting the beginning of each reaction vessel, and the end of the cycle macros (FIG. 43) and the input symbols amino acid and oligonucleotide password (FIG. 44).

图45描述了GUI,显示在合成菜单中的下一级菜单选择。 FIG 45 depicts a GUI, displayed in the menu selection menu under a synthesis.

图46描述了在合成或宏命令气执行期间出现的情况。 FIG 46 describes what occurs during synthesis gas or macro command execution.

图47描述了使用者故障对话箱。 FIG 47 describes the user dialogue box failure.

图48描述了GUI,显示在编辑菜单中的下一级菜单选择。 FIG 48 describes the GUI, displayed in the menu selection in an Edit menu.

图49示意说明在Window环境下的控制软件的主要组件中的数据流量。 FIG 49 schematically illustrates the data flow in the control software of the main components in the Window environment.

本发明一般涉及制备和筛选标记化学库的改进方法。 The present invention generally relates to an improved process for the preparation of chemical libraries and screening marker. 本发明还涉及一种在一批不同分子如上述标记化学库的合成中有用的设备。 The present invention also relates to a number of different molecules useful as synthetic chemical libraries of the marker device.

为了理解改进方法的意义,必须理解不仅制备和使用的标记库的基本工艺,而且各种合成和筛选步骤如何相互配合以及如何选择试剂都影响所获得的结果。 To understand the significance of the improved process, it must be understood not only the basic process tag library preparation and use, and how the various synthetic and screening steps cooperate with each other and how to select agents affect the results obtained. 标记化学库通常在固体载体上合成,载体和接头的选择是成功的关键。 Tag chemical libraries typically synthesized on a solid support, the support and the joint selection is critical to success. 接头可用来将载体与标记物相连,将载体与库分子相连或在没有固体载体的情况下,将标记物与库分子相连。 Linker can be used to connect the carrier and the marker, is connected to the carrier molecule or the library without a solid support, the library is connected to the marker molecule. 与化学结构单元,标记物和合成方法有关的选择可以是同样关键,而且还受可得到的固体载体和接头的特性的影响。 Chemical structural unit, markers and the method of synthesis of the key selection may be the same, but also by the characteristics of the solid support and linker available. 对这些标记库的分析和应用同样影响这些选择,以及可得到的设备和试剂。 Application of these markers and analysis of libraries also affects these choices, as well as available equipment and reagents.

虽然本发明的设备和方法主要根据寡核苷酸和肽的合成来进行说明,但本发明不限于此。 While the apparatus and method of the present invention is mainly based on synthetic oligonucleotides and peptides to be described, but the present invention is not limited thereto. 本发明将寻找在如多糖类,磷脂,聚氨基甲酸乙酯,2,3-苯并二嗪,前列腺素和β-转角拟晶和其它物质的合成中的应用。 The present invention will look as polysaccharides, phospholipids, polyurethane, 2,3-diazine, prostaglandins and β- angle quasi crystals and applications in the synthesis of other substances. 如本文引作参考的美国专利号5,242,974中(Holmes)所公开,可形成环状物质。 As used herein incorporated by reference in U.S. Patent No. 5,242,974 (Holmes) disclosed, may form a cyclic species.

如寡核苷酸和肽的不同物质的使用和合成进一步详细地公开在下列正在审查中的申请中,它们均被本文引作参考:1992年4月29日提出的美国申请系列号07/876,792,1991年9月1 8日提出的美国申请系列号07/762,522和1992年9月16日提出的美国申请系列号07/946,239。 Such as the use of different materials and synthetic oligonucleotides and peptides disclosed in further detail below under review the application, which are incorporated herein by reference: April 29, 1992 raised US Application Serial No. 07 / 876,792 US application Serial No. 07 / 762,522 and 16 September 1992 raised US application Serial No. 18 September 1991 raised 07 / 946,239.

通过下表所示提供本发明的说明。 The table provided by the present invention described in FIG.

I、标记化学库的合成概述II、固体载体A.类型B.接头C.分子载体III、化学结构单元A.寡聚物和单体B.其它结构单元IV、标记物 I, a synthetic chemical libraries outlined tag II, the solid support type A. B. C. linker molecule carrier III, the chemical structure of the monomer unit oligomers and A. B. Other structures of moiety IV, marker

V、合成方法A.寡核苷酸标记肽库B.合成寡核苷酸-标记的肽库的改进方法C.小分子的合成D.制备可溶性库的方法VI、分析方法A.小球库的筛选分析B.筛选可溶性分子C.筛选天然产物库VII、设备和试剂VIII、平行偶合合成反应设备实施例概述结束除了上面概念,提供下面的术语以便于本发发明的描述,一些缩写和术语被规定具有普遍含义被本文用于描述本发明。 V, synthesis of oligonucleotides labeled peptide libraries A. B. Oligonucleotide Synthesis - Method C. Improved method for preparing a soluble library VI D. Synthesis of small molecules, labeled A. Analysis peptide library beads Library filter screening assays B. C. soluble molecule screening natural product libraries VII, VIII equipment and reagents, coupling parallel synthesis reaction apparatus embodiment except the above concepts outlined end, following terms are provided to facilitate the description of the present invention, a number of terms and abbreviations the present invention is defined having ordinary meaning used herein are described.

缩写:HBTV,O-(苯并三唑-1-基)-1,1,3,3-四甲基糖醛六氟磷酸酯;HOBt,1-羟基苯并三唑;HATU,[O-(7-氮杂苯并三唑-1-基)-1,1,3,3-四甲基糖醛六氟磷酸酯;TFA,三氟乙酸;TCA,三氯乙酸;DIEA,二异丙基乙胺;DMF,二甲基甲酰胺;Fmoc,9-芴基甲氧羰基;DMT,二对甲氧三苯甲基;Trt,三苯甲基;Bz,苯甲酰基;Pmc,2,2,5,7,8-五甲基苯并二氢吡喃-6-磺酰;tBoC,叔丁基氧羰基;PBS,磷酸盐缓冲液;BSA,牛血清清蛋白;mAb,单克隆抗体。 Abbreviations: HBTV, O- (benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate uronic; HOBt, 1- hydroxybenzotriazole; HATU, [O- (7-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate uronic; TFA, trifluoroacetic acid; TCA, trichloroacetic acid; DIEA, diisopropylethylamine amine; DMF, dimethyl formamide; Fmoc, 9- fluorenyl methoxycarbonyl; DMT, two pairs of dimethoxytrityl; Trt, trityl; Bz, benzoyl; Pmc, 2, 2,5,7,8-pentamethyl-chroman-6-sulfonyl; tBoC, t-butyloxycarbonyl group; PBS, phosphate buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody .

互补或本质上互补:这些术语指一个化合物与另一个结合的能力,例如配体与它互补受体的结合。 Complementary or essentially complementary: These terms refer to the ability to bind a compound with another, such as a ligand binding to its complementary receptor. 典型地,这些术语用在核酸的核苷酸之间的碱基配对的描述中,例如,双链DNA分子的两条链之间寡核苷酸引物与要测序或扩增的单链核酸上的引物结合位点之间。 Typically, these terms are described by nucleotides between the nucleic acid base pairing, for example, single-stranded nucleic acid molecules between the two strands of a double stranded DNA oligonucleotide primers to be sequenced or amplified between the primer binding site. 互补核苷酸一般为AT(或AU),CG,对于本领域普通技术人员熟知的具结合特性的合成或修饰核苷酸还存在一个广泛的变异。 Complementary nucleotide typically the AT (or AU), CG, those of ordinary skill having the binding properties of known synthetic or modified nucleotides there is a wide variation. “本质上互补”存在于一条RNA或DNA链在杂交条件下杂交至互补核酸中时。 "Essentially complementary" DNA or RNA present in a nucleic acid strand under hybridization conditions to a complementary. 典型地,杂交发生在一条至少有14至25个核苷酸的链存在至少约55%互补性时,但高选择性的杂交将发生在当互补性增加至65%,75%,90%和100%时。 When Typically, hybridization occurs in a presence of at least about 55% complementarity of at least 14 to 25 nucleotides strand, but highly selective hybridization will occur as complementarity increases to 65%, 75%, 90%, and 100%. 见于本文引作参考的Nucl.Acids.Res.12:203,1984。 Found herein incorporated by reference Nucl.Acids.Res.12: 203,1984. 高选择性杂交条件已知为“严紧杂交条件”,定义如下。 Highly selective hybridization conditions known as "stringent hybridization conditions", as defined below.

表位:这个术语用来描述由与已知为抗体的受体亚类相互作用的区域来描述的抗原分子的一部分。 Epitope: term used to describe the interaction region part of the antigen molecule from the receptor is an antibody subclass known to be described.

识别标记物:在最普遍的意义中,这个术语用来表示一种物理特性,它提供一种可识别化学反应的方法,例如在固体载体上的寡聚体合成中一个单独的固体载体经历的单体加成反应。 Identification markers: In the most general sense, the term used to denote one physical property, it may be provided a method of identifying a chemical reaction, for example, oligomer synthesis on a solid support in a single solid support experiences monomer addition reaction. 识别标记物用来记录用于一个化学库的合成中的一系列反应的步骤。 A step for identifying markers for the synthesis of a series of chemical reactions in the library record. 该识别标记物可有任何可识别的特性,包括例如:用显微镜或其它方式可辩别的形状,大小,质量,颜色,光密度等;微分吸光度或光发射;化学反应速度;磁或电学特性;或任何其它能编码所需信息,在一个(或一些)分子水平可翻译出来的鉴别性标记。 The identification marker may have any identifiable characteristic, for example, comprising: a microscope or otherwise discernable shape, size, mass, color, optical density; differential absorbance or emission of light; chemical reaction rate; magnetic or electrical properties ; or any other one (or few) molecular level can be translated identification marker can encode desired information. 识别标记物的优选为寡核苷酸,因为寡核苷酸的核苷酸顺序是被编码信息的坚固形式。 Preferably the identification marker to an oligonucleotide, nucleotide sequence as the oligonucleotide is solid form of encoded information. 无论合成中是否使用固体载体,“识别标记物”可直接偶联到合成的寡聚体上。 Whether or not a solid support synthesis, the "identification marker" may be directly conjugated to a synthetic oligomer. 在后面的实施例中,识别标记物可从概念上认为同样起到寡聚体合成的“载体”的作用。 In the latter embodiment, the identification label can be considered the same function as the synthesis of oligomers "vector" conceptually.

配体:这个术语用来表示典型地通过与一特定受体结合来识别的分子。 Ligand: This term is used to identify the molecule represented typically by binding to a particular receptor. 与受体结合或反应的介质标为“配体”,它只有就对应的受体而论才有明确意义。 Marking Media binding to the receptor or reaction as "ligand", it is only on the terms of the corresponding receptors have a clear meaning. 只要所研究的物质能结合或通过其它方式与受体反应,术语“配体”不包括任何特定的分子大小或其它结构或组成特征。 As long as the study substance capable of binding to the receptor, or by other means the reaction, the term "ligand" does not include any particular molecular size or other structural characteristics or composition. 而且,配体“可作为与受体结合的天然配体,或作为兴奋剂或拮抗剂的功能性类似物。通过本发明可试验的配体包括,但不限于此,细胞膜受体的兴奋剂,拮抗剂,毒素和毒液,病毒表位,激素,糖,辅因子,肽,酶底物,辅因子,药物(如麻醉剂,甾族化合物等)和蛋白质。 Furthermore, ligand "as a natural ligand binding to the receptor as agonists or antagonists, or functional analogue by the present invention can be tested ligands include, but are not limited to, agonists of cell membrane receptors , antagonists, toxins and venoms, viral epitopes, hormones, sugars, cofactors, peptides, enzyme substrates, cofactors, drugs (such as anesthetics, steroids, etc.) and proteins.

单体:这个术语用来表示可被连接在一起形成其它分子或分子组的一组分子的任何成员,例如一组寡聚体或聚合物。 Monomer: This term may be used to indicate any member joined together to form a group of molecules of another molecule or group, e.g. a group of oligomers or polymers. 本发明中有用的单体组,但不限于此,例如对于肽的合成,包括L-氨基酸,D-氨基酸或合成氨基酸。 The present invention is useful in the monomer, but is not limited thereto, for example, synthetic peptides, including L- amino acids, D- amino acids or synthetic amino acids. 本文所用的单体指一个寡聚体合成基本组的任何成员。 As used herein, monomers refers to any member of a basis set of oligomer synthesis. 例如,L-氨基酸的二聚体组成用于多肽合成的400“单体”的一个基本组。 For example, L- amino acid dimer of 400 "monomers" for synthesis of a polypeptide substantially set. 不同的单体基本组可用在聚合物合成中的连续步骤中。 Substantially different monomer groups can be used in successive steps in the synthesis of polymers. 本领域的技术人员将认识到“单体”仅是一类“化学结构单元”,任何类型的化学结构单元可用于本发明中,不管是否正在合成寡聚体或小有机分子或一些其它分子。 Those skilled in the art will recognize that the "monomer" is only one class of "chemical building blocks", any type of chemical building blocks can be used in the present invention, regardless of whether it is synthetic oligomer or a small organic molecule, or some other molecule.

寡聚体或聚合物:这些术语用来表示通过涉及单体亚基的化学或酶加成的方法形成的分子。 Oligomers or polymers: these terms are molecules formed by a method involving chemical or enzymatic subunit monomers used to represent addition. 如寡聚体包括例如核酸,多糖和磷脂的线性,环状和分枝聚合物的及具有α,β或ω-氨基酸的肽,杂聚物,聚氨基甲酸乙酯,聚酯,聚碳酸酯,聚脲,聚酰胺,聚乙烯亚胺,多芳基烯硫醚,聚硅氧烷,聚酰亚胺,聚乙酸酯或其它聚合物,根据本发明公开,它们对于本领域技术人员是显而易见的。 The oligomer includes, for example, and a peptide having α, β ω- amino or nucleic acid, polysaccharides and phospholipids linear, cyclic, and branched polymers, heteropolymers, polyurethanes, polyesters, polycarbonates , polyureas, polyamides, polyethyleneimines, polyaryl alkenyl sulfides, polysiloxanes, polyimides, polyacetates, or other polymers, in accordance with the present disclosure, which are skilled in the art Obvious.

肽:这个术语用来表示其单体为通过酰胺键连接在一起的α-氨基酸的寡聚体。 Peptide: This term is used to indicate which monomers are oligomers joined together by an amide bond α- amino acids. “肽”也可用来指“多肽”。 "Peptide" can also mean "polypeptide." 在本发明的范围内,应理解为氨基酸可以为L-旋光异构体或D-旋光异构体,虽然大于20个氨基酸的肽可能常常被称为“多肽”,肽通常大于2个氨基酸单体长度,经常更多的为大于5至10个氨基酸单体长度,甚至可大于20个氨基酸,采用通用的一个字母缩写来表示氨基酸。 Within the scope of the present invention, it should be understood that an amino acid may be a D- or L- optical isomer optical isomer, although a peptide greater than 20 amino acids may often be referred to as "polypeptide", generally greater than 2 amino acid peptide alone the length, more often greater than 5 to 10 amino acid monomers length, or even greater than 20 amino acids, using a common one-letter abbreviation for amino acids represented. 采用通用的一个字母缩写来表示氨基酸(如P代表脯氨酸)。 Using a common one-letter abbreviation for amino acids expressed (e.g., P for proline). 这些缩写包括在被本文引作参考的Stryer,Biochemistry,Third Ed.(1988)中。 These abbreviations are included in a reference cited herein Stryer, Biochemistry, Third Ed. (1988).

寡核苷酸:用来表示一般通过合成方法制备的单链DNA或RNA分子。 Oligonucleotide: generally used to indicate a single-stranded DNA or RNA molecule prepared by synthetic methods. 虽然不同长度的寡核苷酸在一些条件下为适宜,本发明采用的寡核苷酸通常为50至150个核苷酸长度,优选为80至120个核苷酸。 While oligonucleotides of different lengths nucleotide favorable under some conditions, the present invention employs oligonucleotides typically 50 to 150 nucleotides in length, preferably 80 to 120 nucleotides. 例如,寡核苷酸标记物可用与用于合成寡聚体的单体和单体加成步骤一致的核苷酸和核苷酸的加成步骤来建立。 For example, an oligonucleotide can be used with the marker addition step for the synthesis of monomers and oligomers and addition step nucleotides identical to nucleotides established. 此外,非常短的即有2至10个核苷酸的寡核苷酸可用来扩展现有的寡核苷酸标记物以确定单体偶联步骤。 In addition, very short, i.e., from 2 to 10 nucleotides in an oligonucleotide can be used to extend an existing oligonucleotide tag to identify a monomer coupling step. 可通过被本文引作参考的由Beaucage和Carruthers,1981,Tetr.Lett.22.1859-1862中描述的氨基磷酸酯方法或根据Matteucci et al.,1981,J.Am.Chem.Soc.103:3185中的三酯方法,或通过如使用购置的寡核苷酸自动合成仪的其它方法来制备适宜的寡核苷酸。 . It can be incorporated by reference herein described by Beaucage and Carruthers, 1981, phosphoramidate, or the method described Tetr.Lett.22.1859-1862 according to Matteucci et al, 1981, J.Am.Chem.Soc.103: 3185 in the triester method, or prepared by other methods such as using oligonucleotide purchased automated synthesizer suitable oligonucleotides.

可行地连接:这个术语指一核酸片断与另一核酸片断之间的功能性亲缘关系。 Operably connected: This term refers to a functional relationship between a genetic fragment of a nucleic acid with another nucleic acid fragment. 例如,如果启动子导致或通过其它方式有效地影响了编码序列的转录,启动子则被可行地连接到编码序列上。 For example, if the start or leading to the sub-effectively affect the transcription of the coding sequence by other means, possible promoters were linked to the coding sequence. 一般地,可行地连接意味着被连接的核酸片断或序列为紧接的并且必需与阅读框中的紧接的两个蛋白质编码区连接。 Typically, operably connected nucleic acid fragment or sequence means connected to and immediately two protein coding regions essential reading frame and the immediately connected.

平行偶联:这个短语指二个结构单元化合物同步偶联至底物独立特殊的位点上。 Coupled in parallel: this phrase refers to two structures coupled to the synchronization unit compound on a substrate separate specific site. 该底物可以是具有与这些结构单元相连的特殊基团的固体载体,或形式拥有两个独立地与每一结构单元的特殊基用的其它化合物,同步偶联指在将新的结构单元化合物加入到首先被偶联的结构单元之前,两化合物与这些特殊位点的偶联。 The substrate may be a solid support having a specific group of these structural units connected with, or has the form of two compounds independently from the other groups of each special structural unit with synchronization means coupled to the new compounds of the structural unit prior to addition to the structural unit is first coupled, and the coupling of these two compounds specific sites. 因此,本文所用的术语“同步”并不局限于这两个结构单元精确地同时被偶联。 Thus, as used herein, the term "synchronization" is not limited to these two structural units are coupled at precisely the same.

受体:这个术语指对于所给配体具有特定集和性的分子。 Receptor: This term refers to a ligand for a given set of molecules and the specific resistance. 受体可为自然产生或合成的分子。 Receptor may be a naturally occurring or synthetic molecules. 受体可以它们未改变的天然或分离状态或与其它物质结合的状态使用。 They may receptor unaltered natural or isolated state or in a state bound to other substances. 受体可共价或非共价地与其它物质连接。 Receptor may be covalently or non-covalently linked to other substances. 可采用在本发明中的方法中的受体包括,但不限于此,抗体,细胞膜受体,单克隆抗体,与抗原决定簇反应的抗血清(如病毒,细胞或其它物质),多核苷酸,核酸,外源凝集素,多糖,细胞,细胞膜和细胞器。 The method may be employed in the present invention include receptors, but not limited to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive antigenic determinants (such as viruses, cells or other materials) determined, polynucleotide , nucleic acids, lectins, polysaccharides, cells, cell membranes, and organelles. 受体还已知为“抗-配体”。 Receptor is also known as "anti - ligand." 当两个分子一般为大分子通过分子识别结合形成复合体时,则形成了“配体-受体对”。 When the two molecules are generally macromolecular complexes formed by the binding molecules identified, the formation of a "ligand - receptor pair." 其它受体包括,但不限于此,对于需要抗生素的微生物存活是必需的特殊的运输蛋白质或酶;酶的结合位点,抗体分子中的配体结合位点;核酸;如被本文引作参考的在Lerner et al.,1991,Science 254:659中描述的催化多肽;以及激素受体如胰岛素和生长激素受体。 Other receptors include, but are not limited to, microorganisms needed for the survival of antibiotics is required special transport proteins or enzymes; enzyme binding site, a ligand molecule in the antibody binding site; nucleic acid; are herein incorporated by reference as the, 1991, Science 254 in Lerner et al:. catalytic polypeptides are described in 659; and hormone receptors such as insulin and growth hormone receptor.

底物或固体载体:这些术语表示具刚性或半硬的表面的物质。 Substrate or Solid carrier: These terms represent the surface material having a rigid or semi-rigid. 虽然可采用其它形式,这些物质优选地采用小珠,丸,盘或其它方便的形式。 While other forms may be employed, these materials are preferably employed beads, pellets, disks, or other convenient form. 在一些实施例中,底物至少一个表面可基本上是平的。 In some embodiments, the at least one surface of the substrate may be substantially flat. 优选粗糙的球形表面。 The roughened surface is preferably spherical.

严紧杂交条件:这个短语指高选择性的杂交条件,其中只有当相关序列为完全或高度(即大于80%)互补时,核酸与其它核酸(或相同核酸分子的其它区段)的结合才保持稳定。 Stringent hybridization conditions: this phrase refers to the highly selective hybridization conditions, wherein only when the relevant sequence is complete or high (i.e., greater than 80%) complementary to other nucleic acid binding nucleic acids (or other sections of the same nucleic acid molecule) was held stable. 这些条件一般包括小于盐浓度1M的,如小于500mM,并且通常包括小于200mM的盐浓度。 These conditions typically include salt concentrations of less than 1M, such as less than 500 mM, and typically include salt concentrations of less than 200mM. 寡聚体的杂交温度将一般大于22℃,例如大于约30℃,并通常超过约37℃。 Oligomer hybridization temperature will generally greater than 22 ℃, e.g., greater than about 30 ℃, and usually in excess of about 37 ℃. 对于特殊的杂交长的片断可能要求高的杂交温度。 For special hybrid-long fragment may require higher hybridization temperatures. 因为其它因素可能极大地影响杂交的严紧性(这些因素包括碱基组成,互补链的长度,有机溶剂的情况,碱基错配的程度),所以这些因素的结合比对仅任何一个因素进行绝对的测定要重要得多。 Since other factors may significantly affect the stringency of hybridization (such factors include base composition, length of the complementary strand, the organic solvents, the extent of base mismatching), the combination of these factors than any single factor only absolute Determination much more important.

合成物:当由体外化学或酶合成而制备时,化合物则为合成物。 Composition: When prepared from in vitro chemical or enzymatic synthesis, the compound was composition. 可将本发明的合成库与,例如可在细菌,酵母或其它活的宿主中繁殖的病毒或质粒载体中的那些进行比较。 The libraries of the invention may be synthesized with, for example, can be compared to those propagated in bacterial, yeast, or other living host plasmid or viral vector.

I.标记化学库的合成概述:本发明一般涉及合成和筛选标记化学库的方法。 I. Overview synthetic chemical libraries tag: The present invention generally relates to the synthesis and screening of chemical libraries tag method. 本质上,本发明的化学库的每一本“书”由感兴趣的化学药品或分子,鉴定感兴趣的化学药品或分子或它们的一些重要情况的标记物以及感兴趣化学药品或分子与标记物之间的连接物所组成。 Essentially, the chemical libraries of the invention each of the "book" of chemicals or molecules of interest, to identify chemicals or molecules of interest or a case where some of the important markers of interest and a marker molecule or chemical linker between the object formed. 在一个重要的实施方案中,感兴趣的化学药品或分子为寡聚体,如肽,标记物为寡聚体,如核酸,连接物为固体载体或颗粒,从中寡集体和标记和可选择性地被裂开,如为了便于检测或提供可溶性的库、可筛选这些库来分离与受体结合或具有其他一些所需特性的各个寡聚体。 In one important embodiment, the chemicals or molecules of interest is an oligomer, such as a peptide, an oligomer marker, nucleic acid, linker, or a solid carrier particles, and which collectively labeled oligonucleotides and optionally being split, such as in order to facilitate detection or provide a soluble library, the library may be screened to separate the respective receptor binding or other oligomers having desired characteristics. 通过一大批高度不同的寡聚体的制备来说明制备标记化学库的一般方法,其中每一个库成员针对其它库成员来讲,为具有特定单体顺序的寡聚体(虽然该库一般包括完全相同的“书”)。 Prepared by a large number of different heights oligomers described general method for preparing a labeled chemical libraries, wherein each member of the library for library members in terms of the other, an oligomer having a sequence specific monomer (although the full library generally comprises the same "book"). 这种库或收集品可包含例如,一组单体中的装配到长度为n的寡聚体中的X个不同单体的所有组合产生的Xn个不同化合物。 Such collections or libraries can comprise, for example, a group of monomers, is fitted into a length of Xn n different compounds oligomer in all combinations of two different monomers X generated. 这种收集品还可包含例如仅在一个或少量位置具有不同单体单元,而所有其它位置具有相同顺序的寡聚体。 This collection may comprise, for example, different monomer units having only one or a few positions, while all other positions oligomer having the same sequence.

合成这些寡聚体的一般方法通常包括-随机组合(随机的)过程和单体单元的化学和/或酶装配。 General method for the synthesis of these oligomers generally comprise - random combinations of (random) chemical and monomer units and / or enzymatic assembly. 一种方法包括步骤:(a)将大量固体载体均分至大量反应容器中;(b)运用每一不同反应容器中的第一单体和标记物的不同组合,将第一单体和第一标记物偶联到第一反应容器中的载体上;(c)将载体汇集起来;(d)将载体均分至大量反应容器中;(e)运用每一不同反应容器中的第二单体和第二标记物的不同组合,将第二单体偶联至第一单体上,将第二标记物偶联至固体载体或第一标记物上;用不同标记物和不同单体选择性地重复偶联和均分步骤1至20次或更多。 A method comprising the steps of: (a) the large amount of solid support to average a large number of the reaction vessel; (b) the use of different combinations of each of the different first monomer in a reaction vessel and the marker, the first and second monomer a label conjugated to a carrier in the first reaction vessel; (c) the carrier pooled; (d) will be divided equally to the carrier in a plurality of reaction vessels; (e) using each of the different second single reaction vessel a second body and the different combinations of markers, the second monomer to the first monomer is coupled, the second label is conjugated to a solid support or first marker; selecting different labels and different monomers and coupling both of substeps repeated 1 to 20 times or more. 一般地,基本上等量的固体载体将均分至每一反应容器。 In general, substantially the same amount of solid carrier will average to each of the reaction vessel. 本领域技术人员看到在不同偶联步骤中可采用相同的化学结构单元,相同化学结构单元可采用在不止一个的具简单偶联步骤的偶联反应中(反应容器)。 Those skilled in the art can be used to see the same chemical structural units different coupling step, the same chemical structure unit may employ more than one in the coupling reaction with the simple coupling step (the reaction vessel).

为了更易于检测本发明,可以首先考虑从三种不同单体A,B和C的单体组装配的长度为三个残基的所有寡聚体的未标记库的合成。 For easier detection of the present invention, first consider the unlabeled synthetic oligomer library from all the length of the assembly of three different monomer component monomers A, B and C for three residues. 将三等分的小球均分在三个反应容器中,将单体A偶联至第一个反应容器中,将单体B偶联至第二个反应容器中,单体C偶联至第三个反应容器中。 The average trisection pellets in three reaction vessels, the monomer A is coupled to a first reaction vessel, a monomer B is coupled to a second reaction vessel, the monomer C is coupled to the third reaction vessel. 接着汇集来自所有反应容器的小球。 Then the reaction vessel together from all the pellets. 这种汇集包含大致等量的三种不同类型的小球,每一种小球以偶联至小球上的单体为特征。 This collection contains approximately equal amounts of three different types of pellets, each pellet coupled to the ball to the monomer is characterized. 将这种汇集混合,并重新分配至分别包含A,B或C作为将要偶联的下一步单体的独立的单体反应容器中。 Such mixed together, and redistributed to contain the A, B or C as the next monomer to be coupled to the separate monomer reaction vessel.

在这个偶联反应之后,现在每一反应容器包含带有位置1上的所有三种不同单体的小球和包含在每一特定的第二反应容器中的位置2上的单体。 After the coupling reaction, each reaction vessel now contains all the beads with three different monomers in position one and the monomer contained in each particular second position of the second reaction vessel. 所有的小球被再汇集,这样制备了一个小球的混合物,其中每一小球提供九种可能的二聚体中的一种。 All pellets were then pooled, a mixture of the thus prepared pellets, wherein each pellet is provided a nine possible dimers. 再将这种小球的汇集分配在三个反应容器中,并偶联到这三种不同的单体上,这样制备了三种单体的所有三聚体的完整的组(33=27)。 Such pellets then pooled in three assigned reaction vessel, and coupled to three different monomers, so that a complete set of all trimers of three monomers (33 = 27) was prepared . 很容易理解足够大量的合成小球的使用有助于确保这个组完全代表任意,随机组合的合成方法中采用的单体的各种组合。 Readily appreciate that the use of a sufficiently large number of synthesis beads helps to ensure that the set completely represents any various combination synthetic random combinations of monomers employed.

对这个完全随机方法的修改也是可能的。 Changes to this completely random approach are also possible. 例如,单体组可从一步到另一步而被扩展或缩小;如果偶联化学可达到。 For example, the monomer set may be expanded or reduced from one step to another; if the coupling chemistry can be achieved. 为了下一步可将单体组完全改变(如一个步骤中的氨基酸,另一步骤中的核苷,另一步骤中的糖类)。 The next step may be to completely change the monomer component (e.g., amino acids in one step, nucleosides in another step, carbohydrates in another step). 肽合成的单体单元例如可包括单个氨基酸或较大的肽单元或两者。 Synthetic peptide monomer units may comprise a single amino acid, for example, a larger peptide units, or both. 一种是在固体载体上形成在合成的某些步骤被在不同单体组中分配的各种顺序的一些组合。 One is formed in the order of some combination of the various steps of the synthesis of some of the different monomers are allocated in groups on a solid support. 通过这种方法,还可建立带有相关或非相关顺序的不同长度的寡聚体,并可在改变其它残基的同时将一些单体残基固定在一些位置上来建立寡聚体骨架,其中改变某些残基或区域以形成差异。 In this way, the establishment of further oligomers with different lengths of related and non-related sequence, and the other residues while changing some of the monomer residues at some locations up to establish a fixed oligomer backbone, wherein change of certain residues or regions to form the difference.

标记化学库的合成通常包含这些组合合成步骤。 Synthesis of labeled chemical libraries typically comprise a combination of these synthetic steps. 因为识别标记可容易地被译码来报告每个寡聚体的显著性,然而标记化学库可大大地比未标记库大和复杂。 Because the identification marks can be easily decoded to report the significance of each of the oligomers, but may be greatly chemical libraries tag than unlabeled complex libraries Japan. 事实上,本发明合成化合物的编码合成库的方法使筛选大批由多步骤合成制备的不能测序的化合物成为可能。 In fact, the coding method of synthesizing libraries of synthetic compounds of the present invention enables a large number of compounds screened by a multi-step sequencing can not be prepared in Synthesis possible.

具体地,寡核苷酸标记和寡核苷酸编码的使用为记录通过组合合成制备的限定化合物,特别是肽的巨大的库中的每一成员的结构个性提供了强有力的机制。 Specifically, the use of oligonucleotide labels and oligonucleotides coding for the compound is defined by a combination of a recording synthetically prepared, particularly the structure of each individual member of the huge library of peptides provides a powerful mechanism. 只要平行合成方法保持正交和可配伍,这个方法广泛应用于编码其它非肽结构的组合装配中。 Long and parallel synthesis methods remain orthogonal compatible, this method is widely used and assembled encoding other non-peptidic structure. 仅对于产生极高产量来提供单产物的反应的顺序,明显定义了组合合成的净产量。 Only for extremely high yield to provide a single product of the reaction sequence, clearly defines the net production of combinatorial synthesis. 这种情况与标准肽和DNA合成化学近似,得到的产物结构通过合成中使用的结构单元和/或偶联反应的顺序给予明确的规定。 This situation standard peptide synthesis chemistry and DNA approximation, a structural unit structure of the product obtained by synthesis or used in sequential and / coupling reaction to give clearly defined.

然而大多数合成有机反应为比较特质的,产生变化的产率和常常为多种产物(如区域和立体异构结构)。 However, most synthetic organic reaction is a comparison of the characteristics, and generates the yield often changes in a variety of products (e.g., structural and stereoisomers region). 利用这种化学来合成固体载体上的组合库在库中产生了一个位于每一个小球上的产物的混合物。 With such a chemically synthesized combinatorial libraries on solid support produces a mixture of a product located on each of the small ball in the library. 在最一般情况下,合成的编码不能唯一地确定相关本体的化学结构。 In the most general case, the synthetic coding is not uniquely determine the chemical structure of the relevant body. 反之,该编码方法可更准确地被当作编码-建造库成员的正确的合成条件(例如试剂,反应条件等)。 Conversely, this coding method can be considered more accurately encoded - correct synthesis conditions (such as reagents, reaction conditions, etc.) built library members. 筛选这个库来识别接着可在制备规模上再生产和被分成几份来分离具生物活性的成份的“有效处方”。 Screening of the library can then be identified and reproduced to be divided into several "prescriptions valid" isolated component having biological activity on a preparative scale. 编码库技术对于扩大组合化学的范围和它在发现药物用的应用以及大量有用化合物的开发和分离具有相当大的潜力。 Library encoding technique for expanding the scope of combinatorial chemistry and its applications in the discovery and development and separation of a drug with a large number of useful compounds has a considerable potential. 根据标记分子库的合成,可以更好地理解本发明的重要方面,例如在库合成中的固体载体的使用和选择。 The synthetic libraries of labeled molecule can be better understood with an important aspect of the present invention, for example, using solid support selection and synthesis of the library.

II、固体载体A.类型一般地,本发明的标记化学库由一批固体载体组成,例如小球或颗粒。 II, type A. In general the solid support, chemical libraries according to the present invention is marked by a group consisting of solid carriers, for example, pellets or granules. 这些固体载体可为任何形状,尽管优选为粗糙形。 These solid carriers may be any shape, although preferably rough shape. 这些载体不必为同一大小,形状或组成;虽然这些载体通常和优选为均一的。 These carriers do not have the same size, shape, or composition; While these vectors generally and preferably uniform. 在一些实施方案中,大小非常均匀的载体可特别优选。 In some embodiments, a very uniform carrier size may be particularly preferred. 然而,在其它实施方案中,两组或更多明显不同的固体载体可用于一定目的,即固体载体可由单个颗粒或更多的连结颗粒组成。 However, in other embodiments, two or more distinct solid carriers may be used for certain purposes, i.e., the individual particles or the solid support may be more linked particles.

固体载体可由许多物质组成,主要受衍生至许多化学活性基团中的任何一个的能力,与寡聚体的化学性质或其它分子合成的匹配性以及标记连接的制约。 The solid support may be composed of many substances, mainly due to the ability of any number of chemical derivatives of the active groups, matching the properties of chemical or other molecule of oligomer synthesis and tag attached constraints. 适宜的载体物质包括玻璃,橡胶浆,高度交联聚苯乙烯或类似的聚合物,金或其它胶态金属颗粒和本领域技术人员已知的其它物质。 Suitable carrier materials include glass, rubber, paste, highly cross-linked polystyrene or other material similar polymers, gold or other colloidal metal particles and the person skilled in the art. 除开其它被提到的,可用来衍生这些固体载体的化学活性基团是通常用在各分子或寡聚体的固态合成中的那些,因此它们对本领域技术人员将是熟知的。 Other than those mentioned in the opening, the derivatized solid support can be used to chemically reactive group is usually used in each molecule or solid state synthesis of oligomers, they skilled in the art will be familiar. 本文所用的术语“固体载体”包含带有寡聚体合成以及在一些实施方案中的标记连接和/或合成的适宜位点的颗粒。 As used herein, the term "solid support" includes in some embodiments, labels are attached and / oligomer synthesis and particle with a suitable synthetic or site. 存在许多有用于本发明合成寡聚体库制备的固体载体。 There are many solid support for the preparation of a synthetic oligomer libraries of the invention. 固体载体通常用于例如上面列举的肽,核酸和其它寡聚体的固相合成中,因此为本领域技术人员所熟知。 The solid carriers typically used for solid phase synthesis of peptides, for example, listed above, nucleic acids and other oligomers, and therefore those skilled in the art. 本发明的固体载体不包括活细胞,病毒或如噬菌体载体或质粒的克隆载体。 The solid support of the present invention does not include living cells, viruses, or cloning vectors such as phage vectors or plasmids. MonobeadsTM(从Pharmacia Fine ChemicalsAB,Uppsala Sweden购得)或它们的等效物作为本发明各个方面的固体载体都特别有用。 MonobeadsTM (commercially available from Pharmacia Fine ChemicalsAB, Uppsala Sweden) or equivalents thereof are particularly useful as solid carriers are the various aspects of the present invention. MonobeadsTM提供了良好的大小均一性和10μm的小尺寸。 MonobeadsTM provides a good uniformity of size and the small size of 10μm. 进一步,这些MonobeadsTM在有机或无机溶剂中不凝集,并为寡核苷酸和肽的合成提供适宜的载体。 Further, these MonobeadsTM not aggregated in an organic or inorganic solvent, and provides a suitable carrier for the synthesis of oligonucleotides and peptides. 最后,MonobeadsTM为最初氨基酸(100nmole/mg)提供非常高的负载。 Finally, MonobeadsTM provide very high load initial amino acid (100nmole / mg).

被选择用来完成本发明的特定的固体载体的一个重要方面是载体的大小。 Selected to accomplish a particular important aspect of the present invention, the solid support is the size of the carrier. 如果需要,用足够的固体载体和有效的偶联,可制备某些寡聚体的完整组。 If desired, the solid support with a sufficient and effective coupling, can complete the preparation of certain groups of the oligomer. 一般地,固体载体大小在1nm至100μm的范围,但有时可用达1mm大小的较大质量的固体载体。 Generally, the solid support size is in the range 1nm to 100μm, but may sometimes be the size of the solid support 1mm larger mass. 固体载体的适宜大小依赖于(1)所需寡聚体合成位点的数目和识别标记连接位点;(2)将要合成的不同化合物的数目(和带有每一个需要筛选的寡聚体的固体载体的数目);和(3)固体载体的大小对将要使用的特定筛选策略的影响(例如被荧光活化的细胞分检器)(FACS)。 Suitable size of the solid support depends on (1) the identification number and the desired attachment site labeled oligomer synthesis sites; the number of different compounds to be synthesized (2) (and with each of the oligomers to be screened the number of solid support); and (3) Effect of specific size of the solid support screening strategy to be used (e.g., fluorescence activated cell sorter) (FACS).

作为特殊的例子,直径为1μm的固体载体可用于本发明。 As a specific example, solid support having a diameter of 1μm may be used in the present invention. 如果每一反应容器中包含近0.2ml的固体载体,并且寡聚体由50个单体合成(50个平行反应),那么将需要总共100ml固体载体或近1013固体载体。 If each reaction vessel comprises a solid support near 0.2ml, and oligomer consists of 50 synthetic monomers (50 parallel reactions), then it requires a total of 100ml solid carrier or solid support near 1013. 如果想用这50个单体来制备六聚物,那么存在多于1.5×1010个的可能的顺序,并且那一特定顺序将出现在约103的固体载体上。 If you want to use these monomers to prepare 50 hexamer, it may order more than 1.5 × 1010 th then there, and that a particular order will appear on the solid support of about 103. 根据通常使用的肽分成树脂的容量,每一小球的估算出来的容量为约0.1pg肽/小球。 The peptide capacity of the resin into a generally used, each estimated capacity of about pellets peptide 0.1pg / pellet. 那么通过这个估算,每一固体载体将具约100amol或108寡聚体链。 This estimation By then, each solid support having the 100amol or about 108 oligomer chains.

为了提高洗涤效率,可采用比一般肽合成要少孔的无孔小球或其它固体载体;然而,对于本发明的某些应用,多孔小球或树脂工作良好并通常为优选。 In order to improve washing efficiency, it can be less than the average pore peptide synthesis non-porous beads or other solid carriers; however, for certain applications of the present invention, the porous pellet or a resin generally work well and are preferred. 无孔载体将具有生长链的低密度,但尽管几个数量级的容量降低,为了有效的筛选,可制备足够大的寡聚体密度。 Nonporous support having a low density of growing chains, but although several orders of magnitude reduction capacity, in order to effectively screening, can be prepared sufficiently large oligomers density. 运用少孔载体,更大部分的寡聚体在筛选过程中将可与受体结合。 Using less porous carrier, a greater portion of the oligomers may bind to the receptor in the screening process. 而且,少孔载体将减少从一个反应至下一个反应的标记转移,因此提高了主要(标准)标记的读数的准确性。 Moreover, less porous carrier to reduce the mark transfer reaction from a reaction to the next, thus improving the accuracy of the primary (standard) labeled readings.

如上面所提到,另一实施例包含两个固体载体如小球的使用,它们被物理地连接在一起,其中一个与分子或寡聚体的合成位点(或接头)连接,一个与识别标记的连接位点(或接头)连接。 As mentioned above, another embodiment comprises the use of two solid supports such as beads, that are physically linked together, one of which is connected to the molecule or oligomer synthesis sites (or linkers), and a recognition tag attachment sites (or linkers). 这种结构允许分子或寡聚体分开,识别标记进入不连续“区域”以及允许为了连接使用极大不同的化学反应基和物质的化学组成。 This configuration allows the molecules or oligomers are separated, discontinuous identification mark into the "area" and to allow for the connection using a chemical reactive group greatly different chemical and physical composition. 固体载体可被分别衍生,接着在所有或几乎所有合成固体载体将具有一牵引着的标记连接的固体载体的条件下被连接。 The solid support is attached may be derivatized separately and then the solid support having a pulling marker linked in all or almost all of the solid support synthesis conditions. 固体载体可为不同大小,例如一个大的连接有不同(或许多)小标记连接的小球的合成小球。 A solid carrier can be different sizes, for example, a large connection with a different (or many) smaller tag linked synthetic beads pellet. 在一个实施方案中,第一固体载体将具有至少一个与氨基酸相连的,第二固体载体将具有至少一个与核苷酸相连的小球。 In one embodiment, the first solid support will have at least one amino acid is connected to a second solid support beads having at least one nucleotide linked.

连接两个小球的方法受寡聚体合成化学的限制。 The method of connecting the two beads is limited by the chemical synthesis of oligomers. 连接小球的最显而易见的方法是使用与每一种固体载体上的主要化学反应基反应的杂二官能交联剂(这种试剂的例子见Pierce Immuno Technology Catalog and Handbook PP.E10-E18(1991)。这些交联剂可达到如下文部分描述的各种目的。 The most obvious way is to use a ball connected hetero difunctional crosslinking agent is reacted with a primary chemically reactive group of each of the solid support (see Examples of such agents Pierce Immuno Technology Catalog and Handbook PP.E10-E18 (1991 ). these crosslinking agents may be part of a variety of purposes as described below.

B.接头当被连接至一固体载体上时,寡聚体和它相关的标记通常通用一个或多个分子接头连接到载体上。 B. when connected to a connector on a solid support, the oligomer is usually labeled and its associated one or more universal joints coupled to the carrier molecule. 这个接头分子在连接之前在每一端具有一个适宜的官能基,一个基团适宜于与载体相连,另一个基团适宜于与寡聚体式检记物相连。 Before connecting the linker molecule having at each end a suitable functional group, a suitable group attached to the carrier, the other group is connected to oligomeric style suitable for the subject matter in mind. 在一些实施方案中,采用可裂解的接头以便于分析或检测步骤。 In some embodiments, for analysis or detection steps using a cleavable linker.

提供大量可得到的不同连接试剂,可将识别标记连接到寡聚体或其它感兴趣的库分子上或连接到固体载体或一个预先存在的标记上。 It provides a large number of different linking agents available, identifying indicia may be attached to the oligomer libraries or other molecule of interest or linked to a solid support or on a pre-existing marker. 例如,可将识别标记连接至结合到寡聚体中的单体上,或连接至结合到非寡聚化合物中的结构单元上。 For example, the identification label is attached to the binding of the oligomer to the monomer or to the structural units bonded to the non-oligomeric compounds. 对于肽寡聚体,半胱氨酸残基的侧链为标记的连接提供了一方便的位点。 For peptide oligomers, the side chain of a cysteine ​​residue provides a convenient connection site is marked. 在其它情况下,只要所需寡聚体的净合成的减少量可容易地被允许,标记甚至可被连接来覆盖少量寡聚体链。 In other cases, as long as the amount required to reduce the net synthesis of the oligomer can be easily allowed to be labeled can be connected to cover even a small amount of the oligomer chain. 可将标记直接连接到将寡聚体(或其它感兴趣的化合物)结合至固体载体的接头上。 It can be connected directly to the tag (or other compounds of interest) oligomer bound to a solid support linker. 在这个实施方案中,在连接之前,接头具适宜于识别标记的连接的第三个官能基团。 In this embodiment, prior to connection to a third connector having suitable functional groups attached identification mark.

根据应用和所需效果,当然可以结合大量接头。 Depending on the application and desired effect, of course, may be combined with a large number of joints. 例如,可选择传递疏水性,亲水性或立体部分以获得在例如偶联或结合效率的特性方面的所需效果的接头。 For example, selectively transmitting a hydrophobic, hydrophilic or partially perspective to achieve a desired effect or binding properties of the conjugate, for example, in terms of the efficiency of the joint. 在本发明的一个方面,分枝接头即例如接头Fmoc-Thr(tBu)的带有庞大侧链的接头被用来提供稳定性或用来控制库中固体载体上的分子的间隔或库中分子与标记之间的间隔。 In one aspect of the present invention, i.e., for example, branched linker linker Fmoc-Thr (tBu) a linker with a large side chain are used to provide stability for the control interval or libraries or libraries of molecules on a solid carrier molecule the spacing between the marks.

如上所述,可采用可裂解的接头的获得有效的效果。 To obtain effective results as described above, can be cleavable linker. 本发明优选的可光解的接头包括6-硝基藜芦基氧羰基(NVOC)和其它NVOC有关的接头化合物(见被本文引作参考的PCr专利公开号WO90/15070和WO92/10092,也见于1992年11月2日提出的美国专利申请系列号NO.971,181)。 Preferably the present invention comprises a linker photolysis 6-nitro veratryl oxycarbonyl (NVOC), and other NVOC-related compounds linker (see Patent Publication No. PCr is incorporated herein by reference WO90 / 15070 and WO92 / 10092, also found in US Patent application Serial No. 11 NO.971,181 1992 Nian 2 Yue filed). 在另一个实施方案中,接头为带有一个或多个限制性位点的核酸,这样库成员中的一部分(标记,寡聚体或其它感兴趣的化合物或两者,或固体载体)可通过适宜的限制性酶选择性地从另一个中裂解。 In another embodiment, the linker is a nucleic acid with one or more restriction sites, a portion (or both labeled compound, oligomer or other interest, or a solid carrier) such library member by suitable restriction enzyme cleaved selectively from the other. 这个新的核酸和接头说明了大量可用于获得为了本发明目的的有益效果的接头。 The new linker and nucleic acids described can be used to obtain a large number of purposes of the present invention the beneficial effects linker.

C.分子载体如上所述,本发明还可以一种方法来完成,其中没有固体载体,直接将标记(一般通过一个接头)连接至正在合成的寡聚体或其它分子上。 C. vector molecule described above, a method of the present invention may also be accomplished, where there is no solid support, directly labeled (typically via a linker) to the oligomer being synthesized or other molecule. 用另一种方法,可在固体载体上合成寡聚体或其它分子以及其相关标记,并在筛选或其它使用之前,裂开或通过其它方法从固体载体上除去。 With another method, synthetic oligonucleotides, or other molecules as well as its associated marker on a solid support, and prior to screening or other use, cracked or removed from the solid support by other methods. 这个方法下面将更全面地描述。 This method is more fully described below. 无论固体载体是否存在,库的大小和组成将由偶联和混合步骤,合成中所用到的单体或其它结构单元的数目来决定。 Regardless of whether there is a solid support, the library size and the number of component by coupling and mixing step, the monomers used in the synthesis, or other structural unit is determined.

III、化学结构单元A.寡聚体和单体通过考虑不同寡聚体和聚合物的大库的合成和筛选,可能最容易地理解本发明的广泛适用性。 III, the chemical structure of the monomer unit and oligomer A. by considering the synthesis and screening large libraries of different oligomers and polymers, may be most readily understood in the broad applicability of the present invention. 寡聚体是由单体组成的聚合物;对于生物聚合物,寡聚体中的单体顺序通常确定了重要的生物学特性。 Oligomer is a polymer from a monomer composition; for biopolymers, monomer sequence oligomer is generally determines important biological properties. 感兴趣的优选寡聚体包括肽,寡核苷酸,寡N-取代的甘氨酸和聚碳酸酯。 Preferably oligomer of interest include peptides, oligonucleotides, N- substituted glycines and polycarbonate. 如上所述,为了本发明的目的,单体是可被连接在一起形成-寡聚体或聚合物的一组分子中的任何一个成员,即氨基酸,碳酸酯,砜,亚砜,核苷酸,糖类,尿素,磷酸,类脂,酯,它们的混合物和类似物。 As described above, for the purposes of the present invention, the monomers that may be connected together to form - any member of a group of molecules oligomer or polymer, i.e., amino acids, carbonates, sulfones, sulfoxides, nucleotides , a saccharide, urea, phosphate, lipids, esters, mixtures thereof and the like. 因此,单体可以是可被适当活化来进行化学偶联或能被接受用来进行酶促偶联的任何类型。 Thus, the monomer may be appropriately activated for chemical coupling or accepted can be used for any type of enzymatic conjugation.

从许多类型的单体装配寡聚体的方法要求运用给定的单体单元组或结构单元的适宜的偶联化学性质。 Many types of monomers from the method of assembly requires the use of oligomer to a suitable coupling chemistry properties of a given set of monomer units or structural units. 在逐步的类型中可连接到另一个结构单元上的结构单元的任何组可作为单体组。 Any type of group may be gradually connected to the other structural units the structural unit may be used as monomer. 可通过化学,酶,或其它方法或这些方法的任意组合来传递这种连接。 This connection may be transmitted by any combination of chemical, enzymatic, or other methods or combinations of these methods. 得到的寡聚体可为线性,环状,分枝或采取那些对本领域技术人员为显而易见的各种其它结构。 The resulting oligomers can be linear, cyclic, branched or take those skilled in the art that various other structures is apparent.

B.其它结构单元本文描述的发明主要是关于包含氨基酸序列的分子的制备,但本发明可容易地应用于寡聚体的制备的及可在步骤通过步骤的类型中合成的任何组的化合物中,这些均可被本领域技术人员理解。 Other structural units B. invention described herein primarily for the preparation of molecules containing sequences of amino acids, but the present invention can be easily applied to any set of compounds and can be synthesized in a step by step type of the oligomer prepared in these can be understood by those skilled in the art. 例如,如苯并二氮杂,海因和肽膦酸可通过使用本文来制备(见1993年9月9日提出的美国专利申请系列号08/119,700,它是1993年6月21日提出目前已放弃的为美国专利号5,339,115部分继续申请的系列号为081,577的部分继续申请,这里每一篇均被本文引作参考。 E.g., such as benzodiazepines occlusion, phosphonic acids and peptides hydantoin may be prepared proposed (see September 9, 1993 proposed by U.S. Patent Application Serial No. 08 / 119,700 used herein, which is June 21, 1993 serial number has been abandoned as part of US Patent No. 5,339,115 to continue for some 081,577 filed continuation application, where each one are incorporated herein by reference.

在一个实施方案中,本发明可用于建立分枝聚合物库。 In one embodiment, the present invention can be used to establish a library branched polymer. 然而在许多例子中,线性聚合物库,例如带有大于3-4个残基的肽非常有用,这些线性分子的形状变得长而窄。 However, in many instances, libraries linear polymers, for example greater than 3-4 peptide having residues useful shape of linear molecules is long and narrow. 也许部分因为分子的高度柔韧性大多数药物没有这种扩展的形状。 Perhaps in part because of the high flexibility of most drug molecules do not form such an extension. 分枝主链聚合物可致使分子形状与已知药物类似。 Branched backbone polymer may cause the molecular shapes similar to the known drugs. 因此,在一个实施方案中,本发明涉及单体与至少三种其它单体可被连接至其上的官能基团的结合。 Thus, in one embodiment, the present invention relates to a combined monomer and at least three functional groups may be connected to other monomer thereto.

如果仅用这种单体,那么完全分枝合成将通常导致被偶联的最后单体的高比率(相结于合成中所用的其它单体)。 If only such a monomer, it will generally result in synthesis of completely branched high ratio of monomers to be coupled to the last (junction with the other monomers used in the synthesis). 当然可以掺合不同的分枝单体的混合物来改变这个比率,但是接着在识别感兴趣的化合物的结构上可能有最有困难,即分枝单体的混合物越复杂,标记可提供的关于合成的特定分子的信息就越少。 Of course, mixtures of different branches may be incorporated to change the ratio of the monomers, but then on the structure of the compound of interest may be the most difficult to identify, i.e. the more complex mixture of branched monomers, markers may provide for the synthesis of the less the information of a specific molecule. 在本发明的一个改进方法中,在每一个单体偶联步骤中掺入两种单体的混合物,一种能分枝,一种不能,这样产生了一个包含带有高信息标记的大量不同形状的库。 In an improved process of the present invention, incorporated into the mixture of the two monomers in each monomer coupling step, a branch can be a can, which produces a large number of different tag contains information with a high shape library. 在这种情况下,标记将确定在每一个偶联步骤出现的单体,但不确定单体是否能分枝。 In this case, the marker will be determined for each monomer coupling step occurring, but are not sure whether the branch monomer. 然而,仅使用包含在来自第一库的选择组的化合物中的那些单体的简单再合成将容易识别这些化合物的结构。 However, only those containing simple resynthesis of the compound selected from the group of the first monomer in the library structure of these compounds will readily recognize.

VI、标记识别标记具有一可辨认的特征,即例如在形状,大小,质量,电荷或颜色方面可通过显微镜或其它方法辨别。 Vl, the identification mark having a mark recognizable feature, i.e. for example by microscopy or other methods to identify the shape, size, mass, charge, or color. 这些可辨认的特征可来自于标记的光学,化学,电学或磁特性,或来自于这些特性的组合。 These features may be identifiable from the optical, chemical, electrical or magnetic properties of the tag, or from a combination of these characteristics. 实质上,标记用作在一个(或多个)分子或固体载体的水平上标记分子和编码可辨认的信息。 In essence, the mark used as a marker recognizable molecule encoding information and in the horizontal one (or more) molecules or solid carrier. 通过利用识别标记跟踪化学库的每一成员所采取的路径,可以通过阅读识别标记来推判库中任何化学物质的结构(即任何寡聚体的单体的顺序)。 By tracking the path of chemical libraries each member taken using the identification mark, can be any chemical structure of the library (i.e., the sequence of monomers of any oligomer) to push judged by reading the identification mark.

可将用显微镜可识别的标记建造成具有可辨认的不同大小,形状或颜色或用条型码标记的小小球,这些标记可以为“机器可阅读”的发光或放射性标记。 The microscope may be constructed having identifiable indicia identifying different sizes, shapes, or colors or labeled with bar code small balls, these markers may be "machine readable" luminescent or radioactive labels. 识别标记也可为可编码的分子结构,这些信息可被编码在大小(聚合物长度)或分子组成中。 Identification mark may also be encodable molecular structure, such information may be encoded in the size (length of the polymer) or molecular composition. 也许后面类型的标记的最好例子为核酸序列,即从天然或修饰碱基装配而来的DNA或DNA。 Perhaps the best example of the type of tag is followed by a nucleic acid sequence, i.e. from a base assembled from natural or modified DNA or DNA.

为了证实标记在合成和筛选化学库中所起的作用,考虑例如使用在寡聚体合成中被连接至每一小球上的在显微镜下可辨认的字母数字标记。 To confirm the synthesis and screening of chemical markers in the library in the role, for example, it is connected to consider alphanumeric indicia recognizable under the microscope on each ball in the oligomer synthesis. 标记“A”指在步骤1参加A-单体反应的小球;“C2”指在步骤2参加C-单体反应的小球,“B3”指在步骤3被加入的B-单体,以此类推。 Labeled "A" refers to Step 1 of the reaction participated A- monomer beads; "C2" refers to the reaction of step 2 monomers participate C- beads, "B3" refers to the step 3 is added to the monomer B-, and so on. 在第三步合成结束时,一个小球将具有三种被连接的标记;例如A1,C2和B3,这表明小球上的肽的序列为ACB。 At the end of the third synthesis step, a pellet having a tag the three connected; example A1, C2, and B3, indicating that the sequence of the peptide on the bead is ACB. 这个方法要求许多不同的识别标记至多等于不同单体数目和合成步骤数目的产物(在这个实施例中9),如果标记以这样的步骤顺序A,AC,ACB与另一个相连,识别标记的数目则被减少,在这种情况下只有与单体一样多的识别标记被使用,以加入与何种单体,在哪步加入的记录一致的方式装配识别标记。 This method requires many number of different identification marks at most equal to the number of different monomers and the number of synthetic steps product (9 in this embodiment), if such sequence of steps labeled A, AC, ACB and the other is connected, the identification mark were reduced, and only the identification mark as many monomers to be used in this case, to which was added with the monomers, added to the same step in which the identification mark recording assembly.

在另一个实施例中,标记由一系列光可寻找的分子,例如发荧光的,发磷光的化合物,它们的光谱特性可被改变(例如光漂白)和因此用于贮存信息,这些信息被用于标记库中每一小球或其它固体载体。 In another embodiment, the indicia may look for a series of light molecules, such as a fluorescent, phosphorescent compounds, their spectral characteristics may be altered (e.g., photobleaching) embodiment, and thus for storing information that is used each pellet, or other solid carriers in the tag library. 在这样的一个方法中,一个小球结合多种均可被选择性地光漂白,由此变得不能发荧光或不能减弱发荧光的荧光团。 In such a method, a plurality of pellets may be combined selectively photobleaching, whereby it becomes not fluoresce, or fluoresces not weaken fluorophore. 在每一偶联或化学反应步骤中,照射小球(或没有)使一个或多个特定类型的荧光团光漂白(或没有),因此记录了被合成的寡聚体中的单体个性。 In each coupling step, or a chemical reaction, irradiating pellets (or not) one or more specific types of fluorophores photobleach (or not), thus recording the synthesized oligomers are monomers personality. 见被本文引作参考的Science 255:123(1992年3月6日)。 See incorporated by reference herein, Science 255: 123 (6 March 1992).

因此识别标记识别每一单体偶联或各库成员或固体载体经历的其它反应步骤,并记录了合成系列中每一单体被加入的步骤或完成的其它化学反应。 Thus other identifying indicia identifying each monomer reaction step or in each library members conjugated or solid carrier experienced and record the step synthesis of other chemically reactive monomers are added to each of the series or completed.

为了方便和与识别标记类型,连接方式,和寡聚体化学性质或其它分子合成相适应,可在单体加成或其它反应之前,之中或之后立即将标记连接。 In order to facilitate the identification and the tag type, connection, or other molecules and chemical properties compatible synthetic oligomer, monomer addition may be before or other reactive label is attached immediately, or after. 如上所述,通过各种方式,或直接通过一连接分子,或通过寡聚物在其上被合成的固体载体,可将识别标记与寡聚体相连。 As described above, in various ways, directly or via a linker molecule, or are synthesized on solid support by the oligomer, the identification tag may be attached to the oligomer. 在后面的方法中,还可将标记连接到反过来被连接至寡聚体在其上被合成的固体载体中的另一固体载体上。 In the latter method, the marker may also be connected to the other is in turn connected to a solid support in a solid support on which the oligomer is synthesized. 在经历了特定单体加成或其它化学反应步骤的固体载体被物理性地连接在一起,并且能被标记作为一个基团时,即在下一步汇集步骤之前,将识别标记加入。 Experienced a particular solid support or other chemically reactive monomer addition step are physically connected together, as a group and can be labeled, i.e., before the next step of pooling, the identification mark is added.

在一此情况下,当然仅少量寡聚体的单体单元被改变时,象当想仅仅改变肽中的一此氨基酸时那样,可能需要确定仅仅是在寡聚体中变化的那些单体。 In a case, of course, only a small number of monomer units in the oligomer, when changed, such as when you want to change only one amino acid in the peptide, as this may be needed for only those monomers varies in the oligomer. 例如,可能想改变6至12个氨基酸长度的肽中的仅3至6个氨基酸,或可能想改变长达50个氨基酸长度的多肽中的一些如5个氨基酸。 For example, you may want to change the peptides 6-12 amino acids in length only 3 to 6 amino acids, polypeptide, or may want to change up to 50 amino acids in length, such as 5 amino acids. 通过为每一固体载体提供一个确定仅在每一序列中被改变的氨基酸的识别标记,可唯一地识别每一个肽的序列,这些将容易地被本领域技术人员理解。 By providing a sequence of identification marks it is only changed in each amino acid sequence that uniquely identifies each peptide is determined for each solid support, which will be readily understood by those skilled in the art. 在这种情况下,所有固体载体可保留在为了共同单体单元的加成的相同反应容器中并均分于为了特殊单体单元的加成的不同反应容器中。 In such cases, all solid supports may remain the same for the common reaction vessel the monomer units in addition to sharing and different reaction vessels for the addition of special monomer unit.

合成的寡脱氧核苷酸尤其优选为带有信息的识别标记。 Synthetic oligodeoxynucleotides particularly preferably labeled with identification information. 寡核苷酸为天然,高密度信息贮存介质。 Oligonucleotide natural, high density information storage medium. 与化学合成方法相关的单体类型的个性,加成步骤或任何其它信息可容易地编码在一个短的寡核苷酸序列。 Associated with the chemical synthesis of the individual monomer types, or any other information addition step can be easily encoded in a short oligonucleotide sequence. 寡核苷酸也很容易地与许多固体载体,寡聚体,接头和其它分子相连。 Oligonucleotides also easily with many solid support, oligomers, and other linker molecules are connected. 例如,寡核苷酸可容易地被连接至肽合成小球上。 For example, an oligonucleotide can be readily attached to the peptide synthesis on beads.

使用寡核苷酸编码方法中特有的一个显著好处是通过聚合酶链式反应(PCK,见于PCK Protocols:A Guide to Meth-ods and Applications(Innis,M,Gelfand,D,Sninsky,J.andWhite,T,Academic Press,San Diego 1990);还见于美国专利号4,683,202和4,965,188,每一篇均被本文引作参考)和其它核酸复制和扩增技术得到巨大量的靶扩增的能力。 Encoding method using oligonucleotides specific to a significant benefit is by polymerase chain reaction (PCK, found PCK Protocols: A Guide to Meth-ods and Applications (Innis, M, Gelfand, D, Sninsky, J.andWhite, T, Academic Press, San Diego 1990); also found in U.S. Patent Nos. 4,683,202 and 4,965,188, each incorporated herein by reference are a) and other nucleic acid amplification technology is the ability to copy and huge amounts of target amplification. 虽然最常用于体外DNA扩增方法为PCR,适宜的可选择的扩增方法包括例如核酸序列扩增(Compton,1991,Nature 350:91-92)和扩增反义RNA(见被本文上作参考的Van Gelder etal.,1988,Proc.Nat.Acad.Sci.USA 85:7652-7656)和自续式序列复制系统(见于被本文引作参考的3SR,见Guatelli etal.,1990,Pro.Natl.Acad.Sci.USA 8):1874-1878)。 Although most commonly used in vitro DNA amplification method is PCR, alternative suitable amplification methods include, for example, a nucleic acid sequence amplification (Compton, 1991, Nature 350: 91-92) and amplified antisense RNA (see herein is for the reference Van Gelder etal, 1988, Proc.Nat.Acad.Sci.USA 85:. 7652-7656), and self-sustaining sequence replication system of formula (incorporated herein by reference is found in the 3SR, see Guatelli etal, 1990, Pro.. Natl.Acad.Sci.USA 8): 1874-1878). PCR仅需要少量(运用高选择性和有效方法,甚至一个单拷贝就已足够)DNA模版,这样能使人们使用微观大小的固体载体并获得较大的库。 PCR requires only a small amount (and effective method of using a high selectivity, and even a single copy is sufficient) the DNA template, this enables people to use the solid support and the larger the microscopic size of the library.

使用核酸标记便于远远超过通过其它被限制库方法获得的差异性的合成库的建立和筛选。 And facilitate establishment of a nucleic acid marker screened much more than the difference obtained by the other methods is limited library synthesis library. 而且,这些库采用易控制量的小球原料,因此可被采用切实可行量的生物试剂来分配受体结合。 Furthermore, use of these libraries is easy to control the amount of feed pellets, thus the amount of viable may be employed to assign receptor binding biological agent. 本发明的一个改进方法涉及具有寡核苷酸标记的ESL库的处理中的一个限制步骤——扩增,链分离,和来自各小球的标记的序列测定。 An improved method of the present invention relates to a limiting step in the process labeled oligonucleotides ESL library - amplification, strand separation, and the labeled sequences from each pellet. 这个方法提高了序列测定的效率至少一个数量组,并涉及标记连环(连接)步骤,其中许多一般从选择的库成员扩增而来的不同标记在寡核苷酸标记的克隆或序列测定之前被绑扎在一起。 This method improves the efficiency of the number of sequence determination of at least one group, and to mark chain (connected) step, which is generally different from many prior tag library members selected from the amplified oligonucleotide tag or clone sequencing are banding together.

在本发明方法的一个实施方案中,扩增的标记被连接,并接着克隆为常规序列测定载体中的线性排列的10至20(或更多)的标记。 In one embodiment of the method of the present invention, it is connected to an amplifiable marker, and then cloned as a conventional marker sequencing vector linear array of 10 to 20 (or more). 优选地,适宜的限制性位点被安装在与寡核苷酸标记的“编码区域”(具有信息含量的序列)相邻,在扩增了一组小球上的标记之后,限制性位点被切下,片断被连接形成多联体。 Preferably, a suitable restriction site is mounted in the oligonucleotide labeled "coding region" (information content of the sequence) adjacent to, after amplified indicia on a set of beads, restriction sites under cut, fragments are ligated to form concatemers. 接着将这个多联体克隆降至适宜的序列测定的载体中,只要发现每个模板上具有多于至少10个标记,那么每个模板可用于总共例如500至800个碱基的双向序列测定。 This vector is then reduced to concatamers cloned in suitable sequence determination, if they find on each template having at least more than 10 markers, each template can be used for bidirectional sequence determination Total e.g. 500 to 800 bases. 这种方法还将提供避免用FACS分离各小球的选取方案。 This method also provides isolated to avoid selecting programs of beads by FACS. 小球或标记化合物可被分类至库,用于序列测定的被扩增,连接和克隆的标记的库中。 Pellets or labeled compound may be classified to the library, was amplified for sequencing, ligation and cloning tag library. 此外,因为摆脱了操作各小球的要求,可使用小于1μm(一般这个大小对于常规FACS分板来说太小)的小球来建立和筛选库。 Further, since the operations of the respective requirements out of the ball, using less than 1μm (typically too small for the size of a conventional plate for a FACS) pellets and to establish a library is screened. 这种选择可通过亲和纯化法(扫视,磁性小球),方便地被完成,接着富化的小球被如上所述地扩增和克隆。 This selection can be by affinity purification (panning, magnetic beads), conveniently is completed, and then the enriched pellets were amplified and cloned as described above.

寡核苷酸识别标记可在相应的单体偶联(对于寡聚体合成)或其它化学反应步骤之前,之中或之后,被一个碱基接着一个碱基地装配。 Oligonucleotides can be labeled to identify the corresponding monomers before coupling (for oligomer synthesis) or other chemical reaction steps, during or after, and then a base is a base assembled. 在寡核苷酸标记的碱基接着碱基的合成的一种情况下,每步的标记为一个单核苷酸,或最多为非常少量的核苷酸(即2至5个核苷酸的块)。 In one case the synthetic oligonucleotide is then labeled nucleotide bases, each step of a single labeled nucleotide, or at most very few nucleotides (i.e. nucleotides 2-5 Piece). 在块接着块的方法中,具2至5至10或更多碱基的编码核苷酸(密码子)作为被保护被活化的块加入。 In the process block by block, with 2 to 5 to 10 or more bases encoding nucleotide (codon) as the activated protected blocks added. 每一块携带单体类型或其它信息,一个标记块与下一个的加成顺序代表单体加成或其它反应的顺序。 Each block carries the monomer or other types of information, a marker block sequential monomer addition or other reaction with an addition of a representative of the next sequence. 另外,块可编码寡聚体合成或其它反应步骤数目和单体类型或其它结构单元的信息。 Further, the information may be block or other reactive oligomer synthesis step number and types of monomers or other structural unit of the coding. 这种方法保持了与寡聚体平行生长的寡核苷酸链的线性排列中的步骤顺序。 This method of holding the sequence of steps arranged in a linear oligonucleotide chain grown in parallel with the oligomer in. 为了保持平行合成步骤(例如寡核苷酸和肽)的化学相容性,可以修改标准的合成化学组成的性质,它是下面将要进一步详细讨论的本发明的一个重要方面。 In order to maintain the parallel synthetic steps (e.g., oligonucleotides and peptides) chemical compatibility, can modify the composition of the standard synthetic chemistry properties, it is an important aspect of the present invention will be discussed in further detail below.

还可在每一步连接含有扩增引物位点,单体特定信息和反应顺序信息,长度为50至150个碱基的保护(或未保护)的寡核苷酸。 At each step further comprises connecting amplification primer sites, monomer-specific information, and order information reaction, the length of the protective 50-150 bases (or unprotected) oligonucleotides. 在一系列n个寡聚体合成(单体偶联)或其它化学合成步骤结束时,将有n组不同的被编码的与库中每一寡聚体序列或其它化合物相关的寡核苷酸识别标记。 At the end of a series of n oligomer synthesis (coupling of monomer) or other chemical synthesis steps, you will have a different set of n associated with each oligomer sequence oligonucleotides or other compound libraries are encoded identification mark. 在识别具有配体活性的寡聚体之后,相关的寡聚体可通过PCR来扩增并被进行序列测定来译码寡聚体或其它化合物的情况。 After oligomers with ligand activity in the recognition, the relevant oligomers can be amplified and decoded to a case where other compounds or oligomers sequence determination by PCR.

如下面将更全面的讨论,寡聚体识别标记中用到的碱基的选择是通过寡聚体合成化学或标记准被暴露于其中的其它化学反应条件来决定的。 As more fully discussed below, selected nucleotide oligomers used in the identification mark is exposed to other chemical reaction conditions wherein the registration mark or by synthetic chemical oligomers determined. 因此,当采用需利用强酸的化学过程和化学现象时,由仅嘧啶C和T以及双密码组成的寡核苷酸的使用能表现出有价值。 Accordingly, when the chemical processes and chemical phenomena need to use a strong acid, by the use of only the pyrimidines C and T, and password of the oligonucleotide can be double exhibit valuable. 用类似方法,嘌呤核苷酸对强酸的不稳定可通过使用嘌呤核苷类似物来克服,例如7-去氮杂-脱氧腺苷和7-去氮杂-脱氧鸟苷(见本文引作参考的Barr et al.,1986,Bio Techniques 4:428-432和Scheit,Nucleotide Analogs:Synthesis and Biological Function PP.64-65(John Wiley and Sons,New York)。使用这些或其它类似物将允许使用四元或其它与二元相反的编码方法。因此,在优选的实施方案中,识别标记将是一个有约50至150个核苷酸长度的寡核苷酸,它由嘧啶或嘧啶和嘌呤类似物或在用于装配寡聚体库的偶联条件下不分解的任何类型的核苷组成。寡核苷酸识别标记可包含一个5′和一个3′扩展位点和选择性地包含对于寡聚体合成的每一步骤可能是特定的一个DNA序列测定的引物位点。 In a similar manner, a strong acid of purine nucleotides instability can be overcome by using the purine nucleoside analogs, such as 7-deaza - deoxyadenosine and 7-deaza - deoxyguanosine (herein incorporated by reference see of Barr et al, 1986, Bio Techniques 4:. 428-432 and Scheit, Nucleotide analogs:. Synthesis and Biological Function PP.64-65 (John Wiley and Sons, New York) using these or other analogs would permit the use of four mono- or with a dibasic opposite other encoding method. Thus, in a preferred embodiment, a recognition mark will be from about 50 to 150 nucleotides in length oligonucleotides, which consists of pyrimidine and purine analogs or pyrimidine or it does not decompose under the coupling conditions used to assemble the oligomer library of any type of nucleosides oligonucleotide identifying indicia may comprise a 5 'and a 3' extension contains sites for the oligomer and optionally each step of the synthesis thereof may be specific to a DNA sequencing primer site.

编码带有寡核苷酸的组合成方法为定位在从大库中分离出来的少量配体的直接结构分析中遇到的不定性和灵敏度的主要限制提供了一个方法。 Method of combining with an oligonucleotide encoding a targeting uncertainty is encountered in the direct analysis of the structure of a small ligand isolated from a large library major limitations in sensitivity and provide a method. DNA信息贮存的高容量可被开发归挡一个库结构的精确细节。 DNA Storage The development of high capacity can be a return block to the exact details of the library construction. 在下面的实施例中,采用了一个包括三个碱基(C7dA,dC,T)的2个相邻核苷酸的“密码系”结构,它能编码结合了32=9个氨基酸结构单元的合成(仅七个结构单元用于这个库的合成中)。 In the following embodiments, including uses a three bases (C7dA, dC, T) of two adjacent nucleotides "password-based" structure, which encodes a combination of 32 = 9 amino acids of the structural units synthesis of (only seven building blocks for the synthesis of the library). 如c7dG也被包括在编码模板中,那么采用1000个不同单体的组合成可通过使用仅5个核苷酸(45=1024)的“密码系”大小来供给。 As is also included in the encoded c7dG template, then to be supplied by using only five nucleotides (45 = 1024), "password-based" 1000 using a combination of different size monomers.

信息可被编码在长度中而不是或除了核苷酸序列中或编码在这方面的任何其它聚合寡聚标记中。 Information may be encoded in the length rather than, or in addition to the nucleotide sequence encoding any other polymeric or oligomeric tag in this regard. 只要长度被用来表示每一特定单体与寡聚体的加成,那么译码寡聚体的情况可通过如上所述的扩增寡核苷酸的标记和通过包括聚丙烯酸胺凝胶或毛细管凝胶电泳的按接大小分离的许多技术的任何一种来识别标记得以进行。 Long length is used to represent each specific monomer addition to the oligomer, the oligomer can be decoded where the nucleotide amplified by the oligonucleotides as described above and labeled by including or polyacrylamide gel capillary gel electrophoresis technique in any of a number of separate contact size recognition mark to proceed. 在寡聚体合成的给定步骤或每一不同化学反应步骤中加入的每一不同单体通过独特长度的寡核苷酸标记来表示。 Each different monomer added at a given oligomer synthesis step or steps each of the different chemical reactions is represented by the length of the unique oligonucleotide tag. 寡核苷酸标记包含扩增位点,例如PCR引物序列,它的序列被设计为以寡聚体或其它化学合成中的给定步骤序号为特征。 Oligonucleotide tag comprises an amplification sites, such as PCR primer sequences, which is designed to sequence a given step numbers oligomer or other chemical synthesis is characterized. 那么序列中任何给定位置的寡聚体组成的确定包括运用合成中的步骤为特征的PCR引物序列进行扩增标记和利用本领域熟知的技术如凝胶或毛细管电泳(利用正在标记的寡核苷酸作为标准)对扩增产物进行按大小分离。 Then determining any sequence a given position oligomer composition comprising the step of using synthesis amplified and labeled using techniques known in the art such as gel or capillary electrophoresis (using the feature being labeled PCR primer oligonucleotide sequences nucleotide as standard) The amplified product was separated by size. 当希望制备一个与引导序列有关的化合物库时,这个实施方案特别有用。 When desired to prepare a library of compounds related to the boot sequence, this embodiment is particularly useful. 在合成一个正在被模拟的位点的步骤期间,仅需要一个标记。 During a step of synthesizing being simulated site, only one marker.

除了长度,寡聚体序列信息还可编码在包括寡核苷酸标记的碱基序列中。 In addition to length, oligomer sequence information can also be encoded in a nucleotide sequence comprising the oligonucleotide tag. 这种类型密码不仅在每一偶联步骤中连接一个不同的寡核苷酸标记的实施方案中,而且在每一偶联步骤中扩展一预先存在的寡核苷酸标记的实施方案中都具有价值。 This type of password is not only connected to a different embodiment of the oligonucleotide labeled at each coupling step, and extend in each embodiment the coupling step labeled oligonucleotide in the presence of a previously having value. 例如,可采用达约100个碱基(或更长些)的寡核苷酸,如下所描述,其中每一个具有7个(或更多)区。 For example, up to about 100 bases can be used (longer or more) oligonucleotides, as described below, each having seven (or more) regions.

区1是3′-引物位点(20-25个碱基)。 Zone 1 is a 3'-primer site (20-25 bases). 这个位点被用来与另一个PCR位点一起(在寡核苷酸的5′末端)连接到通过PCR的原始扩增上。 This site is used together (at the oligonucleotide 5'end) connected to the other site to the original PCR amplified by PCR. 也可使用另一个扩增方法。 Another amplification method may also be used.

区2是一个“步骤特有的”DNA序列测定引物位点(15-20个碱基)。 Zone 2 is a "step-specific" DNA sequencing primer site (15-20 bases). 这个位点对于合成系列中的特有的给编号的步骤是特有的。 For this locus synthesis step in the series is specific to a specific number. 在特定步骤加入到所有小球中的全部的寡核苷酸将共同具有这个序列。 In a particular step was added to all of the oligonucleotides in all of the pellet will have in common the sequence. 每一给编号的步骤将具有一个代表那个步骤的高度特殊的引物位点。 Each numbered step will have a highly specific primer site representing that step.

区3是一个间隔区(20-30个碱基)。 Zone 3 is a spacer (20-30 bases). 可定长度的间隔区区段,但优选为20至30个碱基长,将编码位点放置于离序列测定引物位点足够远以提供良好的对单体编码或识别区的阅读。 Spacer section length may be set, but preferably 20 to 30 bases in length, coding site primer site placed sufficiently far sequenced to provide a good reading of the monomer encoding or identification region.

区4是单体识别区(8个碱基)。 4 is a monomer identification region region (8 bases). 在这个说明性实施方案中,在8位信息串的每一个碱基代表二进制密码的一个单位,其中例如T=0和C=1。 In this illustrative embodiment, eight bits per a unit of a base representing a binary information code string, for example, where T = 0 and C = 1. 每一组步骤特有的识别标记由在8个位置的每一个上的带有一个1(C)或一个O(T)的8个碱基组成。 Each set of steps by the unique identifying marks on each of the eight locations with a 1 (C) or a O (T) in 8 bases. 每一个单体类型通过这些“开关”的1至8的混合来编码。 Each monomer type is encoded by mixing the "switch" is 1 to 8.

区5是步骤序号确定区(为3区域的区别在每一边上4个碱基加上2个碱基)。 Region 5 is a step number determining region (region 3 in order to distinguish each side of the 4 bases plus 2 bases). 在这个短的范围中的四个单位编码步骤序号。 Four coding units in this step numbers in the short range. 这对于序列测定引物是多余的,但可用来确认适宜的引物被使用和正确的步骤被译码。 This is for the sequencing primer excess, but can be used to confirm the step of suitable primers are used and are decoded correctly.

区6是单体识别标记(8个碱基)的重复,这个区域具有如区4相同的信息,并被用来确认单体情况。 6 is a monomer identification mark region (8 bases) repeated, this region has the same information as region 4, and is used to confirm monomer situation. 安装这个第二个单体编码区域还增加了获得良好序列测定“阅读”的可能性。 Mounting this second monomer encoding region also increases the possibility of obtaining a good sequencing "read" the.

区域7是5′-PCR引物位点(20至25个碱基)。 Region 7 is 5'-PCR primer site (20-25 bases). 这个位点作为很大用于序列扩增的第二PCR引物的位点。 This site is used as a large amplification sites a second PCR primer. 带有全部七个这些特性,其中一些为选择性的寡核苷酸的长度,将普遍在75和125个碱基之间。 With all seven of these features, some of which are selective oligonucleotide length will generally between 75 and 125 bases.

一个八个二进位数格式可编码256种不同单体类型。 An eight digit binary format can encode 256 different monomer types. 可被编码的步数由现有的步骤特有的组(每组8个)的寡核苷酸决定。 Number of steps can be encoded by the conventional steps specific groups (8) an oligonucleotide decision. 使用10组(80个核苷酸),可编码达256个不同的被装配到达10个单位长度的寡聚体上的单体(因此提供了编码达25610=1.2×1024个寡聚体序列的能力)。 10 using group (80 nucleotides), can encode up to 256 different monomers are assembled on the oligomer reaches 10 units long (thus providing encoding of 25610 = 1.2 × 1024 oligomer sequences th ability). 可采用编码的识别标记,这样每一个单体被指定一个特定的二进制数(例Ala=00000001,Gly=00000110等)。 The identification mark can be encoded, such that each monomer is assigned a specific binary number (for example Ala = 00000001, Gly = 00000110, etc.). 组合适宜的寡核苷酸来提供正确的二进制密码。 Suitable combinations of oligonucleotides to provide the correct binary code.

为了便于寡核苷酸标记识别,有许多选择。 To facilitate oligonucleotide tag identification, there are many choices. 例如,可通过序列测定式杂交直接从球上阅读标记。 For example, the ball may be read from the tag by direct sequencing of formula hybridization. 还可扩增寡核苷酸标记以便于标记识别,由一单一固体载体或寡聚体携带的寡核苷酸识别标记可通过体内克隆或体外例如PCR方法扩增。 Amplification oligonucleotides may also be labeled to facilitate identification mark, by a single solid support or oligomer oligonucleotide carrying identifying indicia may be amplified by PCR and cloned in vivo or in vitro, for example. 如果检测的限制为100个分子的顺序,那么至少将需要球上的每一寡核苷酸标记100或更多的拷贝。 If the detection limit is of the order of 100 molecules, then each oligonucleotide tag on the required ball least 100 nucleotides or more copies. 通过多种方法的任何一种,其中的一些将在下面描述,制备单链寡核苷酸,双链核酸,或单和双链核酸的混合物形式的标记拷贝并且对扩增的物质进行序列测定。 By any of a variety of methods, some of which will be described below, preparation of single stranded oligonucleotides, double stranded nucleic acid, or in the form of a mixture of single and double stranded nucleic acid labeled substance copied and amplified sequence determination . 在本发明的一个实施方案中,其中在每一单体加成步骤加入一分离和特殊的寡核苷酸标记(与每一步扩展现有的标记相反),可立即扩增所有标记,并且接着将扩增的物质与对于每一类型的标记采用不同的序列测定的引物)具有与寡聚体合成步骤一样多的独立的序列测定反应。 In one embodiment of the present invention, wherein each monomer addition step of adding a separate and specific oligonucleotide tag (and extend existing tag at each step opposite), immediately amplify all markers, and then the amplified for each type of substance labeled with different sequencing primers) having synthetic steps oligomer with as many separate sequencing reactions. 在这个实施方案中,通过引物序列的适当选择,还可标记使每一标记可从其它标记分别被扩增。 In this embodiment, by appropriately selecting primer sequences, but also that each mark marking can be amplified separately from the other labels. 完成序列测定反应,在标准序列测定凝胶上进行电泳,寡聚体序列由得到的序列信息揭示的密码推出。 Completed sequencing reaction was electrophoresed on a standard sequencing gel, the oligomer sequence information disclosed in the sequence introduced by the password obtained.

一个供选择的方案是使用普通的PCR引物和普通的序列测定引物(序列测定引物甚至可以全部或部分地与PCR引物位点重叠),通过杂交到至与珠的寡核酸中的每一步骤特有的序列互补的寡核苷酸探针上来识别该步骤。 Scheme An alternative is to use common PCR primers and common sequencing primer (sequencing primer may even be wholly or partially overlap with the PCR primer site), by hybridization to at peculiar to each step of oligonucleotides beads in sequence complementary to an oligonucleotide probe used to identify both the step. 单组的序列测定反应在所有扩增的来自单珠的寡核苷酸上进行,反应产物在凝胶的单组道上电泳,接着将反应产物转移至一适宜的杂交膜上,杂交至单一步骤特有的探针上(见本文引作参考的,Maniatis et al.,Cold Spring Harbor Laboratory,ColdSpring Harboo,NY(1982))。 A single set of sequencing reactions on all of the amplified oligonucleotides from a single bead, the reaction product gel electrophoresis a single set of tracks, then the reaction product was transferred to a suitable hybridization membrane and hybridized to a single step the specific probe (see herein incorporated by reference, Maniatis et al., Cold Spring Harbor Laboratory, ColdSpring Harboo, NY (1982)). 在检测得到的信号后,将探针从膜上洗掉,另一个步骤特有的探针被杂交。 After the obtained detection signal, the probe is washed away from the membrane, another step-specific probe is hybridized. 还可使用描述在本文引作参考的EPO公开号为237,362和PCT公开号为89/11548。 Described may also be used herein incorporated by reference in EPO Publication No. 237,362 and PCT Publication No. 89/11548.

平行杂交为顺序性杂交提供了一种选择。 Hybridization is provided a parallel to sequential hybridization selection. 将序列测定反应分成与肽合成步骤数目相等的许多等分试样,在序列测定凝胶上在每一个的独立的组道上进行电泳。 The sequencing reaction was divided into a number of aliquots equal to the number of peptide synthesis steps, electrophoresed in separate groups of each of the tracks on the sequencing gel. 在将反应产物转移至一适宜的膜之后,该膜被切下来分开各组道,接着每一道组杂交至许多步骤特有的寡核苷酸探针中的一个上。 After the reaction product was transferred to a suitable membrane, the membrane is cut off to separate channels of each group, every group is then hybridized to the number of steps specific oligonucleotide probes in a. (见本文引作参考的“Uniplex DNA Sequencing”and“MultiplexDNA Sequencing,”in Plex Luminescent kits Protuct Catalog,Bed-ford,MA,1990)。 (Incorporated herein by reference see "Uniplex DNA Sequencing" and "MultiplexDNA Sequencing," in Plex Luminescent kits Protuct Catalog, Bed-ford, MA, 1990).

如上所述,单合成固体载体(或一带有标记的连接珠,或在“井”中的溶液中)可仅包括每个寡核苷酸标记的几百个拷贝。 As described above, a single synthesis solid support (or a connection with the mark beads, or "well" in a solution) may include only each oligonucleotide tag hundreds of copies. 通过PCR或其它本领域技术人员熟知的方法可将这些标记扩增以提供足量的将要被准确地进行序列测定的DNA。 By PCR, or other methods well known to those skilled in the art may be labeled to provide a sufficient amount of amplification to be accurately DNA sequence determination. 译码寡聚体的能力依赖于可获得的寡核苷酸识别标记的数目,从可获得的标记能达到的扩增量以及对那个扩增的DNA进行序列测定的准确性。 Ability to decode the oligomers depends on the number of oligonucleotides available identification mark, obtainable from the amount of amplified marker can be achieved as well as for the accuracy of the amplified DNA sequence determination.

如果采用寡核苷酸识别标记的PCR扩增,可能遇到“PCR产物污染”,它由PCR反应的产物引起,污染用来扩增其它具有相同PCR引物结合位点的标记的后来的PCR反应混合物。 If the identification tag oligonucleotide PCR amplification, may encounter "PCR product contamination," which is caused by the product of the PCR reactions, to amplify other contamination subsequent PCR reactions with the same numerals PCR primer binding site of mixture. 通过将不稳定性引入产物序列,处理后来反应以致消灭从前面反应带下来的可能的污染可预防这个问题。 By introducing instability product sequences, then the reaction process such that the reactions may eliminate contamination brought down from the front can prevent this problem. 这种方法的一个特殊的实施例是将dUMP引入产物,其中购得的全套由PECI and Life Techndogies出售。 A particular embodiment of this method is the introduction of dUMP product, wherein a full set of available sold by PECI and Life Techndogies. 用尿嘧啶N-糖苷酶处理每一新的PCR反应降解了出现的任何含dUM的DNA,这样防止了污染的扩增。 N- glycosidase treatment with uracil each new PCR reaction containing degraded DNA dUM any occurring, thus preventing contamination of amplification. 不包含dU(仅含dT)的模板DNA不受影响,当然,在扩增开始前将糖苷酶去除或失活。 It does not contain dU (containing only dT) template DNA will not be affected, of course, prior to the start of amplification glycosidase is removed or inactivated.

上面描述的一些肽合成标记具仅有包含嘧啶的独特特点。 Some synthetic peptides having the above-described marker comprising only the unique characteristics pyrimidine. 这意味着尿嘧啶糖苷酶方法(本文引作参考的PerkinElmer Cetus Instruments(PECI)Catalog,Alameda(1991))将在产生的这些链的仅仅一半中即那些含T′S(或U′S)的中起作用。 This means that the uracil glycosidase method (herein incorporated by reference PerkinElmer Cetus Instruments (PECI) Catalog, Alameda (1991)) will be contained in those i.e. only half of the chains produced by T'S (or U'S) of play a role. 不能将dUMP引入到互补的仅有嘌呤的链中;然而嘌呤链极易受酸脱嘌呤作用和主链的碱引起的裂开的损伤。 dUMP can not only be introduced into the strand complementary to the purine; however easily cleaved purine strand damage acid depurination and backbone caused by alkali. 这些处理的组合可大大地降低产物污染带来的问题。 The combination process can greatly reduce the problems caused by contamination of the product. 防止携带的污染的另一种方法包括在怀疑被标记污染的反应扩增之前将一限制性位点(EarI可用作多嘧啶标记)掺入到寡核苷酸标记中,并用相应的限制性酶消化。 Another method of preventing contamination carried suspected comprising labeled prior to amplification reaction contaminated with a restriction site (EarI multi-pyrimidin useful marker) is incorporated into the oligonucleotide tag, with the corresponding restriction enzymatic digestion. 这个方法仅当将要扩增的标记不被酶所裂开时才起作用,它通常是单链寡核苷酸标记的情况。 This method only when the mark is not to be amplified is cleaved by an enzyme function, it is usually the case of a single-stranded labeled oligonucleotide.

对于序列测定的扩增的DNA,通常期望产生单链模板。 For sequencing amplified DNA is usually desirable to generate single-stranded template. 可通过任何一种方法来产生。 It may be produced by any method. 其中之一的方法是不对称PCR,其中使用过量的一种引物来扩增一条链至高于其它的10至100倍的量(见本文引作参考的例如US专利号为5,006,584)。 The method is one wherein asymmetric PCR, where an excess of one primer to amplify one strand to an amount other than 10 to 100 times (see, for example, incorporated herein by reference to US Patent No. 5,006,584). 提供一单链模板的另一种方法是通过生物素标记一种引物,通过吸附至固定化链毒亲和素来纯化或去除所得链(本文引作参考的Pierce Immunotechnology Catalog andHandbook,1991)。 Another method for providing a single-stranded template is labeled by biotin one primer, strand by adsorption to immobilized drug affinity purification or removal always resulting chain (herein incorporated by reference Pierce Immunotechnology Catalog andHandbook, 1991). 然而另一种方法包括从RNA聚合酶启动子产生的RNA转录物(代表仅一条链)以及用反转录酶序列测定转录物(本文引作参考的Sommer et al.,Chapter 25,InPCR Protocols:A Guide to Method and Applications,Supra)。 Yet another method includes RNA transcripts (representing only one strand) and determining the reverse transcriptase transcripts using sequences (incorporated herein by reference Sommer et al generated from the RNA polymerase promoter, Chapter 25, InPCR Protocols.: A Guide to Method and Applications, Supra). 如果标记仅由嘧啶核苷酸组成,那么所有嘌呤可通过酸/碱基处理被消去,留下序列测定的嘧啶链。 If only marked by pyrimidine nucleotides, then all purine by acid / base treatment is eliminated, leaving the pyrimidine strand sequence determination.

每一步骤特有的寡核苷酸的独立的序列测定引物的使用要求每一步骤特有的引物的独立方便的序列测定反应。 Independent of each step-specific oligonucleotide sequence was determined using primers to facilitate independence primer sequences specific for each step of the reaction was measured. 被区别性标记的引物的使用允许来自单一固体载体的识别标记在单一反应中被序列测定,并在凝胶上的单道组(当仅为多嘧啶时为2个道;如果使用4个不同碱基则为4个道)中电泳。 Used distinctive labeled primers allow identification tag from a single solid support to be sequenced in a single reaction, and a single channel group on the gel (when only two channels of the multi pyrimidine; if four different 4, compared with the nucleotide Road) electrophoresis. 目前具有适于此目的的用可辨别的荧光基团标记的可购得到的引物(被本文引作参考的ABI Catalog)还可使用目前可购得到的一系列化学发光标记(本文引作参考的Bron-stein et al.,BioTechniques 8:310-314(1990)被扩增的产物可被容易地进行序列测定或通过其它方法被识别来译码球上的肽或其它分子的情况或通过其它方法连接至寡核苷酸的标记上。为了此目的,可使用多种序列测定方法中的一种,其中包括通过序列特有的探针杂交的序列测定。本发明中可采用的DNA序列测定酶包括Taq DNA聚合酶,E.Coli DNA聚合酶(或Klenow片断),T7聚合酶,SequenaseTM测序酶和Sequenase IITM测序酶(被修饰的T7DNA聚合酶),BSt DNA聚合酶和反转录酶(来自本文引作参考的AMV,MMLV,RSV,etc,see USB Enzymes for DNA Sequencing,VS BiochemicalCorp,1991,Clevaland OH)。寡核苷酸标记的序列还可通过高可靠性DNA杂交技术来 May also be obtained using the current commercially available series of chemiluminescent labels currently used having distinguishable fluorophore tag suitable for this purpose may be commercially available primers (incorporated by reference herein ABI Catalog) (incorporated herein by reference Bron-stein et al, BioTechniques 8:. 310-314 (1990) was amplified products can be easily sequenced or otherwise identified to the case of a peptide or other molecule coding on the ball by other methods or by other methods coupled to the oligonucleotide tag. for this purpose, an assay method may be used in a variety of sequences, including a sequence determined by sequence-specific probe hybridizes. the present invention can be employed in DNA sequencing enzymes include taq DNA polymerase, E.Coli DNA polymerase (Klenow fragment or), T7 polymerase, SequenaseTM and Sequenase IITM Sequenase Sequenase (modified T7DNA polymerase), BSt DNA polymerase and reverse transcriptase (from herein incorporated by reference AMV, MMLV, RSV, etc, see USB Enzymes for DNA Sequencing, VS BiochemicalCorp, 1991, Clevaland OH). the sequence of the oligonucleotide may be labeled by a high reliability DNA hybridization techniques 识别,为此,大量带有寡核苷酸的固定化聚合酶合成可能是有益处的(见本文引作参考的PCT专利公开号92/10587和92/10588)。 Identifying, for a large number of immobilized polymerase synthesis with oligonucleotides may be beneficial (see herein incorporated by reference, PCT Patent Publication No. 92/10587 and 92/10588).

无论标记是寡核苷酸还是一些其它分子结构,标记的选择依赖于组成库的分子的属性和合成这些分子的方法,它们将在下面讨论。 Whether labeled oligonucleotides or other molecular structures, methods that rely on the choice of label attributes and synthetic molecular libraries of these molecules, which will be discussed below.

在合成化学过程包括使用与上述寡核苷酸标记不相容的试剂和反应条件时,可望使用替代的标记方法。 In the synthesis of chemical processes include the use of labeled reagents and reaction conditions incompatible with the above-described oligonucleotides, expected to use an alternative labeling method. 因此,合成本发明的标记分子库的方法还期望利用通过一系列方法如色谱方法能单个地解析的化学惰性烃标记分子。 Thus, the method of synthesizing labeled molecule libraries of the present invention is further desirable to use a chemically inert hydrocarbon molecule labeled by a series of chromatographic methods such as a single method can be resolved.

分子库中的这些隋性烃标记的使用已得到了描述。 Using these inert hydrocarbon labeled molecule library has been described. 见均被本文引作参考的Michael HJ Ohlmeyer,et al.,Proc.Nat′1A-cad.Sci.90:10922-26(1993年12月)和已公开的PCT申请号为WO94/08951。 See are incorporated herein by reference Michael HJ Ohlmeyer, et al, Proc.Nat'1A-cad.Sci.90:. 10922-26 (December 1993) and published PCT Application No. WO94 / 08951.

描述的标记运用象本文所述的二进制编码方法,二进制密码被用来规定每一化学结构单元,即在合成中要加入的氨基酸。 Labeled as described herein using methods described in binary-coded, the binary code is used to define the chemical structure of each amino acid units, i.e., to be added in the synthesis. 密码的长度可依赖于加入的结构单元的总数目。 The password may depend on the total number of structural units added. 例如,在仅有总共七个要加入的结构单元时,可用三位二进制密码。 For example, when only a total of seven structural units to be added, the available three-digit binary code. 这允许七个独立不同的密码,从001至111,每一个用来规定七个结构单元的一个,例如赖氨酸=001。 This allows independent seven different passwords, from 001 to 111, for each of a predetermined one of the seven structural unit, such as lysine = 001. 结构单元的数目越大,可使用的密码越大,例如,如本文所述的一个八位二进制密码。 The larger the number of the structural units, the greater the password can be used, e.g., as described herein, an eight-bit binary code.

运用色谱方法制备许多标记,其中每一标记具有与其它标记不同的分辨或分离图形。 Numerous labels prepared using chromatographic methods, wherein each labeled with other markers having a different resolution or separation pattern. 如果出现一特殊标记,它将在给定合成分子的最终标记密码的每一个位置上显示一个“1”。 If a particular mark appears, it displays a "1" in the final position of each code mark given on the synthetic molecules. 因此,在一个分子具有四个结构单元,以三位编码系统编码时,具有12个可能的密码数字,每一个结构单元具有三位数。 Accordingly, in one molecule, a structural unit having four, three encoding to the coding system, having 12 possible digital code, each structural unit having three digits. 对于合成中的每一步,标记分子将要在其上被合成的固体载体,以致不仅显示加入的结构单元,而且显示加入时所在的步骤。 The solid support at each step in the synthesis, labeled molecule to be synthesized thereon, so that not only the structure of the display unit is added, but the step where the display is added. 例如,标记是1或“T1”的出现表明在第一步加入的结构单元的二进制标记密码的第一位上有一个“1”。 For example, a marker or "T1" indicates that there is an occurrence of a "1" is added in the first step on a first binary code marks structural units. 类似地,T7的出现表明在所有密码的第七位上有一个“1”,在一个三个数字的密码中,它将同样对应于第三个加入的结构单元的第一个位置。 Similarly, there appear T7 indicate a "1" on the seventh of passwords, in a three-digit password, it also corresponds to the third position of the first join structural unit. 因此规定为密码111的结构单元,如果在位置1加入,将由出现标记T1,T2和T3来编码。 Therefore predetermined password 111 structural units, if added at 1 position, by a flag T1, T2 and T3 encoded. 换句话说,如果在步骤2加入,相同的结构单元将由出现标记T4,T5和T6来编码。 In other words, if added in step 2, marked by the same structural units T4, T5 and T6 appear to encode.

由Ohlmeyer描述的特殊烃标记具有下列结构: Particular hydrocarbons which may be described Ohlmeyer marked by the following structure:

其中n为1至10。 Wherein n is 1 to 10. 烃链变化的长度和被改变的卤代基团使其能通过色谱方法,具体地为俘获氯相色谱法进行标记的物理分离或分辨。 And varying hydrocarbon chain length is changed so as to pass halo groups chromatographic methods, in particular physical separation or resolution of labeled capture chloro chromatography.

然而,这些烃标记的检测受检测方法灵敏度的限制。 However, these are hydrocarbons labeled by limiting detection sensitivity. 因此,为了确保标记的检测,必须用大量的标记。 Therefore, to ensure the detection of the marker, the marker must be large. 这要求大量分级的微球,这样降低了可被合成,并被增加了反应时间的保证标记最大量地偶联至固体载体上的所有库的大小。 This requires a large number of grading microspheres, which can be synthesized is reduced, and the reaction time increases the size of the largest amount of labeled conjugate to ensure all the libraries to a solid support. 最后,在筛选较大分子的地方,更多的烃标记被加入至固体载体中,在固体载体上的大量烃的掺入,例如,具有大量标记的大合成化合物,由于空间,疏水或离子相互作用将有可能对连续合成和/或固体载体上的化合物的筛选产生不利影响。 Finally, where the screening of larger molecules, more markers are added to the hydrocarbon solid support, the incorporation of a large amount of hydrocarbons on the solid support, e.g., having a large number of compounds synthesized labeled, due to space, another hydrophobic or ion effect will likely adversely affect the screening of compounds in the continuous synthesis and / or solid carriers.

与这些烃标记相关的可检测性,标记时间,和筛选或合成干扰的问题可运用本发明的方法来防止发生。 These hydrocarbons associated with detectable markers, time markers, and screening or interference problems can be synthesized using the method of the present invention to prevent. 特别地,在本发明的一个实施方案中提供了一种动用烃标记的标记方法,其中这种标记具有一“分子钩”来代替如Ohlmeyer等描述的可检测电泳,这种“分子钩”在本文被定义为允许连接可扩增,可检测基团的标记上的官能基团,因此使固体载体上的标记的检测成为可能。 In particular, there is provided in one embodiment of the present invention use a method of marking marks a hydrocarbon, wherein such mark has a "hook elements" instead of as described in Ohlmeyer et electrophoresis may be detected, such "molecular hook" in herein is defined to allow connection amplifiable, detectable functional groups on the labeling group, and therefore the detection mark on a solid support becomes possible. 这个钩一般包括一稳定的官能基团或分子,这个分子与可扩展可检测的基团形成共价键或与那个基团的一部分具有高度的亲和性。 The hook includes a generally stable molecule or a functional group, this group may be extended molecule detectable form a covalent bond or a part of that group having high affinity. 这种钩包括例如生物素,连接了可扩增可检测的基团的链霉亲和霉可被结合到其上,或高结合肽的补体。 The hook comprises a biotin e.g., streptavidin connected mold amplifiable groups may be detectably incorporated thereto, or high-complement binding peptides. 这种高结合肽一般包括对另一个具高亲和性的肽的互补对。 Such a peptide generally comprises a high binding affinity for another peptide with high complementarity yes. 因此,补体指的是这样一对中的一个肽。 Thus, complement refers to a peptide of such a pair. 高结合肽被概括地描述在1994年10月12日提出的美国申请系列号为08/321,933中,它是1993年5月24日提出的美国申请号为08/067,387的部分继续申请,每一篇均被本文引作参考。 High binding peptides are generally described in the October 12, 1994 raised US Application Serial No. 08 / 321,933, which is 24 May 1993, submitted as part of US Application No. 08 / 067,387 a continuation, each articles are incorporated herein by reference. 另一方面,钩可包括被保护的可活化基团,它可被活化共价性地将可扩增可检测基团连于标记上。 On the other hand, the hook may include protected activatable groups, which can be activated to covalently the exemplary amplifiable detectable group attached to the tag. 被光保护的活化基团在运种应用中特别有用。 Activating the light protective groups are particularly useful in a variety of applications run. 活化基团在本领域中普遍熟知,包括这些基团,如羧基,羟基,氨基,硫羟和类似物。 Activating group generally known in the art, including such groups, such as carboxyl, hydroxyl, amino, thiol and the like. 动用对光不要的保护基团例如在为所有目的被本文引作参考的已公开的PCT申请号WO93/22680中的描述的那些可保护这些基团。 Do not use the light in, for example, protecting groups are incorporated for all purposes by reference herein disclosed has been described in PCT Application No. WO93 / 22680, those groups may be protected by these groups. 得到的基团为可光敏化的。 The resulting group is photoactivatable.

除上所述,分子钩可包括多官能基团。 In addition to the above, the molecular hook may include a plurality of functional groups. 例如,分子钩可包括两或多个能被偶联至两个不同本体上的不同官能基团。 For example, the molecular hook may comprise two or more different can be conjugated to two different functional groups on the body. 可为这种情况,例如为了检测目的希望将标记再偶联至另一个固体载体上,例如微量滴定盘中的反应井。 This may be the case, for example, for detection purposes desired mark and then coupled to the other solid carriers such as reaction wells of a microtiter plate. 第一个官能基团可用来选择性地结合固体载体上的互补基团。 The first functional group can be used to selectively bind a complementary group on the solid support. 一旦被偶联,第二个官能基团可用于可扩增可检测基团的选择性偶联上。 Once coupled, a second functional groups can be used for selective coupling may amplify a detectable group. 这种钩一般包括本文描述的官能基团或能被选择性地结合到其它这种基团上的其它基团的组合。 Such functional groups include a hook generally described herein can be selectively coupled to or in combination with other groups on other such groups. 例如一个实施例,这一钩可包括正交性地连接至烃标记的生物素和digoxin。 One embodiment for example, this may include a hook connected to the orthogonality hydrocarbon labeled biotin and digoxin. 一旦被分离,这些标记可与固体载体例如微量滴定井接触,这种固体载体被覆盖一种能与标记上的一个官能基团例如抗digoxin的抗体结合的基团,并被允许与它们结合。 Once isolated, these markers may be, for example, microtiter wells in contact with a solid carrier, this solid carrier can be one kind of group is covered with a functional group on the marker, for example, anti-digoxin antibody binding, and allowed to bind to them. 在重复洗涤步骤之后,将固体载体与寡核苷酸接触,寡核苷酸被偶联至能结合到第二个官能基团如前面所述的链霉亲和素连接的寡核苷酸上的基团上。 After repeating the washing step, contacting the solid support with an oligonucleotide, the oligonucleotide is capable of binding to the conjugated to a second functional group as previously described and streptavidin-linked oligonucleotide of the group. 接着如前所述检测结合的寡核苷酸。 Followed by detection of the bound oligonucleotides as previously described. 本发明的烃标记的合成可通过本领域熟知的方法来进行。 Labeled synthetic hydrocarbons of the present invention can be carried out by methods known in the art. 参见例如March,Advanced Organic Chemistry(John Wiley& Sons,3rd Ed.,1985),Larock,Comprehensive Organic Trans-formations CVCH Pubishers,1989)。 See, for example, March, Advanced Organic Chemistry (John Wiley & amp; Sons, 3rd Ed, 1985.), Larock, Comprehensive Organic Trans-formations CVCH Pubishers, 1989).

由于本发明的惰性烃标记提供了更为灵敏的烃标记的检测,固体载体上的特殊标记的量可在不影响它的可检测性的情况下被降低。 Since the present invention an inert hydrocarbon marker provides a more sensitive detection of hydrocarbons labeled specific amount of label on the solid carrier can be reduced without affecting the case of its detectability. 进一步,通过降低固体载体上的标记的量。 Further, by reducing the amount of label on a solid support. 将标记偶联至载体上所需的时间被缩短。 The time required label is conjugated to the carrier is shortened.

本发明中有用的标记将一般地包括可得到的烃区和分子钩。 Useful labels in the present invention generally will include hydrocarbons available area and molecular hook. 更优选地,这种标记将包括将标记与固体载体相连的可裂解的接头,所述的分子钩和将分子钩与接头相连的变化长度的烃链。 More preferably, such a marker would include a cleavable linker attached to a solid support and labeled, the hooks of the hydrocarbon chain molecules of varying length and connecting the linker and molecular hook. 不同标记将具有不同长度的烃链或不同的分子钩以便于它们的物理分离和检测。 Hydrocarbon chains having different labels or different molecules of different lengths so that the hooks to their physical separation and detection.

具有下列通式结构的标记为优选的: Tag having the following general structure are preferred: 其中n为1至10,X为可裂解的接头,R为分子钩。 Wherein n is 1 to 10, X is a cleavable linker, R is a molecular hook. 优选的分子钩包括例如生物素,高结合肽和可活化基团,例如可光敏化的基团或它们的组合。 Preferred molecules include hooks such as biotin, peptides and high binding activatable group, for example a photosensitizer group or a combination thereof.

本发明中有用的可裂解的接头包括例如为所有目的被本文引作参考的1994年6月23日提出的美国申请号08/265090中描述的可光裂解的接头。 Useful in the invention may comprise, for example, be cleavable linker photocleavable U.S. Application No. 08/265090 June 23, 1994 proposed for all purposes by reference is incorporated herein described linkers. 这种可光裂解的接头包括具有下列结构的那些物质: Such photocleavable linkers include those having the following structure: 在合成和标记之后,通过例如接头的光解将标记从固体载体上去除。 After synthesis and tag, the tag is removed from the solid support by a linker e.g. photolysis. 接着用维持标记分离图形的方法,例如对部分收集品的HPLC,或例如凝胶或毛细管电泳的其它色谱方法将标记各自分开。 Followed by separation method for maintaining mark pattern, for example, the HPLC fraction collection, or other chromatographic methods, for example gel or capillary electrophoresis each separately labeled. 因为标记通常以常规方式如吸光度等不能检测的量出现,它们必须以允许后续检测,与它们分离图形相关的方式被分离。 Since the amount of labeled generally as absorbance can not be detected occurs in a conventional manner, they must allow subsequent detection, separated from their separate graphics-related manner. 作为一个实施例,从固体载体上裂解的标记在HPLC柱上被分离,并收集在部分收集器中。 As one example, cleavage of the tag from the solid support is separated in the HPLC column, and collected in the fraction collector. 当标记被进行最后的检测时,下面将进一步详细描述,对于用在标记/合成的所有标记,表示标记存在的部分与已知的洗脱分布型或分离图形相关。 When the flag is the final detection, will be described in further detail below, with respect to the mark / synthesizing all markers, the presence of a mark part with a known separation or elution profile related graphics.

接着根据它们的分离图形将分离的标记固定。 Then according to their fixed pattern separating the separated marker. 这种固定可采取点样各部分,为凝胶为基础的分离点样,或在反应井即在微量滴定盘中固定的形式。 This fixing can take spotted portions, gel-based separation spotted, or immobilized form i.e. in the reaction wells in the microtiter plate.

一旦被固定,标记可被用钩连结到可扩增增可检测的基团上,可扩增可检测基团一般包括能被扩增或产生能被扩增的信号的化合物或结构。 Once fixed, the indicia may be coupled to a hook on the groups may be detectable by amplification, amplifiable typically include detectable group can be amplified or amplified signal to produce a compound or structure. 能指数级扩增的化合物为优选的。 Compound capable of exponential amplification is preferred. 特别有用的可扩增可检测基团是一个寡核苷酸序列。 Particularly useful amplifiable detectable group is a oligonucleotide sequence. 可扩增可检测的基团的用钩连结可采取多种形式。 Detectable group can be amplified coupled with a hook may take many forms. 例如,在钩包括一个生物素基团时,将寡核苷酸偶联到将紧紧地与生物素结合的链亲和素上。 For example, the hook comprises a biotin group was coupled to an oligonucleotide bound tightly to the biotin-streptavidin. 另一方面,可在后面接着加入生物素标记的寡核苷酸的一个中间步骤中加入链霉亲和素。 On the other hand, an intermediate step may be followed by a biotin-labeled oligonucleotide was added biotin streptavidin later. 在一个选择实施方案中,标记可包括高结合肽的补体。 In an alternative embodiment, the indicia may include complement high binding peptides. 在这种情况下,寡核苷酸与肽的其它补体结合,这样寡核苷酸可紧紧地与标记结合。 In this case, the oligonucleotide complement in combination with other peptides, oligonucleotides may be so tightly bound labeled. 然而在另一个实施例中,在标记包括由如以前被引作参考的已公开的PCF申请号为WO93/22680中描述的对光不稳的保护基团保护的活化基团,这个基团可通过对光不稳基团的光解被活化,使接着寡核苷酸通过本领域熟知的方法被偶联至标记上。 Embodiment, the tag includes a PCF published application number as previously incorporated by reference for the photolabile protecting group of activating groups in WO93 / 22680 is described, however, this group may be in another photolysis by light unstable group is activated, so that the oligonucleotide is then coupled to the marker by a method known in the art. 在这种情况下,如果使用可期望选择具有与可光裂解的接头不同的光解特性的对光不稳的保护基团。 In this case, if the desired choice using photolabile protecting groups and having a photocleavable linker photolysis different characteristics. 这将允许在不活化分子钩的情况下标记选择性地从固体载体上裂解。 This will allow the mark to selectively cleaved from the solid support without the activated molecules of the hook.

一旦被钩连接到标记上,使用本文描述的PCR技术可扩增寡核苷酸序列,当固定采用印迹形式时,必须进行扩增以保持局部浓度和避免扩增的寡核苷酸的扩散,因此使它能检测并与分离方法相应。 Once hooking to the tag, using PCR techniques described herein can be amplified oligonucleotide sequence employed when the fixed blot form must be amplified to maintain the local concentration and diffusion-amplification oligonucleotides, Thus it can be detected and corresponding to the separation process. 例如,这些可通过进行印迹的凝胶重叠中的扩增反应来完成。 For example, the amplification reaction can be obtained by overlapping these gels were blotted in to complete.

被钩住的寡核苷酸和由此的标记的检测可通过将一标记掺入到被扩增的寡核苷酸中或通过探测其中这个序列为已知的被扩增的寡核苷酸序列来完成。 Snagging the oligonucleotides and thus may be detectably labeled by a label incorporated into the amplified oligonucleotide probe or by sequence wherein this is known to be amplified oligonucleotide sequence to complete. 标记的检测与已知的标记分离方法相对应。 Detecting labeled markers known separation methods, respectively. 再将如此识别的标记掺入到被合成分子的所有标记密码中。 Tag so identified then are incorporated into the password all labeled synthetic molecule. 仅为实施例目的,如果根据HPLC分离,用钩连接,扩增和探测,数码#17表示标记相对应。 Example embodiments purposes only, if the corresponding separated by HPLC, connecting hooks, amplification and detection, # 17 represents a digital mark. 例如,如果这是标记#15,那么“1”可用来表示特殊载体上的被合成分子的所有二进制密码的位置#5。 For example, if it is labeled # 15, the "1" may be used to represent the binary code are all synthesized molecules on a particular carrier position # 5. 本文描述的这种编码方法是仅为了实施例的目的,本领域的技术人员将理解为多种编码方法可被用于本发明的方法中。 This coding method is described herein only for the purpose of embodiments, those skilled in the art will appreciate that various methods of encoding methods can be used in the present invention.

本领域的技术人员还将认识到不需要将报导的合成分子的序列的密码包含在各标记的单一聚合序列中。 Those skilled in the art will also recognize that a synthetic coding sequence need not be reported in the molecule contained in a single polymerization sequence of each tag. 相反,密码可通过固体载体上的各个不同标记的存在不存在来体现。 In contrast, the absence of the password can be embodied by various marks present on the solid support. 虽然这些标记可能被顺序地互相偶联,技术人员将理解使每个不同标记单独地连接至固体载体上的好处。 Although these markers may be sequentially coupled to each other, the art will understand that each separately connected to a different label on the solid support benefits. 特别地,对任何和所有的标记步骤仅需要单偶联化学过程。 In particular, for any and all markers requires only a single step conjugation chemistry. 进一步,可避免具有不同反应基团的标记的保护/去保护的复杂约定。 Further, different markers may be avoided with the protection of the reactive group / deprotection complex convention.

V合成方法本发明的方法可用在制备不同化合物的程序中进行的任何组的合成化学反应中。 Any of the synthetic chemical group V method of synthesis of the present invention can be performed in a different procedure for the preparation of compounds. 虽然本发明一般使用化学结构单元来说明,更一般地用单体结构单元,应理解本发明的普遍实质。 While the present invention is generally used of chemical building blocks will be described more generally with monomer units, generally to be understood that the spirit of the present invention. 大多数合成化学反应的进行与一般单体偶联反应有很大不同;一般有机化学反应提供可变的产率并产生多种产物,例如区域和立体异构结构。 Usually monomers conjugated with most synthetic reaction chemistry is quite different; typically organic chemical reactions to provide a variable yields and produces a variety of products, such as regional and stereoisomeric configuration. 本发明可用来识别这样一系列化学反应的有用产物,因为可利用这个方法来使标记编码合成产物的信息来代替清楚确定反应产物结构。 The present invention may be used to identify useful products such a series of chemical reactions, because this method can be used to make synthetic product tag encoding information instead of the reaction product of clearly defined structure.

然而,为了简化讨论,本发明差不多被看作是一系列单体偶联步骤。 However, to simplify the discussion, the present invention is almost be viewed as a series of monomer coupling step. 因为本发明的各种偶联步骤可在分离时间在单别的反应容器中进行,甚至例如单元的具有极大不同的偶联化学过程的结构单元也可用于库中感兴趣化合物的装配。 Since various coupling step of the present invention may be carried out in a single reaction vessel at the other separation time, for example, even a structural unit having a different conjugation chemistry greatly unit may also be used in the assembly of the library compounds of interest. 虽然本发明可通过将固体载体同时暴露于结构单元和识别标记中,或顺序地(先暴露于结构单元,再暴露于标记中或先暴露于标记中再暴露于结构单元中),根据偶联化学这个有顺序性的方法允许了一附加的柔韧性。 While the invention may be prepared by the solid support and simultaneously exposed to a structural unit of identification marks, or sequentially (before exposure to the structural unit, and then exposed to a label or tag before re-exposure to the exposure to the structural unit), according to the coupling the chemistry of the sequential method allows an additional flexibility. 在任何情况下,进行偶联反应优选的安排是平行地进行不同的偶联反应。 In any case, the coupling reaction is preferably arranged parallel to different coupling reactions.

在每一个平行系列的偶联步骤完成后,在重新分配至下一偶联步骤的各容器中之前,寡聚体和库的其它化合物在其上合成的固体载体被汇集和混合。 After each series of parallel coupling step is completed, before each container reallocated to the next coupling step, the oligomer libraries and other compounds synthesized on the solid support which were pooled and mixed. 这种混合方法制备了带有不同固体载体上的库的每一个不同成员的化合物的一个大库。 This method of mixing a large library of compounds with a different member of each of the different libraries on solid supports were prepared. 如果每一合成步骤均具有高的偶联效率,那么基本上所有单一固体载体上的化合物具有相同结构,或如果化合物为寡聚体,则具有相同的单体序列。 If each synthesis step has high coupling efficiency, then substantially all of the compound on a single solid support have the same structure, or if the compound is an oligomer, the monomers having the same sequence. 通过在合成末期的任何给定的固体载体的合成路径决定结构或序列。 Determine the structure or sequence given by any route of synthesis solid support in the synthesis of late. 寡聚体的最大长度一般小于约20个,通常为3至15个单体长度,但在一些情况下8至12个单体(残基)的长度为优选的。 The maximum length of the oligomers is generally less than about 20, typically from 3 to 15 monomer length, but the length of 8-12 monomers (residues) is preferred in some cases.

被给适宜于本发明的用途的不同数目的标记和结构单元,有许多可制备本发明化学库的化学方法。 It is suitable for use according to the invention a different number of markers and the structural unit, there may be many chemical methods of producing a chemical library according to the present invention. 然而,必须确保无论是标记或寡聚体的每一偶联步骤不能产生不合格的量的不需要的反应或损坏已在载体上出现的标记或寡聚体。 However, it must be ensured either damaged or unwanted reactions in each step of coupling markers or oligomer not produce unacceptable amount has occurred on a support or labeled oligomer. 在一个实施例中,必须确保通过使用带有标记和寡聚体连接的化学反应基团的固体载体,仅发生所期望的反应,其中标记和寡聚体的连接通过使用两个不同或“正交”类型的保护基团被保护。 In one embodiment, must be ensured by using a solid support with a chemically reactive group and a labeled oligomers connection, only the desired reaction occurs, and wherein the connector labeled oligomer by using two different or "n cross "type protecting group is protected. 固体载体被暴露在第一个去保护剂或活化剂中,从例作为寡聚体合成位点的化学反应基团中去除了第一种类型的保护基团。 The solid support is exposed to a first deprotection agent or activator, chemically reactive group from the synthetic examples of the oligomer site in addition to a first type of protecting group. 在与第一个单体反应之后,在任一选择性的保护步骤之后,接着将固体载体暴露于去除第二种类型的保护基的第二活化剂中,暴露例如作为识别标记连接位点的化学反应基团。 After reaction with the first monomer, after either a selective protection step, the solid support is then exposed to a second activator to remove a second type of protective group, for example as an identification mark is exposed chemical attachment sites reactive group. 再将标记偶联,重复这些步骤,一般为1至约20次。 Then the label is conjugated, these steps are repeated, typically from 1 to about 20 times.

A.寡核苷酸标记的肽库在一个重要的实施方案中,本发明涉及不同肽的大型库的合成。 A. Oligonucleotides labeled peptide libraries In one important embodiment, the synthesis of large libraries of the present invention relates to the different peptides. 虽然许多其它化合物和寡聚体可通过方法(见被本文引作参考的Gait Oligonucleotide Synthesis:A Practical Ap-proach,IRL Press,Oxford(1984),Friesen and Danishefsky,1989,J.Amer.Chem.Soc.111.6656;and Paulsen,1986,Angew.Chem.Int.Ed.Engl.25:212)来制备,但肽的固相合成方法为特别重要和众所周知(见被本文引作参考的Mer-rffield,1963,J.Am.Chem.Soc.85:2149-2154)并且肽库对于各种目的都极为有用。 Although many other compounds and oligomers can be obtained by the method (see reference cited herein Gait Oligonucleotide Synthesis: A Practical Ap-proach, IRL Press, Oxford (1984), Friesen and Danishefsky, 1989, J.Amer.Chem.Soc .111.6656; and Paulsen, 1986, Angew.Chem.Int.Ed.Engl.25: 212) was prepared, but the method of solid-phase peptide synthesis is particularly important and well-known (see are incorporated herein by reference Mer-rffield, 1963 , J.Am.Chem.Soc.85: 2149-2154) and peptide libraries are extremely useful for various purposes. 在Merrifield方法中,氨基酸被共价地结合到由不溶聚合物制成的载体上。 In the Merrifield method, an amino acid is covalently bound to a support made of an insoluble polymer. 另一个带有α-氨基保护基团的氨基酸与被以共价键结合的氨基酸反应形成一个二肽。 Another amino acid with an amino acid is covalently bound to the α- amino protecting group to form a dipeptide. 去除保护基团,将带有α保护基团的第三个氨基酸加1到二肽中。 Removing the protecting group, with the third amino acid protecting group α plus 1-2 peptide. 继续这个步骤直至肽获得了期望的长度和序列。 Continue this procedure until a desired peptide length is obtained and sequences. 可用本领域技术人员已知的保护基团来防止假的偶联(见本文引用参考的The Peptide,Vols.1&3(eds.Gross,E.,and J Meinhofer,Academic Press,Orlando(1979 & 1981))或允许一个来控制偶联。为了本发明的各种目的对光不稳定,对碱不稳定和对酸不稳定的保护基团和它们的组合可被都是使用。 Known to those skilled in the available protecting groups to prevent false coupling (see reference cited herein The Peptide, Vols.1 & amp; 3 (eds.Gross, E., and J Meinhofer, Academic Press, Orlando (1979 & amp ; 1981)) or to allow a controlled coupling of the present invention for various purposes photolabile, alkali labile and acid-labile protecting group and all combinations thereof can be used.

此外,L型和D型氨基酸都可被采用在本文描述的肽合成方法中。 In addition, L and D amino acids can be employed in peptide synthesis methods described herein. 如为所有目的被本文所引作参考的1994年9月21日提出的美国申请系列号08/309,45/中描述的,采用D-氨基酸可在“向后-反向肽”(retro-inverso peptide)的合成中有用。 Was cited for all purposes as described September 21, 1994 raised US Application Serial No. 08 / 309,45 / in the reference, using D- amino acids in the "backwards - reverse peptide" (retro- synthesis inverso peptide) useful in. 这些向后-反向肽将包括相同的氨基酸序列,但具有与运用L-氨基酸合成的肽相反的立体化学。 The rearward - backward peptide comprising the same amino acid sequence, but with the use of the L- amino acid peptide synthesis opposite stereochemistry.

当本发明用来制备和筛选肽库时,选择的标记为核酸。 When the present invention is used to prepare peptide libraries and screening, selection marker is a nucleic acid. 对于肽合成和寡核苷酸识别标记一个循环接着一个循环的连接存在许多相容的化学性质和过程。 For the synthesis of peptides and oligonucleotides recognition mark followed by a loop connected to a loop and there are many chemical properties compatible with the process. 然而,为了保持肽合成期间寡核苷酸标记的完整性,可能需要使用保护基团和/或合成核苷酸的不同组合来避免合成的标记或寡聚体的降解。 However, to maintain the integrity of an oligonucleotide tag during peptide synthesis, use of protecting groups may be necessary and / or different combinations of synthetic nucleotides to avoid degradation of the tag or synthetic oligomer. 一般地,多嘧啶寡核苷酸标记在一般的肽合成条件下相对稳定,这与包含天然嘌呤核苷酸的寡核苷酸标记相反,但多嘧啶核苷酸标记可多少耐熔PCR的扩增。 In general, polypyrimidine oligonucleotide tags under typical peptide synthesis conditions is relatively stable, which is opposite to the natural purine nucleotides comprising an oligonucleotide tag, but more pyrimidine nucleotides may be labeled PCR many refractory expansion increase. 可能需要将嘌呤基或类似物如7-去杂氮-脱氧腺苷和7-去杂氮-脱氧鸟苷,它们被测试经受肽偶联条件的能力,掺入到标记中的获得扩增的期望效率。 Purinyl may need to go, or the like such as 7-aza - deoxyadenosine and 7-deaza aza - deoxyguanosine, they were subjected to test the ability of peptide coupling conditions, tag incorporated into amplified obtained expected efficiency. 为了本发明的目的,标记选择性地包含10%至90%,较优选地为35%至50%,更优选地为33%-35%的嘌呤或嘌呤类似物核苷酸。 For purposes of the present invention, the selective marker comprises from 10 to 90%, more preferably 35% to 50%, more preferably 33% -35%, purine or purine analog nucleotides. 寡核苷酸标记可选择性地掺入生物素或其它报道基团以便于纯化,杂交,扩增或检测(见被本文引作参考的Pierce ImmunoTechnology Catalogand Handbook,1991)。 The labeled oligonucleotide is selectively incorporating biotin or other reporter group to facilitate purification, hybridization, amplification, or detection (see is incorporated herein by reference Pierce ImmunoTechnology Catalogand Handbook, 1991).

因此,在选择用于制备本发明寡核苷酸标记的肽库的化学过程和性质中,必须(1)选择带有适宜官能基团的固体载体;(2)选择氨基酸偶联化学;(3)选择寡核苷酸标记偶联化学;(4)选择各种标记,单体和寡聚体的保护基团和(5)选择去保护和在一些实施例中的裂解化学(对于标记或肽)。 Thus, the selection process for the chemical nature and preparation of oligonucleotides of the present invention labeled peptide library, must (1) selecting a solid support with a suitable functional group; (2) selecting an amino acid coupling chemistry; (3 ) coupling chemistry selected oligonucleotide tag; (4) selection marker variety of protecting groups, monomers and oligomers, and (5) selecting the chemical deprotection and cleavage in some embodiments (or labeled peptide for ). 本领域技术人员认识到在每种情况下不是所有上面选择都需要作出,因为一些应用不可能提出与其它的相同的问题。 Those skilled in the art recognize that not all of the above options are need to be made in each case, because some applications can not raise the same problem as other. 例如,对于所有的应用不可能都要求一个或多个保护基团。 For example, for all possible applications require one or more protecting groups. 在一般情况下,这些选择中的一个是重要的。 In general, these options one is important.

考虑与偶联化学,寡核苷酸标记的肽库的合成的保护基团的选择有关的因素,需考虑用标准的Merrifield化学偶联购得的Fmoc保护的氨基酸和用标准的氨基磷酸酯化学偶联寡核苷酸的合成。 Consider coupling chemistry, synthesis of selected factors related protecting groups oligonucleotide labeled peptide library, Fmoc protected amino acids using standard Merrifield be considered available and chemically coupled using standard phosphoramidite chemistry conjugated synthetic oligonucleotides. 这个方法可认为具有以下步骤:(1)从连在小球的接头或肽上去除氨基末端的Fmoc保护基团;(2)将Fmoc保护(侧链也可被保护)的氨基酸偶联至步骤(1)中产生的游离氨基上;(3)对任何未反应的游离氨基进行选择性覆盖;(4)将DMT保护基团从小球或核苷酸标记被连接在其上的标记上的羟基中除去;(5)偶然带有5′-DMT保护基团和磷酸酯上的保护基团和碱基的环代胺的核苷酸氨基磷酸酯;(6)选择性地覆盖任何未反应的游离羟基;(7)寡核苷酸标记的三价磷的氧化;和(8)肽和寡核苷酸标记的去保护。 This method can be considered as having the following steps: (1) removing Fmoc protecting group from the communication terminal amino group on the linker or peptide beads; (2) the coupling of Fmoc protected amino acids (side-chain may be protected) to step the free amino group (1) produced; (3) selectively to cover any unreacted free amino group; (4) the DMT protecting group from the pellet or labeled nucleotide is connected to the mark thereon hydroxy removed; (5) with occasional cyclic phosphoramidate nucleotide 5'-DMT-generation amine protecting groups and base protecting groups and the phosphate ester; (6) to selectively cover any unreacted free hydroxy; oxide (7) labeled oligonucleotide trivalent phosphorus; and (8) and peptides labeled oligonucleotide deprotection. 下面讨论,这些步骤中每一步。 Discussed below, these steps each step.

(1)在连接下一个氨基酸单体之前,必须从与小球相连的接头或肽中去除氨基末端的Fmoc保护基团。 (1) prior to a single amino acid, the Fmoc protecting group must be removed from the amino terminus of the peptide linker or beads coupled at connection. 一般地,在DMF中用30%的哌啶处理约1小时来完成这种去保护(还见于步骤8),但本发明的一个方面涉及为了寡核苷酸标记的肽库的合成,使用降低浓度的哌啶或缩短的去保护时间。 Generally, in DMF with 30% piperidine about 1 hour to accomplish this deprotection (also found in step 8), but one aspect of the invention relates to the synthesis of oligonucleotides labeled peptide library using reduced concentration or shortened piperidine deprotection time. 哌啶可导致甲基三酯保护的寡核苷酸标记的去保护,O-甲基磷的酯保护团因比已知对哌啶裂解敏感的标准的β-氰乙基基团具有较大的碱基稳定性。 Piperidine methyl triesters can cause protected oligonucleotide tag deprotection, O- methyl ester protecting group due to the large ratio of phosphorus are known to be susceptible to piperidine cleavage standard β- cyanoethyl group base stability. 本发明优选的Fmoc去保护条件为5%至15%,优选为10%哌啶处理5至60分钟,优选地为10至20分钟,15%至30%哌啶为15至30分钟。 The present invention preferably Fmoc deprotection conditions 5% to 15%, preferably 10% piperidine for 5 to 60 minutes, preferably from 10 to 20 minutes, from 15 to 30% piperidine 15 to 30 minutes. 已知影响Fmoc去除的另一个处理为用DBU处理(1,8-二氮杂二环〔5.4.0〕十一-7-烯),例如5%DUB处理5分钟。 Another treatment known to affect removal of Fmoc treated with DBU (1,8-diazabicyclo [5.4.0] undec-7-ene), e.g. 5% DUB 5 minutes. 然而,被本文引作参考的Palom et al.,Tetr.Lett.34:2195-2198的报导认为这种处理能导致胸苷的N-3的甲基化作用。 However, herein incorporated by reference Palom et al, Tetr.Lett.34:. 2195-2198 reports that this process can lead to methylation of N-3 of thymidine. 虽然DBU-介导的Fmoc的去除在一些应中的能够有效,但应承认碱基修饰的可能。 Although the removal of Fmoc DBU- mediated in some applications can be effective, it should be possible to recognize a modified nucleotide.

(2)用常规BOP偶联化学(见The Peptides,Supra可将Fmoc保护的氨基酸偶联至小球或肽的游离氨基基团上。一般地,采用由1∶1 DMF/DCM组成的溶液中的Fmoc保护的氨基酸(110mM),HBTV(100mM),HOBt(100mM)和DIEA(300mM)的混合物来氨基酸的偶联。其它活化化学过程也可被用在这种情况中,例如用HATV替代HBTV/HOBT。然而在本发明的一个实施方案中,反应混合物由3∶1DMF/DCM组成的溶液中的55mM Fmoc保护的氨基酸,50mMHBTV和150mMDIEA组成;这个实施方案优选使用可省略试剂运送瓶的设备,侧链也可被保护;由于结构单元的商业可获得性,为多种目的,优选Fmoc/tBu保护。其它带侧链保护的有用的氨基酸结构单元包括Arg(Pmc),Gln(Trt),HB(Trt),Asn(Trt),Asp(OtBu),Glu(OtBu)和Lys(tBOC)和由对光不稳的保护基团提供的带侧链保护的氨基酸。 (2) using conventional BOP coupling chemistry (see The Peptides, Supra Fmoc amino acids may be coupled to the protection of free amino groups of the peptide or pellets. Generally, the use solution 1:1 DMF / DCM consisting of Fmoc-protected amino acid (110mM), HBTV (100mM), HOBt (100mM) and a mixture of DIEA (300mM) to amino acid coupling. other chemical activation process may also be used in this case, for example HATV alternative HBTV / HOBT However, in one embodiment of the present invention, the reaction of protected amino acid Fmoc solution mixture of 55mM 3:1DMF / DCM consisting of, 50mMHBTV and 150mMDIEA composition;. this embodiment is preferred to use a reagent bottle conveying apparatus omitted, side chain may be protected; commercial availability since a structural unit, for a variety of purposes, preferably Fmoc / tBu protected other useful sidechain protected amino acid building blocks include Arg (Pmc), Gln (Trt), HB. (Trt), Asn (Trt), Asp (OtBu), the amino acid Glu (OtBu) and Lys (tBOC) and provided with a side chain protected by photolabile protecting groups.

(3)通过用乙酸酐和1-甲基咪唑处理或其它本领域已知的方法可获得对未反应的游离氨基基团进行选择性覆盖。 (3) selective coverage of unreacted free amino group by treatment with acetic anhydride and 1-methylimidazole, or other methods known in the art can be obtained.

(4)通过用三氯乙酸(TCA)即CH2Cl2中10%TCA处理可将DMT保护基团从核苷酸标记将与其相连的小球或标记上的羟基中除去。 (4) a hydroxyl group may be removed on the DMT protecting group from the labeled polynucleotide beads or connected thereto by a mark with trichloroacetic acid (TCA) in CH2Cl2 i.e., 10% TCA treatment. 如果使用磷酸酯中的对酸不稳定保护基团和核苷酸的环代胺(即脱氧胞苷,7-去杂氮-脱氧腺苷,和7-去杂氮-脱氧鸟苷),那么这些基团将足够坚固地抵抗5′-O-脱三苯甲基作用中用到的TCA(一般为1-3%)。 If the phosphoric acid-labile cyclic amine Generation protecting groups and nucleotides (i.e. deoxycytidine, 7-deaza aza - deoxyadenosine, and 7-deaza aza - deoxyguanosine), then these groups will be strong enough to resist TCA 5'-O- detritylation used in (typically 1-3%).

(5)偶联带有5′-DMT保护基团的核苷酸氨基磷酸酯可通过使用常规的氨基磷酸酯化学来获得,虽然必须考虑需要磷酸酯氧上和寡核苷酸标记的碱基的环代胺上的保护基。 Nucleotide phosphoramidates (5) coupled with 5'-DMT protecting group may be obtained by using conventional phosphoramidite chemistry, although the need to be considered on the phosphate oxygen and the oligonucleotide labeled bases the protecting group on behalf of the cyclic amine. 对于核酸的对光不稳的保护基团,见被本文引作参考的PCT专利公开WO92/10092和Baldwin et al.,1990,Tetr.Lett.46:6879-6884。 For photolabile protecting groups of the nucleic acid, herein incorporated by reference, see PCT Patent Publication WO92 / 10092 and Baldwin et al, 1990, Tetr.Lett.46:. 6879-6884. 如上所述,适宜的磷酸酯保护基团包括O-甲基和β-氰乙基,但O-烯丙基和/或IV-烯丙氧羰基(即引用的3-(烯丙基N,N′-二异丙基)氨基磷酸酯)也可用来保护磷酸酯氧和核苷碱基的环代胺,分别(见被本文引作参考的HgyaKawa et al.,1990,J.Amer,Chem.Soc.112:1691-1696。通过使用含有三(联苄基亚基丙酮)二钯氯仿复合物,三苯基膦和正丁胺/甲酸,接着进行THF洗涤,含水N,N-二乙基二硫化氨基甲酸钠和水洗涤可去除烯丙基保护基团。氨基磷酸酯偶联通过如1H-四唑;4-硝基苯四唑;吡啶鎓氢氯化物/咪唑。近来的氨基磷酸酯活化剂在以肽或寡核苷酸上的氮上的假反应的低水平为代价导致了选择性的5′-O-亚磷酸化(见被本文引作参考的Gryaznov andLetsinger,1992,Nuceic Acids Research 20:1879-1882)。 As described above, a suitable phosphate protecting groups include 3- (N-allyl-O- methyl and β- cyanoethyl, but O- allyl and / or allyloxycarbonyl group IV- (i.e. reference, N'- diisopropyl) phosphoramidate) may also be used to protect the generation of cyclic amine phosphates and nucleobases of oxygen, respectively (see reference cited herein HgyaKawa et al., 1990, J.Amer, Chem .Soc.112: 1691-1696 by containing tris (dibenzyl ylidene) dipalladium chloroform complex, triphenylphosphine, and n-butylamine / formic acid, followed by washing of THF, aqueous N, N- diethyl. washed with water and sodium disulfide amino allyl protecting group may be removed by coupling, such as phosphoramidate 1H- tetrazole; 4- nitrophenyl-tetrazole; pyridinium hydrochloride / imidazole recent activation phosphoramidate. agents at low levels of false reactions on the nitrogen on the peptide or oligonucleotide resulted expense 5'-O- phosphite selectivity (see, herein incorporated by reference Gryaznov andLetsinger, 1992, Nuceic Acids Research 20: 1879-1882).

(6)通过用乙酸酐和1-甲基四唑处理或通过用乙酸酐/二甲基吡啶/DMAP处理可达到对任何未反应的游离羟基的选择性覆盖。 (6) or a tetrazole by treatment with acetic anhydride / lutidine / processing of DMAP can be achieved selectively cover any unreacted free hydroxyl groups by using acetic anhydride and 1-methyl.

(7)通过用碘和吡啶处理或用I2,可力丁,水中的MeCN处理可达到寡核苷酸标记中的三价磷的氧化。 (7) by treatment with iodine and pyridine or with I2, collidine, MeCN treated water can reach trivalent phosphorus oxide oligonucleotide tag. 另一方面,通过采用氧化三价磷的温和氧化剂tBuOOH,可减少氨基酸甲氨酸,色氨酸和组氨酸的氧化(见被本文引作参考的Hayakawa et al.,1990,Tetr.lett.27:4191-4194)。 On the other hand, by using a mild oxidant tBuOOH trivalent phosphorus oxide can be reduced methanesulfonic acid, tryptophan and histidine oxide (see herein are incorporated by reference Hayakawa et al., 1990, Tetr.lett. 27: 4191-4194).

(8)通过顺序地用二氯甲烷中1%的TCA,再用苯硫酚/NEt3/二噁烷(1∶2∶2),接着用1,2-乙二胺/EtOH(1∶1)在55℃下处理从标记中去除保护基团,接着使用三氟乙酸(95∶5TFA/水,阳离子清除剂)来去除对酸不稳定的保护基团可实现肽和寡核苷的标记的去保护。 (8) sequentially with 1% methylene chloride in the TCA, and then thiophenol / NEt3 / dioxane (1:2:2), followed by ethylenediamine / EtOH (1:1 ) for removing the protecting group from the marker at 55 ℃, followed by trifluoroacetic acid (95:5TFA / water, a cation scavenger) to remove the acid-protecting groups labeled labile peptides and oligonucleotides can be achieved in the to protect. 嘌呤核苷酸对强酸(如TFA)的不稳定性可通过利用嘌呤核苷类似物7-去氮杂-2′-脱氧腺苷和7-去氮杂-2′-脱氧鸟苷的氨基磷酸酯来避免(见被本文引作参考的Barr et al..1986.BioTechniques4:428-432,and Scheit,Nucleotide Analogs:Synthesis and Bio-logical Function PP.64-65(John Wiley and Sons,NewYork)。 Purine nucleotides to strong acid instability (e.g., TFA) may be prepared by using the purine nucleoside analogs 7-deaza-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyguanosine amino acid to prevent the ester (see reference cited herein Barr et al..1986.BioTechniques4: 428-432, and Scheit, Nucleotide Analogs: Synthesis and Bio-logical Function PP.64-65 (John Wiley and Sons, NewYork).

下面的部分说明合成寡核苷酸标记的肽库的一个优选实施例。 The following sections describe a synthetic oligonucleotide labeled peptide libraries preferred embodiment.

B.合成寡核者酸标记的肽文库的改进方法建立一种实用的小球基寡核苷酸编码肽文库的方法需要满足几个关键技术标准。 Establishment of a practical method yl pellets oligonucleotides encoding peptide libraries and improved method B. Synthesis of oligonucleotides were labeled acid peptide library need to meet several key technical standards. 这些标准包括(i)平行装配肽和寡核苷酸的相互匹配化学方法的开发;(ii)具有适当物理性质的小球,材料的选择;(iii)方便地分离载有配体的微型小球其中配体与靶受体结合;和(iv)从单一球粒成功读码标记物,即通过PCR扩增和从单一小球排序模板标记物DNA。 These include (i) the development of chemically matched parallel assembly of oligonucleotides and peptides; (ii) having physical properties suitable pellets, choice of material; (iii) separating conveniently contain ligands miniature wherein the ball to the target ligand binding receptor; and (iv) the successful reading labels from a single pellet, i.e., by PCR amplification and sequencing template DNA markers from a single pellet. 本发明提供一种合成这类文库的改进方法,正如此部分和实施例1所描述,其表明如何使用单链寡核甘酸标记物在10μm直径聚苯乙烯小球上编码组成肽的合成。 The present invention provides an improved method for the synthesis of such libraries, so the positive portion as described in Example 1, which indicate how to use synthesis on 10μm diameter polystyrene beads encoded peptide consisting of a single-stranded oligonucleotide nucleotide marker.

在该改进方法中,肽和核甘酸以平行交替合成方式进行装配,以便每一粒小球都带有一条单链肽序列和唯一的寡核苷酸识别标记物。 In this improved process, the peptides and nucleotides to be assembled synthetically alternating parallel, so that each ball with a small single-chain peptide sequence and a unique identification marker oligonucleotide. 这些寡核苷酸共享共同的5′-和3′-PCR引发位点;因此这些小球可作为PCR模板。 These oligonucleotides share a common 5'- and 3'-PCR priming sites; thus these beads can be used as a template for PCR. 为说明该方法,合成并筛选约8.2×105七肽的编码合成文库以与抗-强啡肽B单克隆抗体D32.39(Cull等,1992,Proc.Natl.Acad.Sci.USA 89:1865-1869,此处引为参考文献)结合,使用荧光激光激活细胞分选(FACS)仪选择与抗体强烈结合的个别小球。 To illustrate the method, encoded synthetic library synthesized and screened with the anti approximately 8.2 × 105 heptapeptide - dynorphin B monoclonal antibody D32.39 (Cull et, 1992, Proc.Natl.Acad.Sci.USA 89: 1865 -1869, incorporated herein by reference) in conjunction with a laser using a fluorescence activated cell sorting (FACS) instrument strongly bound to the antibody selected individual pellets. 在选择小球上进行寡核苷酸标记物PCR扩增后,对DNA进行排序以确定肽配体的特性,这些更详细地描述于下文。 After PCR amplification of the marker oligonucleotides on beads selected, the DNA was sorted to determine the characteristics of the peptide ligand, which is described in greater detail below.

该方法一个重要方法描述于下面实施例1的附加细节部分,其是为肽和标记物合成选择的固相载体。 This method is an important method of Example 1 described the additional details in the following examples, which is a solid support for the peptide synthesis and selection marker. 优选固相载体,即10μm直径小球由大孔苯乙烯-二乙烯共聚物制成并用十二烷胺接头衍生。 Preferably a solid support, i.e., a 10μm diameter beads macroporous styrene - divinylbenzene copolymer made with dodecyl amine and derivatized linker. 通过以Fmoc-甘氨酸完全酰化,随后哌啶除去Fmoc基固及分光光度法定量释放的哌啶二苯并富烯(piperidine-dibenzofulvene)加成物(e302=7,800 1mol-1cm-1,估算这些小球上的氨基为~100μmol/g。5×109小球/g,其相应的最大肽载量为~20fmole/球。使用适当保护的氨基酸和Ω羟基酸的混合物进行小球酰化得到正交分化的氨基和羟基,由此可分别发展肽和核苷酸链。通过放变偶联于初始小球量(参见下文)的氨基酸和羟基酸的比例来控制每粒小球肽对寡核苷酸的平均化学计量。使用标准Fmoc化学方法测试在带有三氟乙酸可裂解Knorr接头(Bernatowicz等,1989,Fetr.Lett.30:4645-4648,这里引为参考文献)的小球上的肽合成(5肽至12肽,发现其具有高度的重现精度,与在常规肽合成树脂上进行的合成没什么区别,正如粗制的裂解肽酰胺HPLC分析所测定的结果。 Fmoc- glycine completely acylated by, followed by removal of the Fmoc Solid piperidinyl and quantified spectrophotometrically released dibenzofulvene piperidine (piperidine-dibenzofulvene) adduct (e302 = 7,800 1mol-1cm-1, these estimates amino group on the beads is ~ 100μmol / g.5 × 109 beads / g, corresponding to a maximum loading of peptide ~ 20fmole / ball. mixture Ω suitably protected amino acid and hydroxy acid is acylated to give pellets positive differentiation cross amino and hydroxyl groups, whereby the development of peptide and nucleotide chains, respectively (see below) of the amino acid and hydroxy acid ratio to control each piece ball core peptides of the initial oligonucleotide beads by placing an amount of change coupling the average stoichiometry nucleotide using standard Fmoc chemistry tests cleavable with trifluoroacetic acid Knorr linker (Bernatowicz et, 1989, Fetr.Lett.30: 4645-4648, incorporated herein by reference). peptides on beads synthesis of (5 peptides to peptide 12, was found to have a high degree of fidelity, with no synthesis performed on a conventional peptide synthesis resins distinction, as cleavage of the crude peptide amide of the measured results of HPLC analysis.

平行合成策略要求在相互正交的氨基酸和核苷酸结构单元上使用一套保护基团,并且每一条聚合物链对合成中所使用的及第二链脱保护中所使用的试剂而言是稳定的。 Parallel synthesis strategy requires a protecting group orthogonal to each other in amino acid and nucleotide structural units, and each polymer chain of the second strand synthesis and used in the de-protection reagents is used in terms of stable. 虽然原则上可使用各种保护/脱保护方案(如上所述),但优选在肽结构单元上使用Fmoc/tBu保护,因为这种方式保护的天然和非天然氨基酸可广泛购买得到。 While various protection / deprotection scheme (as described above) in principle, but preferably Fmoc / tBu protected peptide structural unit, as in this way protected natural and unnatural amino acids are widely commercially available. 然而,tBu-基的肽侧链保护基团要求使用强酸(通常为三氟乙酸)进行处理以脱去,条件是能够引起寡核苷酸包括2′-脱氧腺苷(dA)或2′-脱氧鸟苷(dG)的快速脱嘌呤作用(见Capon,1969,Chem.Rev.69:407-498,这里引为参考文献)。 However, the peptide side chain protecting group requires tBu- group using a strong acid (typically trifluoroacetic acid) to be treated off, that can cause an oligonucleotide include 2'-deoxyadenosine (dA) or 2'- deoxyguanosine (dG) fast depurination (see Capon, 1969, Chem.Rev.69: 407-498, incorporated herein by reference). 围绕这个问题的就是在模板寡核苷酸标记物中以7-脱氮-2′-脱氧腺苷(c7dA)取代dA。 Around this problem it is to label the template oligonucleotide to 7-deaza-2'-deoxyadenosine (c7dA) substituted dA. 脱氮嘌呤核苷的配糖键抗酸催化的水解作用(参见Scheit,1980,Nucleotide Analogs:Synthesis and Biolgical Func-tion(John Wiley和Sons,New York)PP.64-65,这里引为参考文献),通过PCR中使用的热稳定性聚合酶完全复制掺有这些单体的寡核苷酸(参见Mc Conlogue等,1988,Nucl.AcidsRes.16:9869,和Barr等,1986,Bio Techniques 4:428-432,其中每一篇在此都作为参考文献),抗酸鸟苷也可被包含在模板DNA中。 Deazapurine nucleosides acid-catalyzed glycosidic bond hydrolysis (see Scheit, 1980, Nucleotide Analogs: Synthesis and Biolgical Func-tion (John Wiley and Sons, New York) PP.64-65, incorporated herein by reference ), thermostable polymerase by PCR using oligonucleotides doped with completely duplicate these monomers (see Mc Conlogue etc., 1988, Nucl.AcidsRes.16: 9869, and Barr et al., 1986, Bio Techniques 4: 428-432, each of which are herein incorporated by reference in one document), guanosine acid may also be included in the DNA template. 在所有平行合成中均使用5′-O-二甲氧三苯甲基2′-脱氧核苷3′-(O-甲基-N,N-二异丙基)氨基磷酸酯。 Using both 5'-O- dimethoxytrityl 2'-deoxynucleoside 3 'in all the parallel synthesis - (O- methyl -N, N- diisopropyl) phosphoramidate. 在DNA合成方案中用来将核苷酸亚磷酸酯中间体转化为磷酸三酯的试剂(I2/可力丁/H2O/乙腈),未发现其对易氧化残基Trp和Met或其它被保护氨基酸具有不利影响。 DNA synthesis scheme nucleotide phosphite intermediate to be converted to the phosphate triester reagent (I2 / collidine / H2O / acetonitrile), which was found to be protected or other easily oxidized Met and Trp residues amino acids have an adverse effect. 使用二氯甲烷中1%的三氟乙酸(TCA)在~40秒内将5′-O-DMT基团从正在增长的寡核酸链上完全除去,而所有常规用于Fmoc/tBu化学方法中的酸不稳定性侧链保护基,除酪氨酸衍生的tBu醚外,对1%TCA处理1小时均呈惰性。 Dichloromethane 1% trifluoroacetic acid (TCA) to 5'-O-DMT group is removed from the growing nucleic acid strand oligonucleotides in fully to 40 seconds, while for all conventional Fmoc / tBu chemistry in acid-labile side-chain protecting groups, in addition to tyrosine-derived tBu ether, 1% TCA showed an inert 1 hour. Fmoc-Tyr(OBz)证明是含酪氨酸的肽合成中合适的替代,该O-苯甲酰脂对肽合成中用于除去α-N-Fmoc保护基的TCA和哌啶均是稳定的。 Fmoc-Tyr (OBz) proved to be a suitable alternative to the synthetic tyrosine-containing peptide, the peptide O- benzoyl lipid synthesis and for removing TCA piperidine α-N-Fmoc protecting groups are stable . α-氨基残基的定量脱保护要求以哌啶/DMF(10%v/v)处理5-10分钟,并还会导致被保护的多核苷酸磷酸三酯的部分脱甲基作用(t1/2~45min)。 Α- amino residue quantitative deprotection requirements for 5-10 minutes piperidine / DMF (10% v / v), and also result in demethylation of the protected portion of the polynucleotide phosphate triester (t1 / 2 ~ 45min). 对照实验表明,在随后的核苷酸链增长过程中产物磷酸二酯类的任何异常的亚磷酸化作用会被最终寡核苷酸脱保护步骤改变回去(参见Lehmann等,1989,Nucl.Acid Res.17:2379-2390,这里引为参考文献)。 Control experiments indicated that the nucleotide chain in a subsequent growth process of any unusual effect phosphite diester phosphate product will be a final deprotection step change oligonucleotides back (see, Lehmann et al., 1989, Nucl.Acid Res .17: 2379-2390, incorporated herein by reference). 平行合成完成时,使用苯硫酚酯(thiophenolue)(磷酸酯O-脱甲基作用)然后使用乙醇乙二胺(被保护胞苷和7-脱氮-腺嘌呤残基的脱苯甲酰作用)使DNA完全脱保护。 When the completion of parallel synthesis, using thiophenol ester (thiophenolue) (O- demethylation phosphate) ethylenediamine and ethanol (protected cytidine and 7-deaza - debenzoyl effect adenine residues ) reacting fully deprotected DNA. 这些温和的无水氨解条件不对被保护的肽序列产生不利影响(参见Juby等,1991,Tetr.Lett 3:879-882,这里引为参考文献)。 These mild conditions for ammonolysis in anhydrous protected peptide sequence does not adversely affect (see Juby et, 1991, Tetr.Lett 3: 879-882, incorporated herein by reference). 在标准条件下使用TFA会使被保护的肽序列脱保护。 Using TFA under standard conditions would be protected peptide sequences deprotection.

先前已表明阿片样肽强啡肽B(YGGFLRRQFKVVT)(SEQ ID NO:1)的羧基末端区代表了抗-强啡肽B mAbD32.39(参见Cull等,上述)的表位:可溶性七肽RQFKVVT(SEQ ID NO:2)与D 32.39以高度亲和力(Kd~1nM)结合。 Previously been shown that opioid peptide dynorphin B (YGGFLRRQFKVVT) (SEQ ID NO: 1) represents the carboxy-terminal region of an anti - dynorphin B mAbD32.39 (see Cull et al., Supra) epitope: Soluble heptad RQFKVVT (SEQ ID NO: 2) D 32.39 and binding with high affinity (Kd ~ 1nM). 在带有一个酸可裂解的Fmoc保护的碳酰胺(Knorr)接头的正交分化的小球上进行此肽和69碱基寡氧核苷酸的平行合成。 For parallel synthesis the peptide and oligonucleotide 69 bases in the nucleotide oxygen carboxamide with Fmoc protection of a cleavable acid (Knorr) perpendicular to the differentiation of the joint ball. 在加入起始的20个核苷酸之后,用哌啶/DMF和偶联于游离胺的第一个肽残基(Fmoc-Thr(tBu)-OH)处理小球。 After addition of the initial 20 nucleotides, pellets piperidine / DMF and the peptide coupled to a first free amine residues (Fmoc-Thr (tBu) -OH) treated. 然后小球处于氨基磷酸酯化学反应过程和偶联下一个氨基酸(Fmoc-Val-OH)的双循环中。 Then the ball is in two-cycle phosphoramidite chemistry reactions and coupling one amino acid (Fmoc-Val-OH) in. 重复这一过程,直至七肽序列和核苷酸编码区被完全合成出来,然后再以下面的35个核苷酸延伸DNA以提供一个间隔区和PCR的5′-引发位点。 This process is repeated until peptide sequence and seven nucleotides of the coding region is completely synthesized, and then in the following 35 nucleotides extend 5 'DNA to provide an initiation site and a spacer region of PCR. 最后将小球曝露于全部寡核苷酸和随后的肽脱保护条件中,含裂解下的肽的TFA上清液以反相HPLC分析。 Finally, the pellet is exposed to all of the oligonucleotides and subsequent deprotection conditions of a peptide, peptide-containing supernatant TFA cleavage at the reverse phase HPLC analysis. HPLC结果表明,平行合成的粗制肽由一种单一的主成分组成(用真正的RQFKVVT(SEQ ID NO:2)共洗脱),这一粗产品与没发生寡核苷酸化学反应的对照肽合成所产生的肽相比没有明显区别。 HPLC results showed crude peptide synthesized in parallel from a single main component composition (with real RQFKVVT (SEQ ID NO: 2) co-elution), the crude product is not a chemical reaction occurring oligonucleotide control no significant difference as compared to synthetic peptide produced.

含T,dC和c7dA的模板DNA的平行合成化学的稳定性与含标准嘌呤核苷酸dA的类似靶物相比。 Parallel-containing template DNA T, dC and c7dA of synthetic chemical stability as compared to a standard containing purine nucleotides dA similar targets. 利用荧光激活细胞分选仪正细胞计数器(FAC Star Plus cytometer)的单一球粒克隆能力,来自双合成的个别脱保护小球被分选入微型离心(microfuge)管中,并且,被束缚的寡核酸模板通过PCR45个循环进行扩增。 Using a fluorescence activated cell sorter positive cell counter (FAC Star Plus cytometer) single pellets clonality, individual deprotection pellets from two synthetic were sorted into microcentrifuge (a microfuge) tube, and is bound oligonucleotide nucleic acid template was amplified by PCR45 cycles. 仅从含脱氮嘌呤的模板制得所期望大小和核苷酸序列的“干净的”扩增产品。 Only deazapurine containing template was prepared by "clean" amplification products and nucleotide sequence of a desired size. 因此在平行肽合成的过程中保持了该寡核苷酸的完整性,证明来自单一小球的模板可被容易地进行扩增和排序。 Thus maintaining the integrity of the oligonucleotide during synthesis of the peptide in parallel, from a single template proof pellets can be easily sorted and amplified.

使用7个氨基酸结构单元Arg,Gln,Phe,Lys,Val D-Val和Thr通过组合合成方法构建编码文库,设计该文库含823,543(77)个不同的连接于10μm小球上的七肽。 Use seven amino acid building blocks Arg, Gln, Phe, Lys, Val D-Val and Thr encoded library constructed by a combination of synthetic methods, the design of the library contained 823,543 (77) is connected to seven different peptides on beads 10μm . α-N-Fmoc-Thr(叔丁基-羟苯并三唑(被保护的苏氨酸)和4-O-DMT-氧化丁酸琥珀酰亚胺酯残基首先与所有的小球偶联,得到用于合成的正交分化的氨基和羟基。通常,每颗小球带有每分子DNA标记物20个分子的单链肽序列。通过建立特有的二核苷酸单元编码每一加入的氨基酸,在肽偶联的第七个循环之后,合并小球,DNA合成完成。初始所用总球量为35mg(1.75×108个小球),确保了文库中每一肽序列存在于~200个不同的小球上。一等份文库的肽显微排序分析确证,七个氨基酸随机地分布于降解的七肽混合物(注意L-缬氨酸和D-缬氨酸在Edman降解过程中是不可区分的)每种情况中。 α-N-Fmoc-Thr (t-butyl - hydroxyphenyl benzotriazole (protected threonine), and 4-O-DMT- oxide succinimidyl butyrate ester residue first coupled with all of the balls , for the synthesis of amino and hydroxyl groups obtained orthogonal differentiation. typically, every single ball chain peptide sequences with a single DNA molecule per molecule marker 20 by establishing a unique coding of each unit dinucleotide added amino acids, a seventh cycles after the peptide coupling, with small balls, the DNA synthesis is complete. the initial amount of the total ball 35mg (1.75 × 108 balls), to ensure that the library is present in each of the peptide sequences to 200 different beads peptide sorting microscopic analysis confirmed a library aliquot, seven amino acids randomly distributed degradation heptapeptide mixture (note D- and L- valine-valine in the Edman degradation process is not ) in each case distinguished.

通过流动细胞计数分析mAb D32.39与对照小球和与小球文库的结合。 Analysis of binding control mAb D32.39 beads and beads with a library by flow cytometry. 带有正向对照序列RQFKVVT(SEQ IDNO:2)和69无寡核苷酸标记物的小球用抗体进行强烈染色,而对空白小球不染色。 Control sequences with the forward RQFKVVT (SEQ IDNO: 2) and 69 non-small oligonucleotide marker balls intensely stained with the antibody, but do not stain blank pellet. 通过对比,只有一小部分的编码文库结合有D32.39。105次分析表明~2%。 By contrast, only a small portion of the encoded library bound D32.39.105 secondary analysis showed ~ 2%. 文库染色高于背景水平。 Library staining above background levels. 显然,这种在结合位点与D32.39的结合是特异的,因为它能通过mAb与可溶性RQFKVVT(EDY ID NO:2)预温育而被完全阻断。 Obviously, in this binding site it is specific for the D32.39, because it by mAb to soluble RQFKVVT (EDY ID NO: 2) is completely blocked by preincubation. 来自文库的个别小球具有可与正向对照小球相比的荧光强度,其被分选入微型离心(microfuge)管中作为标记物通过PCR扩增(收集顶部有荧光的球,占总量的0.17%)。 From individual library beads having a fluorescence intensity compared to the positive control beads, which are classified into a micro-centrifuge (a microfuge) tube was amplified by PCR as a marker (fluorescent collected at the top of the ball, accounting for the total the 0.17%). 扩增反应含duTP和尿嘧啶DNA糖苷酶以防止遗留的杂质,带有来自前面扩增过程的可溶性产品(参见Longo等,1990,Gene 93:125-128,这里引为参考文献)。 DuTP amplification reaction containing Uracil DNA glycosylase and to prevent the remaining impurities, the soluble products from the front with the amplification process (see Longo et, 1990, Gene 93: 125-128, incorporated herein by reference). 从12颗分选的小球获得核苷酸序列,推断出的肽序列列于表1。 12 nucleotide sequences obtained from the sorted beads, deduced peptide sequences are listed in Table 1. 从单一球得到的代表性肽序列带有的荧光不是明显强于背景,作为对比其也被列成表格。 Obtained from a single representative peptide sequences with fluorescent balls are not significantly stronger than the background, which is also tabulated for comparison.

表1高荧光密度小球序列 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低荧光密度小球序列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 Table 1 High fluorescence intensity of beads 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 of beads 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

表1中的数据与较早期研究相一致,证明了D32.39的优选识别序列位于强啡肽B的RQFKVV六个氨基酸片断(参见Cull等,上文)。 The data in Table 1 is consistent with earlier studies demonstrated recognition sequence is preferably located D32.39 dynorphin B segment of RQFKVV six amino acids (see Cull et al., Supra). 优选正电荷残基精氨酸和赖氨酸处于该表位的第一位和第四位,苯丙氨酸表现为该基序所独有的第三位残基。 Preferably the positively charged arginine and lysine residues in the first and fourth epitope, showed that phenylalanine unique motifs third residue. 在该文库中优选第二位为谷氨酰胺残基而明显地优选脂肪族b-支链的氨基酸缬氨酸(仅L-异构体)和苏氨酸作为第5和第6位残基。 In the second library is preferably a glutamine residue and significantly preferably an aliphatic amino acid valine branched b- (L- isomer only) and threonine as the 5th and 6th residue . D-缬氨酸表现为共同基序之外位置上最被接受的残基。 D- valine residue showed a position outside the most accepted common motif. 根据所设计的结合分析实验,所选择的肽亲和性范围(Kd~0.3-1400nm)是可预知的带有被标记的另一抗体二价初级抗体的检测。 Peptide binding assay The affinity of test range, the selected design (Kd ~ 0.3-1400nm) is predictable with another antibody detection bivalent primary antibody is labeled. 结合价的操作(例如,使用直接标记的一价受体)和洗涤条件的严格性会提高仅分离最高亲和力配体的能力。 Stringency will bind monovalent operation (e.g., using directly labeled monovalent receptor) and wash conditions to improve the separation ability of only the highest affinity ligands. C.小分子合成虽然最初从肽合成或其它大的聚合物文库角度描述了本发明各个方面,正如前所述,本发明各方面可等同地适用于其它化学合成。 C. Although initially small molecules synthesized from peptide synthesis or other high polymers angle libraries described various aspects of the present invention, n described above, aspects of the present invention is equally applicable to other chemical synthesis. 特别地,本发明的装置和方法可用来在许多合成方案中完成各种化学合成步骤,包括如小分子合成。 In particular, the apparatus and method of the present invention can be accomplished in a variety of chemical synthesis steps a number of synthetic schemes, including small molecules such as synthetic. 这些装置可被用于化学合成方案中以选择性添加试剂,与用于特定分子的合成方案一致。 These devices can be used to selectively add reagents consistent with the synthetic scheme for the chemical synthesis of a specific molecule schemes.

进一步地,可平行地进行不同的合成方案,如与上述进行的肽合成一起进行,其中在不同步骤中加入不同的试剂从得到固体载体上多种小分子文库。 Further, in parallel may be different synthetic scheme, performed together as described above for the synthesis of the peptide, wherein the different reagents were added to give a solid support from a plurality of small molecule libraries in different steps. 这些多种文库可用这里所述的方法筛选,以具有所需性质。 These various available libraries screening methods described herein to have the desired properties. 此外,当需要此小分子多种文库时,可对该文库进行标记以对合成步骤进行编码,这些合成步骤包含在分子文库的每一分离子的合成中。 Further, when the need of diverse libraries of small molecules, the library may be labeled to encode the synthesis step, the synthetic steps in the synthesis of each separated sub-containing molecule in the library.

这种小分子在固体载体上合成的例子包括,如噻唑烷酮(thiazolidinones),间噻唑烷酮及其衍生物,正如这里包括的实施例2所述,及US专利申请No.08/265,090,1994年6月23日申请,在此引为参考文献,用于所有目的。 Examples of small molecule synthesis on a solid support include, as thiazolidinone (thiazolidinones), inter-thiazolidinone and its derivatives, as used herein the embodiment 2, embodiment and US Patent Application No.08 / 265,090 includes, June 23, 1994 application, herein incorporated by reference for all purposes. D.生产可溶性文库的方法为某些应用,人们可设想一种“无球”或“可溶的”分子文库。 Methods D. production of soluble libraries for certain applications, one can imagine a "no ball" or molecule libraries "soluble." 可溶性分子,标记的和未标记的,可用于各种目的,包括分析化合物的活性(参见下面的VI.B部分)及扩增标记物。 Soluble molecules, labeled and unlabeled, may be used for various purposes, including the activity of the compound was analyzed (see below VI.B portion) and amplification marker. 有各种方法产生可溶性分子文库,标记的和未被标记的,及溶解合成于固体载体上的化合物,标记的和未被标记的。 There are various methods of producing libraries of soluble molecules, labeled and unlabeled, and the synthesis of the compound is dissolved in a solid support, labeled and unlabeled. 通常,在这种方法中采用可裂解的接着。 Typically, a cleavable Next in this method.

例如,如上述部分II.B所示,可裂解的接头能用于从小球或其它固体载体上裂解被标记的或未被标记的分子,由此使所感兴趣的分子溶解。 For example, as shown in section II.B of the above, a cleavable linker can be used to lyse labeled or unlabeled on a small ball or other solid carrier molecules, whereby the molecules of interest were dissolved. 为生产可溶的被标记的分子,可裂解接头将连接于小球或其它固体载体上并具有至少两个官能团:一个是用于合成感兴趣的分子,另一个用于合成标记物。 For the production of soluble molecules are labeled, a cleavable linker attached to beads or other solid carriers and having at least two functional groups: one for the synthesis of the molecule of interest, for the synthesis of the other markers. 由此将分子和标记物合成连接于同一接头上,该接头反过来与固体载体结合。 The molecular markers and thereby connected to the same synthetic linker that in turn bound to a solid support. 一旦合成此分子和标记物,便裂解掉接头得到一可溶的被标记的分子。 Once the synthetic molecular tag, then cleaved to obtain a soluble linker molecule being labeled.

利用极大规模固定化聚合成(VLSIPSTM)技术,并在筛选之前从载体上可裂解合成物。 Using Very Large Scale Immobilized polymerized to form (VLSIPSTM) technology, and may be cleaved from the carrier composition prior to screening. 参见US专利No.5,143,854和PCT专利申请No.92/10092,其在此均引为参考文献。 See US Patent No.5,143,854 and PCT Patent Application No.92 / 10092, which are hereby incorporated by reference. 在一实施方案中,在VLSIPSTM片上合成一组寡核苷酸,并且每一寡核苷酸通过一可裂解接头与片连接,接头如二硫化物(参US专利申请No874,849,1992所4月24日申请,这里引为参考文献)。 In one embodiment, a sheet is synthesized on VLSIPSTM set of oligonucleotides, and each oligonucleotide via a cleavable linker connecting piece, such as a disulfide linker (see US Patent Application being No874,849,1992 4 filed May 24, cited herein by reference). 寡核苷酸标记物具有一个游离的官能团,如胺,用于被标记分子的连接,该分子通常为寡聚体,优选为肽。 The labeled oligonucleotide having a free functional group, such as an amine, for being connected to the labeled molecule, which is generally an oligomer molecule, preferably a peptide. 该标记物可选择性地仅含有嘧啶或含有嘧啶和嘌呤的类似碱基。 The marker may optionally contain only contains pyrimidine or pyrimidine and purine bases similar to. 该标记物也可含用于扩增的结合位点,即PCR引物位点,可选拔的排序引物位点,及含有单纯编码被标记寡聚体的单体序列的短部份。 The label can also contain binding sites for amplification, i.e., PCR primer sites, selection can sort primer site, and the short portion comprising pure monomer sequence encoding the labeled oligomer is. 然后,合成寡聚体,即从标记物的自由末端氨基或连接于标记物的接头进行合成,以便使每寡聚体与标记物连接。 Then, a synthetic oligomer, i.e. an amino group or a linker connected to the free end of the marker from marker synthesized, so that each label is connected with the oligomer. 收集的被标记寡聚体可通过裂解接头而从片上被释放出来,产生一可溶性的被标记的寡聚体文库。 Collection of labeled oligomers can be released by cleavage of the linker out from the sheet, to generate a library of oligomers of a labeled soluble.

通过产生分子的可溶性文库可认识到其它优点。 Libraries generated by soluble molecules may recognize other advantages. 在任意一种球基文库中,小球的大小(数量)将会实际限制可装配的文库的大小。 In either library ball group, the size (number) will limit the practical size of the pellets may be fitted library. 例如,可需要几克小球以装配一个含109个不同被标记分子的文库。 For example, pellet may require a few grams to fit a different library 109 containing the labeled molecules. 本发明提供了一种合成被标记分子文库的改进方法,以使得人们可获得实用得多,大得多的文库。 The present invention provides an improved method of synthesizing a library of labeled molecules, such that it can be obtained much more practical and much larger library. 这种改进方法提供了一种手段,其中在混合步骤之前化合物从固体载体上释放,但在每次偶联步骤之前又连结于固体载体上。 This improved method provides a means, which is released from the solid support prior to the step of mixing the compound, but prior to each coupling step and is connected to the solid support.

在该方法中,被标记分子以一种可逆方式被固定于一固体载体上,在该方法的每一混合步骤中允许从载体上释放被标记的分子。 In this method, the labeled molecule in a reversible manner is immobilized on a solid support, allowing the release of the labeled molecules from the carrier at each step of the mixing method. 在一实施方案中,通过一种超滤膜(适合的膜,参见如“Commercial Compatibilty Chart”在Millipore目录中,其表明了对用于合成方法的各种溶剂和化学品稳定的膜)提供此可逆结合。 In one embodiment, by means of a ultrafiltration membrane (for a film, see, e.g., "Commercial Compatibilty Chart" Millipore directory in which indicate the various solvents used in chemical synthesis methods and stable films) provides this reversible binding. 截留分子量约2,000至10,000 daltons的膜(如Amicon YM5膜)适于大多数文库,在偶联步骤中,文库的分子被膜保留,而偶联剂和其它试剂会通过真空抽吸穿过膜。 Cutoff molecular weight of about 2,000 to 10,000 daltons membrane (e.g. Amicon YM5 membrane) is suitable for most libraries, the coupling step, a library of molecules retained by the membrane, while the coupling agent and other agents will pass through the membrane by vacuum suction. 在混合步骤中消除真空,以便分子混合。 Elimination of the vacuum in the mixing step, so that molecular mixing.

在该方法另一实施方案中,在偶联步聚中可逆的共价连接被用于将被标记的分子与载体连接。 In another embodiment of the method, the reversible coupling Buju covalently linked to the carrier molecule are used to be marked is connected. 合适的可逆化学连接例子包括(1)由,如硫醇化(thiolated)被标记分子和N-羟基-琥珀酰亚胺基载体提供的硫酯键,该连接作用可由NH2OH浓度控制;和(2)由如硫醇化(thiolated)被标记的分子和2-吡啶基二硫化物载体(如硫代琼脂糖,来自Sigma)提供的二硫键,该连接可由DTT(二硫苏糖醇)浓度控制。 Suitable examples of reversible chemical linkage include (1), such as thiolated (thiolated) and N- hydroxy labeled molecule - succinimidyl thioester bond provided by the vector, the ligation may be NH2OH concentration control; and (2) as a thiolated (thiolated) labeled molecules and 2-pyridyl disulfide carrier (e.g., agarose thio, from Sigma) disulfide provided, which is connected by DTT (dithiothreitol) concentration control. VI.分析方法利用大的组合文库发现配体,强烈依赖于稳定的亲和敏感的生物化学分析方法的效力。 Vl. Analysis found with a large combinatorial libraries of ligands, strongly dependent on the potency and stability of the affinity sensitive biochemical analysis. 本发明提供了大量与已编码的合成分子文库一起使用的新分析方法,该分子文库反正来也具有广泛的应用。 The present invention provides a number of new analytical methods with a library of synthetic molecules encoded, anyway, to the library of molecules also has wide application. 例如,该文库可用于分析测定中以鉴别结合受体的配体,如与蛋白质结合的肽和核酸,与治疗的靶受体结合的药物,由抗体识别的表位(天然的与合成的),以及识别各种化合物,具有药学,农业和治疗诊断应用。 For example, the library can be used for analysis of ligand binding assays to identify a receptor, such as binding to the protein and peptide nucleic acids, the target receptor binding drug therapy, the epitope recognized by an antibody (natural and synthetic) , and identifying various compounds having pharmaceutical, diagnostic and therapeutic applications agriculture. 相应于所给的这些多种应用,有大量的分析方法相关于本发明。 To those corresponding to the plurality of applications, a large number of analysis methods associated with the present invention. 分析的两种重要类型,虽有某些重叠,包括小球基分析和可溶性分子分析。 Analysis of two important types, although some overlap, pellets and group analysis comprises analyzing soluble molecules.

然而通常这两类分析一般包括下列步骤。 However, usually these two types of analysis generally includes the following steps. 通过分析对文库进行筛选,其中对文库中每一不同的分子测定其与感兴趣受体结合的能力。 Libraries were screened by analysis of the library wherein each of the different molecules was measured its ability to bind with the receptor of interest. 该受体与合成分子文库接触,在受体和文库中分析条件下能与受体结合的任意分子间形成结合产物。 Contacting the receptor with a library of synthetic molecules, between any molecule capable of binding under binding receptors form receptor and the product was analyzed in the library. 然后通过相关于此分子的标记物检查鉴别结合分子,在一实施方案中,文库在结合条件下曝露于其中的受体是一种受体混合物,每个受体与特异于该受体类型的鉴别标记物相关联,所以结合分析之后检查两标记物。 Binding molecules and markers related thereto checking identification molecule, in one embodiment, the library is exposed to a receptor wherein the receptor mixture under binding conditions, with each receptor type receptor specific for the identification of markers associated, so check after two marker binding assay. A.小球基文库的筛选分析当在受体筛选中分离出特异的小球时,可通过各种方式分隔开球粒呈独立形式,包括无限稀释,显微操作或优选地流动细胞计数,在结合分析中用可溶性被标记的受体最有效地评估被束缚的配体文库,通过采用细胞大小的固体载体或小球,可使用流动细胞计数进行了灵敏度受体结合分析及快速球操作。 Screening Analysis A. When the ball-yl libraries isolated receptor-specific screening pellets, may be separated by various means tee tablets were independently forms including infinite dilution, micromanipulation or flow cytometry preferably in binding assays with a soluble labeled receptor most effectively assess ligand library to be bound by the solid support by cell size or pellets, it may be performed using a flow cytometry analysis of the sensitivity and fast ball receptor binding operation .

流动细胞计数,通常是指荧光激活细胞分选成FACS,被认为等同于为达到本发明目的所进行的“荧光激活细胞分选”或“荧光激活小球分选”。 Flow cytometry, generally refers to fluorescence activated cell sorting FACS, it is considered equivalent for the purposes of the present invention to carried out "fluorescence activated cell sorting" or "fluorescence-activated sorting pellets." 熟悉FACS方法的技术人员能很容易地实施本发明的分析方法,这里FACS应用于表达细胞表面抗原或受体的克隆的哺乳动物细胞。 FACS methods familiar in the art can easily implement the analysis method of the present invention, FACS applied herein express cell surface antigen or receptor cloned mammalian cells. 通常,这些分析测定包括标记有荧光标记物的受体与小球混合物的结合,该小球混合物显示了分子文库中各种分子。 Typically, these analyzes assays include binding receptor labeled with a mixture of small balls fluorescent marker, the pellets showed a molecular mixture of various molecular libraries. 洗涤掉未结合的或非特异结合的受体之后,再利用FACS仪来分选小球并鉴别和物理分离有高度荧光的个别小球。 After non-specific binding of unbound receptor was washed away, and then sorted by FACS analyzer to identify and pellets and physical separation of individual highly fluorescent beads. 参见Methods in Cell Biol-ogy,Vol.33(Darzynkievic2,2.and Crissman,HA,编,Aca-demic Press);和Dangel和Herzenberg,1928,J.ImmunolMeth.52:1-14,均在此引为参考文献。 See Methods in Cell Biol-ogy, Vol.33 (Darzynkievic2,2.and Crissman, HA, ed, Aca-demic Press); and Dangel and Herzenberg, 1928, J.ImmunolMeth.52: 1-14, both incorporated herein by by reference. 一旦所需的小球已被分离,人们例可确认标记物以例确定小球上所感兴趣的分子的特性(或分子结构),组成,或合成条件)。 Once the desired beads have been isolated, it can be confirmed that Example Example marker to determine the characteristics of the pellets molecule of interest (molecular structure), consist of, or synthesis conditions).

标准FACS仪允许小球(细胞)荧光分析速率为~104次/秒,并且当以单个球克隆模式操作时,分选速率会降低5-10倍。 Standard FACS fluorescence analysis instrument allows pellets (cell) rate to 104 times / second, and the ball when operating in a single clone mode, the sorting rate is decreased 5-10 times. 在分析非常大的文库时(如>>107个球粒),在使用细胞分选仪分离个别小球之前,可用选择亲和性的一些预筛形式。 In the analysis of very large libraries (e.g., >> 107 pellets), prior to isolation of individual sorter using a cell pellet, some of the available selective affinity prescreened form. 例如,包有受体的亚微细粒大小的超顺磁颗粒经常用来从大的混合群体中通过磁激活分选亲和纯化特异性细胞(参见Miltenyi等,1990,Cytometry 11:231-238,这里引为参考文献)。 For example, packages have submicron sized superparamagnetic particles receptors magnetically activated sorting is often used to affinity purification of specific cells (see Miltenyi the like from a large mixed population, 1990, Cytometry 11: 231-238, incorporated herein by reference). 为高概率探测极少有的结合情况,文库中每种不同的化合物均应存在于文库的许多小球上。 There is a high probability of detecting rare combination of circumstances, each of the different compounds in the library should be present in the library of many small balls. 对于大小为从10μm颗粒构建的已编码文库,假设有100倍的多余度,实际的上限为约1010~1011颗小球上合成108~109个化合物,使用较小的小球可制备出甚至更大的文库,但是常规的细胞计数器看来不能探测或操作远远小于~1μm直径的颗粒。 For the size of the encoded library constructed from 10μm particles, assuming excess of 100 times the actual upper limit for the synthesis of compounds 108 ~ 109 1010 ~ 1011 about beads, use of smaller beads can be prepared even more large libraries, but the conventional cell counters or operation seems not detect much smaller than the particle diameter of ~ 1μm. 当然,正如这里各处所注释的,本发明提供了这种小球在分子文库的合成和筛选中的各种用途。 Of course, as used herein throughout annotated, the present invention provides various uses of such pellets in the synthesis and screening of molecular libraries. 例如,通过使用本发明寡核苷酸标记物串联法,人们不必使用FACS技术来分选文库中的分子。 E.g., by using oligonucleotide marker cascade process of the present invention, it is not necessary using FACS sorting techniques in molecular libraries.

尽管如此,人们也不应过低估计用于本发明目的的FACS仪的效力。 Nevertheless, it should not be underestimated the efficacy of the present invention for purposes of FACS instrument. 在本发明的一个分析方法中,被标记的分子文库合成于荧光小球上。 In an analysis method according to the present invention, libraries of synthetic molecules labeled on the fluorescent beads. 该小球比细胞小,且由荧光材料组成。 The cell pellet is smaller than, and a fluorescent material. 此文库与细胞悬浮液一起温育,此细胞是高水平表达所感兴趣的细胞表面受体的,如连结G-蛋白的受体。 This library of cell suspension was incubated with high levels of expression of this cell is a cell surface receptor of interest, such as G- protein coupled receptors. 当然,人们还可以做许多对照实验,如在所有步骤中使用没有高水平表达细胞表面蛋白的细胞,以及利用这些对照确认错误的阳性细胞(positives)。 Of course, one can do a number of control experiments, as used in all the steps is not expressing high levels of cell surface proteins, and the use of these controls to confirm positive cells (positives) error.

在任一种情况中,表达受体的细胞都能与具有受体配体的任一文库分子结合。 In either case, the receptor-expressing cells can be combined with any text library molecules having receptor ligands. 使用FACS仪根据光散射或另一种荧光信号如来自细胞核内的信号能很容易地识别荧光标记的细胞,并将其从未结合荧光的文库小球中分离及从未标记的细胞中分离。 FACS instrument using light scattering or isolated according to another fluorescent signal. The signal from the nucleus can easily identify the fluorescent labeled cells, and separating unbound fluorescence library pellets and unlabeled cells. 分选后,检测与细胞相连的小球上的标记物,以确认特异于受体的配体,根据此用途,人们可通过如仅选择最明亮的细胞分选出最高水平表达所需受选择最明亮的细胞,并且可调整结合条件以使特异结合情况最大化。 After sorting, the label on the associated detectors and cell pellets to confirm the ligand specific for the receptor, according to this use, it can be obtained by selecting only the brightest The sorted cells expressing the desired level by selecting the highest brightest cells, and the binding conditions be adjusted such that the specific binding of maximized. 为区别开特异于所感兴趣的受体配体与特异于其它细胞表面受体配体,人们可检测与小球相关的标记物,这些小球结合有高水平表达所感兴趣的受体的细胞及非高水平表达的细胞。 To distinguish specific for the receptor of interest and a ligand specific for other cell surface receptor ligands, one can detect the correlation with the marker beads, these beads bound cells expressing high levels of receptor of interest and high levels of non-expressing cells.

本发明的方法也可使人们能够利用FACS仪分选在小球上合成的被标记的分子文库,该小球比当前使用的FACS仪所能分选的最小球粒要小得多。 The method of the present invention also allows people to sorting by FACS analyzer labeled molecule libraries on synthetic beads, the beads can be sorter than the FACS analyzer currently used is much smaller minimum pellets. 在此方法中,筛选已编码的合成文库在信号转换途径的有效活性。 In this method, the encoded synthetic library screening potent activity in the signal conversion pathway. 用几种修饰方法构建此合成文库:(a)小球为1μm或更小,且在FACS中不必是可分选的,这就允许更小的球粒用于一些实例中;(b)标记物是对胞内环境有抵抗性的(特别是对核酸酶有抗性的)寡核苷酸,硫代磷酸酯(physphorothioates)是实现此目的的优选物质;和(c)肽(或其它多种化学实体)通过一接头与小球载体相连接,该接头在胞内环境裂解掉。 This library was constructed in several synthesis modification methods: (a) the ball is 1μm or less, and not necessarily in FACS sortable, which allows for smaller pellets for some examples; (b) labeled material is resistant to the intracellular environment (particularly resistant to nucleases) oligonucleotides, phosphorothioate (physphorothioates) is a preferred material for this purpose; and (c) a peptide (or other multiple chemical entities) connected by a ball joint and a carrier, the linker cleaved intracellularly environment. 这类接头包括通过细胞无害的外部因素如光的使用可裂解的接头,和对胞内环境敏感的接头,如磷酸二酯键或二硫键,但在任一种情况下,可裂解的接头必须对平行合成过程是稳定的。 Such linkers include harmful cells by external factors such as the use of light cleavable linker, and linker sensitive intracellular environment, such as phosphodiester bond or a disulfide bond, but in either case, the cleavable linker parallel synthesis process must be stable.

优选通过机械方法如brolish计划将文库小球导入报道细胞,在一些情况下,可利用导致内在化的生物化学介导的途径,但这种途径通常会导致不符合需要的细胞区室(即溶酶体定位)的掺入。 Preferably by mechanical means such as a library program brolish introduced reporter cell pellet, in some cases, it can lead to the use of biochemical pathways mediated internalization, but this approach often leads to cellular compartments do not meet the (desired instant proteasome positioning), incorporated. 一旦这些小球处在细胞中,所感光趣的肽或其它化合物就会释放出来。 Once the pellets in the cells, the photosensitive peptide or other compounds of interest will be released. 如果10μm的小球已证明具有容纳1010个肽(或其它)合成位点,且容量规模以体积表示,则1μm同样材料的小球将容纳107个分子的合成肽。 If 10μm beads have proven receiving peptide 1010 (or other) site of synthesis, and is expressed in volume capacity size is 1μm same material pellets 107 accommodated synthetic peptide molecule. 如果所有合成的肽在10μm直径(体积为~0.5pl)的单个(球形的)细胞中被释放,那么将会得出~30μm的自由肽的浓度。 If all of the synthetic peptides (spherical) are released in a single cell diameter of 10 m (volume ~ 0.5pl), then the results will be the concentration of free peptide of ~ 30μm. 此浓度可通过小球上的合成密度来控制,并且较低的装载密度会规定更为严格的筛选形式(即筛选活性更高的化合物)。 This concentration can be controlled by the density of synthesis on beads, and the lower loading densities will be more strict screening form (i.e., more active compound screening). 制备受体细胞,在所感兴趣的途径激活或灭活产生荧光信号。 Preparation of recipient cells, activation or inactivation of the pathway in the fluorescent signal of interest. 通过FACS仪选择产生所需效果的个别细胞,并对仍连接于小球和含在细胞内的标记物进行扩增和排序,以确认有活性的合成化合物。 By FACS analyzer selecting cells to produce the desired effect in an individual, and still attached to the beads and the cells contained within the marker amplification and sequencing to confirm that there is active synthesis of the compound.

在另一实施方案中,采用巨球并用其筛选表达受体(即β半乳糖苷酶)的细胞群,该受体能够产生荧光或其它可探测信号,即通过裂解底物产生一种可检测的化合物。 In another embodiment, using macroglobulin and used to screen a population of cells expressing receptors (i.e., β-galactosidase), and the receptor is capable of generating fluorescence or other detectable signal is generated by cleavage of the substrate, i.e., one detectable compound of. 然后这些小球与细胞群混合,细胞群可与小球连接。 Beads are then mixed with the cell population, the cell population may be connected with the ball. 如果用小球上的化合物刺激细胞表面的受体,那么便可产生可探测的化合物,从而提供了从未激活细胞中分选连接于小球上的激活细胞的基础。 If the compound on the bead stimulation cell surface receptors, then the compound can produce a detectable, thus providing a basis for sorting the unactivated cells connected to the activated cell pellet. 人们可使用合适的试剂(即未标记的或没被标记上的)最大限度的选择高亲和性配体。 One can use a suitable reagent (i.e., not being unlabeled or labeled on) the maximum select high affinity ligands.

当然有许多替代流动细胞计数的选择方法用以筛选和选择所感兴趣的分子文库。 Of course, there are many alternative methods of flow cytometry for library screening and selecting molecules of interest. 在一实施方案中,筛选一种编码合成文库,具有细菌活性,以发现能抑制细菌或任意其它微生物生长或杀死细菌或其它微生物的化合物,这些微生物能以二维形式铺平板,如病毒感染的细胞,许多真核细胞包括癌细胞,和一些原生动物。 In one embodiment, an encoding synthetic library screening, having bacterial activity to bacteria found to inhibit the growth of microorganisms or any other compounds or killing of bacteria or other microorganisms, such microorganisms can be plated in two dimensions, such as viral infections cells, many eukaryotic cells, including cancer cells, and some of the protozoa. 通过肽或化合物从其被合成的小球上的控制释放,可筛选相关的或不相关的化学结构的大文库,作用于琼脂培养物中的细胞。 By controlled release from a peptide or compound synthesized on the beads can be screened large libraries of related or unrelated chemical structure, the role of cell cultures in agar.

此方法步骤如下:(1)在琼脂平板上对所感趣的细胞铺平板;(2)用另一层琼脂在细胞上面重叠辅层,其中第二层琼脂中以足以提供悬浮液的稀释度悬浮着带有合成肽/药物的小球,这样的例如使用毛细管可从固体琼脂上挑选出个别的小球;(3)从小球上初始释放肽/药物;(4).培养平板,以使肽/药物的扩散从小球被固定的琼脂扩至周围的琼脂中及下面含指示细胞的琼脂中;(5)读出来自个别小球的已扩散的测试化合物对指示细胞的生长/形态/表型的影响程度;(6)选择指示细胞指示出所需反应(如细菌菌苔死亡)的区带,并使用毛细管或类似工具选挑出含原始小球的琼脂区,其中测试药物从该小球扩散;(7)读码标记物,如通过对个别小球上编码材料的PCR扩增来读出,以确定给出所需反应的肽/药物的结构;和(8)可选择地化学合成适当的药物/肽,并证实所需效果。 This process step as follows: (1) on agar plates of the sensed cells were plated interest; (2) a secondary layer superimposed on another agar above the cell, wherein the second layer agar dilution sufficient to provide a suspension suspension the beads with the synthetic peptides / drugs, such as for example a capillary may be selected from a solid agar individual pellets; (3) the initial release from small peptides ball / drug;. (4) culture plates, so that the peptide / diffusion of the drug from the pellet is fixed to the surrounding agar agar and expanded in the following agar containing the indicator cells; (5) reads out the test compound has diffused from individual pellets of indicator cell growth / shape / phenotype the degree of influence; (6) the cell selection indication indicates that the desired reaction (e.g., bacterial lawn death) zone, and a capillary pick or similar tool is selected from agar-containing region of the original pellets, wherein the pellets from test drug diffusion; (7) reading a marker, such as the individual by PCR amplification coded material pellet is read out to determine the structure of the peptide is given / drug desired reaction; and (8) optionally chemically synthesized appropriate drugs / peptides, and confirmed the desired effect.

有多种方式从小球中释放测试化合物,例如,使用TFA从小球上部分裂解肽/药物,并且允许裂解下的肽在小球表面干燥,这种形式使随后在水中(琼脂)中的重新悬浮允许肽/药物的释放以及将被释放化合物定位于特定小球周围的琼脂区。 There are several ways the test compound released from the pellet in, for example, using TFA cleavage from the small ball portion peptides / drugs, and allow the peptide cleaved surface of the pellet was dried, then re-enable this form suspended in water (agar) of allow release of the peptide / drug compound will be released and positioned around a specific region agar beads. 使用对小球环境的特定变化敏感的化学手段人们可将药物/肽与小球连接,其中这种特定的变化可由在琼脂上铺平板和指示细胞引发或在铺平板和琼脂固化后被引发,如光敏连接,硫醇敏感的连接,高碘酸盐敏感的连接等等,如果必要的话,这些化学试剂本身可被扩散入另一薄的琼脂重叠层中。 Pellets using a specific environmental changes sensitive chemical means one can drug / peptide pellets connecting this particular variation may be plated on agar plates initiator and initiator or in indicator cells and plating agar after curing, photosensitive connection, thiol labile linker, periodate labile linker, etc., if necessary, these chemical agents can themselves be diffused into the agar overlay layer of another film. 这种释放化学过程必须与测试物质的完整性,小球上en-cryption的完整性及下部指示细胞的健康状况具有相容性。 This chemical process must release and integrity test substance on the integrity and the lower part of the small ball en-cryption indication of the health of cells with compatibility. 当然采用的特殊的释放化学过程也会依赖于用于合成文库的化学方法的类型及指示细胞的性质。 Of course, the chemical process using the special release will depend on the nature of a chemically synthesized libraries and indicates the type of cell. 特别优选的是筛选β-内酰胺抗生素文库的方法,用于鉴别新抗生素,这种新抗生素可能会杀死新发展的对已存在β-内酰胺有抗性素的细菌菌株,及优选筛选肽文库的方法,该肽文库是已知抗菌肽如瓜蟾抗菌肽的类似物的文库。 Particularly preferred is a method for screening a library of [beta] -lactam antibiotics, for the identification of new antibiotics, the new antibiotic may kill the bacterial strain is the new development of resistant pigment existing lactam [beta], and preferably screen peptide library method, the peptide library is a known antimicrobial peptides such as melon toad antimicrobial peptide analogs library.

也可使用其它方法筛选小球基分子文库。 Other methods may also be screened pellets yl molecule libraries. 可将亲和吸附技术与本发明文库一起使用。 Affinity adsorption techniques can be used with the libraries of the invention. 例如,小球的混合物可被暴露于受体被固定化的表面(参见PCT专利申请No。91/07087,这里引为参考文献)。 For example, a mixture of pellets may be exposed to a surface receptor is immobilized (see PCT patent application No.91 / 07087, incorporated herein by reference). 洗涤底物从除去未结合的小球之后,便可使用能降低寡聚体/受体相互作用的抗体亲抗原性的条件(如低pH,酸处理或碱处理)洗脱结合于表面的小球。 After the substrate was washed to remove unbound beads, can be used to reduce oligomers conditions (e.g. lower the pH, acid treatment or alkali treatment) of avidity / receptor interaction eluted bound to the surface of the small ball. 如果需要的活,可使用被洗脱的小球重新进行亲和吸附过程。 If desired live, it can be eluted using affinity beads and re-adsorption process. 这些方法及相关的变异体,如上述的磷选择的使用均可以多种方式实施;例如固体载体可以是装入色谱柱的树脂。 These methods and related variants, such as phosphorus can be selected by the above-described embodiment in various ways; for example, a solid carrier may be loaded into the resin column.

本发明的另一种方法中,将被束缚的化合物文库用作结构多样性的来源,所呈的形式适于一族相关分子的亲和性纯化,如药物学上令人感兴趣的受体族。 Another method of the present invention, the source of diversity in the compound libraries as bound structure, the affinity purification was adapted to form a family of related molecules, such as receptor family pharmaceutically interesting . 通常,这种方法涉及使用一种被标记的束缚分子文库筛选另一种未被标记的分子文库。 Typically, this method involves the use of a labeled bound molecule libraries for screening libraries of molecules to another unlabeled. 被标记的、被束缚的文库分子用作一种亲和性纯化试剂,筛选复杂的混合物可溶性蛋白,寡核苷酸,糖、抗体等。 Labeled, bound library molecules are used as a reagent affinity purification, screening complex mixture of soluble proteins, oligonucleotides, sugars, antibodies and the like. 亲和纯化之后,通过洗脱适当的分离及鉴定方法来鉴别与组合文库成员多结合的分子。 After affinity purification, eluting by suitable separation and identification to identify library members in combination with the plurality of binding molecules. 然后,将组合文库分为组合合成的化合物的小部分,通过必需的重复循环过程以少量实验次数准确地测定哪种化合物介导结合过程。 Then, the combinatorial library is divided into smaller sub-combinations of the compounds synthesized, which compounds bind mediated processes accurately measured by the cycle is repeated the necessary number of experiments in a small amount.

用相似的方式,使用组合化学文库鉴别和克隆的受体。 In a similar manner, a combinatorial chemical library used to identify and clone receptors. 许多受体是具有序列同源性(通常反映了来自一个祖先母体的多种进化形式)但呈现不同特异性/亲和性的蛋白质家族成员,其中特异性/亲和性是针对于一组结构相关的配体/同族受体的。 Many receptors are having sequence homology (typically it reflects a variety of forms of evolution from a common ancestor parent) protein family members but presents different specificity / affinity, specificity where / is an affinity group structure specific to Related ligand / cognate receptor. 受体家族(Rn)的每个成员都可代表针对特异药物作用的分离的靶,并且由此通过利用它们不同的性质即身体中的位置,特异性,与配体的亲和性等等来进行药物的发现和开发。 Each receptor family (Rn) can be representative of a separate target for specific drug action, and thus by utilizing their different properties i.e. the position of the body, specificity, affinity and the like ligand discovery and development of drugs. 如果鉴别出一个受体(R1)其结合性质足以令人感兴趣,以致于同一家族其它受体的鉴别将是有利的,那么便可采用下述方法鉴别与R1相关的受体在其结合位点的性质。 If you identified a receptor (R1) which binds sufficiently interesting properties, such that identify other receptors of the same family would be advantageous in that it can use the following method for identifying a binding site for R1 related receptor nature points. 人们首先鉴别与R1结合的配体,然后生产出结构上与该配体非常相关的被标记的组合化合物分子文库。 It first identified ligand binding to the R1, and then produce a combined structure of the compound molecule libraries is associated with the labeled ligand.

下一步,从认为能表达受体家族其它成员的细胞制备多核糖体制剂。 Next, that prepared from other members of the receptor family cell capable of expressing the polysome preparations. 这种多核糖体包含与mRNA结合的核糖体,在以新生肽至几乎完全制备好的蛋白质这一蛋白质合成的各个阶段此mRNA上带有下垂的受体。 This comprises a polysome ribosome binding and mRNA, with pendant receptors in various stages to almost completely prepared nascent proteins to the protein synthesis of this mRNA. 合成近乎完成时的受体蛋白会表达与一种或多种组合文库成员结合的特异的受体性质,使用束缚于固体载体的组合文库,通过与组合文库任一成员间的亲和力亲和纯化带有受体的多核糖体。 Receptor-specific nature in synthesizing nearly complete expression of the receptor protein will bind with one or more members of a combinatorial library, use of combinatorial libraries bound to a solid support, the affinity between the affinity purified by a combination with any member of the library there are more than ribosomal receptor. 这种亲和纯化可包括柱层析方法,从液相中批量分离固定化成分,或水溶液双相分离法;以分离带有连接受体的固相和从非粘附多核糖体编码受体的相关mRNA。 This affinity purification by column chromatographic methods may include, bulk liquid phase is separated from the immobilized component, or an aqueous biphasic separation; in connection with separating the solid phase from the non-receptor and receptor-encoding adhesive polysome related mRNA.

下一步,使用标准技术(逆转录酶等)从编码同源受体群的mRNA进行cDNA合成,并将cDNA群克隆入适于快速序列分析的载体中。 Next, using standard techniques (reverse transcriptase, etc.) for cDNA synthesis from mRNA encoding homologous receptor population, and population of cDNA cloned into suitable vector in rapid sequence analysis. 根据可能的受体序列的现有知识及期望的序列保留程度,可使用PCR或另一种扩增方法扩增以该方法富集的cDNA。 According to the prior knowledge and the degree of retention of the desired sequence may receptor sequences, using PCR or another amplification method to amplify cDNA enriched in this way. 通过对适当数目cDNA克隆的,可鉴别与已知受体R1序列具有足够的序列同源性的cDNAs(无论是全部长度或不是全部长度的),代表公认的相同受体家族(Rn)的其它成员。 By appropriate number of cDNA clones can be identified with known receptor R1 sequences have sufficient sequence homology to cDNAs (whether or not all the entire length of the length), the same as the other representative of a recognized family of receptors (Rn) of member. 通过标准克隆方法,选择性地制备这些新cD-NAs的全长cDNA克隆(或其相关部分,如编码相关于配体结合的胞外区区域部分),并通过标准方法表达这些cDNA(即在真核表达系统中表达为适当的可溶性或膜结合蛋白)。 By standard cloning methods, these new length cDNA clone of the cD-NAs (or a relevant portion, in relation to the encoding region of the ligand binding portion of the extracellular domain) is selectively prepared, and expressed the cDNA by standard methods (i.e. eukaryotic expression systems suitable for the expression of soluble or membrane-bound proteins). 使用检测受体配体相互作用的标准形式,检测来自组合文库混合化合物的群体结合成个别化合物的结合。 Detected using a standard form of receptor ligand interactions, detecting a mixed population of compounds from a combinatorial library bind to binding of individual compounds. 以这种方法可准确鉴定来自文库的那个化合物与新鉴别的受体结合。 In this method can accurately identify the compound from the new library with the identified binding receptor.

例如通过有限稀释法或那些将细胞与偶联于小的超顺磁球粒的受体一起温育的类似方法,可物理分离个别小球,然后使用高功率磁力提取表达受体配体的细胞(参见Mil-tenyi等,1990,Cytometry 1.1:231-238,这里引为参考文献)。 For example, by the limiting dilution method or methods analogous to those coupled to the cells and small superparamagnetic pellets incubated with receptor, can be physically separate individual pellets, then extracted using a high-power magnetic cells expressing receptor ligands (see Mil-tenyi etc., 1990, Cytometry 1.1: 231-238, herein incorporated by reference). 正如上所示,使用FACS可进一步分析和分选磁选择细胞。 As shown in the above, and may be further analyzed using FACS sorting magnetic cell selection. 放射性核苷酸也可用于标记受体,这样可以通过选择被放射活性标记的小球来鉴别和分离小球。 Radionucleotides may also be used to label the receptor, which can be identified and isolated by selecting the pellets radioactively labeled pellets. B.筛选可溶性分子人们也可采用被标记的分子文库有效用于本发明新的分析中,其中配体在与感兴趣的受体结合之前被溶解呈标记的或未标记的形式。 B. Screening of soluble molecules may also be used by people labeled molecule libraries useful in the present invention, a new assay in which the ligand is dissolved in the form of labeled or unlabeled form before binding to the receptor of interest. 为筛选可溶性(不带有小球)被标记分子的特大文库,在弱亲和条件下优选使用亲和层析。 Screening of soluble (without pellets) is large library of labeled molecule, under conditions of low affinity and preferably using affinity chromatography. 例如,用含几百μg所感兴趣受体的10mL亲和层析柱的样品可筛选108个分子的30mg文库。 For example, an affinity chromatography column 10mL sample of interest containing the receptor can be screened several hundred μg 30mg library 108 molecules. 寡核苷酸是该文库优选的标记物,其能很易被PCR扩增并克隆入商业可购的TA克隆载体(In-vitrogen,公司)该载体是DNA序列分析之前用于分选标记物信息的一种方便形式。 Oligonucleotide library is the preferred label, which can be very easily amplified by PCR and cloned into the TA cloning vector commercially available (In-vitrogen, Inc.) which is a vector DNA sequence analysis before sorting markers for a convenient form of information. 此外,寡核苷酸标记物可按如上所述的方式串联起来,从而允许人们收集可溶性被标记分子库(pools),克隆所选库的被串联的标记物,然后测定比标记序列鉴别所需的化合物。 Further, the embodiment described above, the oligonucleotide tag can be linked together, thereby allowing it to collect the label is soluble in series labeled molecule libraries (Pools), the selected library clones, and determination of sequence identification mark than desired compound of.

使用固定化受体也可筛选可溶性被标记的分子。 Immobilized receptor can be screened soluble labeled molecule. 将被标记的分子与固定化受体接触并洗去非特异结合的分子之后,用各种方法中的任何一种方法将结合的被标记的分子从受体上释放出来。 After the labeled molecule to be labeled receptor molecule in contact with the immobilized molecule and washing away non-specific binding, by any of a variety of methods will be released from the bound receptor. 选择扩增标记的分子从受体上释放出来。 Selecting amplifiable marker molecules released from the receptor. 选择扩增标记物,然后对其进行检定,解码以鉴别与受体特异结合的分子结构。 Amplification selection marker, and then subjected to assay, to identify and decode the molecular structure of the receptor specific binding. 使用一种通过与小球连接面被固定化的受体可分析测定溶液中的被标记的寡聚体,例如用一种荧光标记的配体重复测定。 By use of a ball connection surface immobilized receptors Determination labeled oligomers in the solution, such as repeated measurement a fluorescent labeled ligand. 可将带有固定化受体的小球复原,并使用FACS分选小球以鉴别阳性小球(减弱的荧光是由于文库分子与被标记配体竟争而造成的)。 The pellets can be of the receptor with a fixed recovery, and sorted using FACS to identify positive beads pellet (diminished fluorescence is due to the library molecules are labeled ligand competition caused). 然后将相关的识别标记扩增,解码。 Then identifying indicia associated amplification, the decoding.

在小球上可合成文库的可溶性分子;然后分析之前将分子裂解下来。 In the synthetic library beads can be soluble molecules; and then cleaved molecules prior to analysis. 在一实施方案中,分子文库的显微小球被置于非常小的个别室或孔中,这些是在硅或其它适合的表面上“毫微级制造”出的室或孔。 In one embodiment, the molecule libraries microscopic beads are placed in individual chambers or very small holes, which are on the surface of silicon or other suitable "nano-create" the cells or pores. 通过将小球分散于足以产生每孔一个球粒的装载缓冲液中,使小球装载于小孔中。 By dispersing the pellet loading buffer sufficient to produce pellets of each hole, so that the ball in the loaded wells. 在一实施方案中,小球溶液被置于小孔上部的池中,允许小球沉降入小孔中。 In one embodiment, the solution is placed on top of the ball apertures in the pool, allowed to settle into the wells pellets. 使用化学或热系统可完成寡聚体从小球上的裂解,但优选光裂的系统。 Using chemical or thermal systems can complete cleavage of the oligomer from small ball, but is preferably the light split system. 所感兴趣的分子可从小球上裂解下来,在溶液中产生未标记分子(标记物仍连接于小球上)或在溶液中或产生标记分子。 The molecule of interest may be cleaved from small ball, generating unlabeled molecules in solution (label still attached to the bead) in solution or generated or labeled molecule. 在任一种情况下,所感兴趣的分子从小球上裂解,但仍与小球和识别标记一起保留于小室中。 In either case, the cleavage of small molecule of interest on the ball, but the ball is still retained in the recognition mark and the cell together.

在一实施方案中,小孔的表面或部分表面覆盖有受体,在小孔中加入结合缓冲液和荧光标记的已知的受体配体,以进行配体的溶液相竞争分析,这里配体是特异于受体的。 In one embodiment, the surface or a part of the pores is covered with a receptor, the addition of a known receptor ligand binding buffer and fluorescence-labeled in the wells, compete for the ligand solution is analyzed here with body is specific for the receptor. 通过单层固定化受体的同焦图象可估计一实例中荧光标记配体与受体的结合。 Single confocal images through the same receptor can be immobilized binding estimate an example fluorescently labeled ligand to the receptor. 受体表面呈减弱荧光的孔表明被释放的配体与标记配体产生竞争,检查孔中呈现竞争的小球或标记物,以揭示竞争性配体的特性。 Receptor surface was reduced fluorescence indicates release hole ligand compete with the labeled ligand, competition presented manhole pellets or marker, to reveal the characteristics of competing ligands.

通过显微操作器从孔中取走个别小球,可选择性地对阳性孔的识别标记小球进行复原。 Individual pellets removed from the wells by a micromanipulator, the identification mark may be selectively pellets of positive wells for recovery. 另一种方式包含在小球生产过程中或标记过程中使用结合有荧光分子的小球,仅在阳性孔中使用合适波长的激光漂白存留的小球,然后将所有小球全部移去,并用FACS分选,以鉴别被漂白的阳性小球,之后相关标记物可被扩增,解码以鉴别与受体特异结合的分子。 Another embodiment included in the pellet production process or during the use of labeled molecules bound fluorescent beads, using only laser light of appropriate wavelength in the bleached pellets remaining positive wells, and then all the pellets removed all, and with FACS sorting to identify positive bleached pellets after related markers may be amplified, decoded to identify molecules that specifically bind to the receptor.

在本发明另一实施方案中,采用相对较大的被标记小球,其中所感光趣的分子在一系统反应中从小球上被裂解下来。 In another embodiment of the present invention, a relatively large beads are labeled, wherein the photosensitive interest is cleaved from small molecules on the ball in a reaction system. 在该方法中,小球直径为50至500μm,容量相当于每粒球100至500pmol的肽,优选地如果构建肽文库,使用容量的为200pmol的100μm小球。 In this method, pellets having a diameter of 50 to 500 m, the capacity of each piece ball 100 corresponds to a peptide 500pmol, preferably constructed if the peptide libraries, the used capacity 200pmol of pellets 100μm. 这种文库的典型大小是从106至108,优选107个不同分子。 A typical size of such libraries is from 106 to 108, preferably 107 different molecules. 文库被分为100个库,每库含约100,000颗球粒。 Library was divided into 100 library, the library comprising about 100,000 particles per pellet. 例如在肽文库的情况下,从库中裂解某一百分比约25%的所感兴趣的分子,产生每1mL体积500nM的肽。 For example, when a peptide library, from the library as a percentage of cleavage of about 25% of the molecules of interest is generated per volume of 1mL 500nM peptide.

然后对初裂解的库进行竞争分析或功能分析测试。 Then early cleavage library competition analysis or functional analysis test. 鉴别出具有最高活性的库,然后恢复原始中的存留分子,并将存留分子等分成100个库,每库1000颗球粒,重复进行此过程,直至每库具有一颗球粒,从该球粒读出标记物并鉴别出所感兴趣的分子。 Library having the highest activity were identified, and then restore the original remaining in the molecule, and the molecules remain divided into 100 pools, each library 1000 pellets, repeating this process until each having a library pellets, from which the ball tablets and read markers identified molecule of interest. 这种方法避免了Houghten法的再合成和框架限定,优点在于库是随机的而不是相关的化合物。 This method avoids the re-synthesis and the frame defining Houghten method, is advantageous in that the compound library is random and not relevant. 由于许多低亲和力相关分子的累积作用,活性混合物的机会减少了。 Because of the cumulative effect of many low-affinity associated molecules, the chances activity of the mixture is reduced. C.筛选天然产物文库由于具有即可获得的自动高通量分析,现在在筛选天然产物时的限制条件首先是获得和操纵(分散,溶解,标记等等)样品的能力;第二是定性阳性样品的活性成份所要求的实际操作。 Since the screening of natural product libraries C. automatic high-throughput analysis can be obtained, in the present limitations of screening natural product first obtained and manipulated (dispersion, dissolution, label, etc.) the ability of the sample; second positive qualitative the actual operation of the sample required for the active ingredient. 本发明提供了产生和筛选天然产物的文库的方法,该文库提供了大量的呈易被筛选形式的样品并鉴别样品中活性化成份。 The present invention provides methods for producing and screening libraries of natural products, the libraries provide large numbers of samples as a form readily screened and identified in a sample activated ingredient. 该方法的基础是生物化学和化学多样性的结合,具有来自“天然产物”即来自自然的代谢作用的多样性,最简单的例子包括给微生物培养物补料肽收集物。 The method is based biochemical and chemical diversity of bound, i.e. from the metabolism of diversity from the natural "natural products", the simplest examples include microbial cultures fed to a peptide collection. 每一微生物菌株会产生许多修饰的肽(代谢物文库)。 Each produce many strains of microorganisms modified peptide (metabolite library). 因为每种培养物都会(潜在地)含有非常复杂的代谢物混合物,所以需要一种有效的筛选方法。 Because each culture are (potentially) complex containing a mixture of metabolites, the need for an effective screening method.

可采用几种途径,且这些途径可被正交分类为因子化的或被标记的,和可溶性的或被束缚的。 It can be used in several ways, and these pathways can be classified as a factor of orthogonal or marked, or is bound and soluble. 为清楚描述,考虑作为原料的可溶性肽文库,将该文库的等份样与每种微生物发酵筛选程序中典型的菌株一起温育,且筛选典型形式的培养基,然后阳性培养物与文库的集合一起温育并再筛选,继续分解因子(factoring)过程,直至鉴别到产生最多活性代谢物的肽的输入,然后根据可能前体分子的知识进解活性代谢物的定性。 For clarity of description, consider the soluble peptide libraries as a starting material, the typical strain aliquot incubated with each library screening programs microbial fermentation, and the typical form of the filter medium, then the set of positive cultures with library and then incubated with screening, continued factoring (to factoring) process until the identification of the peptide generating an input to the most active metabolites, and then into the solution in accordance with the qualitative knowledge of active metabolites thereof may be pre molecules. 因此,第一次筛选鉴别活性生物体,随后的步骤鉴别活性前体,最终通过标准分析手段鉴别活性代谢物。 Thus, the biological activity of the first differential screening, a subsequent identification step the precursor activity, the final identification of the active metabolite by standard analytical means.

然而在所有形式中,分解因子是最繁琐的过程,由切断合成产生的和从树脂上裂解下来自由的文库产生可溶性化合物,这些化合物用于细胞摄取和完整生物体代谢作用。 However, in all forms, is the most tedious factoring process, soluble compounds produced by the cutting synthetically produced libraries cleaved from the resin and free of these compounds for cellular uptake and metabolism of whole organism. 然而,个别化合物的浓度相当低(相反地与培养收集物的多样性有关),导致无效的酶转化和极低浓度的结果代谢物,通过生物产文库子集并将每一子集单独与每种微生物分离物一起发酵,可提高化合物的浓度。 However, relatively low concentrations of individual compounds (diversity conversely related to the collection of the culture), resulting in an invalid and enzymatic conversion of a very low concentration results metabolites produced by biological library subset and each subset separately with each isolates fermented with microorganisms, can increase the concentration of the compound. 通过固定一个或多个位置并使剩余的位置随机化来构建子文库,例如,有500个五肽子文库,其使用了50个结构单元包含20个固定位置的所有排列。 Sublibrary constructed by fixing one or more positions and the remaining positions randomized, e.g., 500 sub-pentapeptide library using all permutations of the structural unit 50 comprises a fixed position 20. 这些子文库中的每一个都包含125,000个化合物。 Each of these sub-libraries are contained 125,000 compounds. 标记文库的使用在便利程度和灵敏度方面其有很大的优越性,但对将化合物收集物暴露于代谢活性的方法,要求有所改变。 Library using labeled convenience in terms of their degree of sensitivity and has a big advantage, but were exposed to the compound was collected by metabolic activity requires a change. 组合原料不必仅仅是肽,但可由任意一种组合化学收集物组成。 Composite materials is not necessary that the peptide, but can be any one of chemical composition composed collected.

寡聚体和其它分子文库可构建于在组合过程中,每步以识别标记物编码,这可通过与标记物直接连接和寡聚体平行合成来进行。 Oligomer and other molecule libraries may be built in the combination process, each encoding step in order to identify the marker, which can be performed by direct connection to the parallel synthesis of markers and oligomers. 如果寡核苷酸被用作标记物,那么复合体会相对大些,但仍小得足以插入细胞中,呈活性形式,这通过脂质体融合,电穿孔,溶剂透化等进行。 If the oligonucleotide is used as a marker, the complex experience relatively large, but still small enough to be inserted into a cell, in an active form, liposome fusion, electroporation, permeabilized solvent like. 一旦进入细胞中,该复合体将对与细胞的代谢机制,通过使用修饰的核苷酸和核苷酸连接可避免寡核苷酸标记物易受损害而降解。 Once inside the cell, the composite will have the metabolism of cells, by use of modified nucleotides and nucleotide linkers may be avoided oligonucleotide marker vulnerable to degradation. 通过回收溶解细胞的培养物中的活性代谢物,筛选样品并解码标记物以揭示前体化合物。 Recovered by dissolving the active metabolite in the culture of cells, a marker screening samples and decoded to reveal the precursor compound. 和活性前体一起进行的活性生物体扩大发酵将产生足够量的活性代谢物用于定性测定。 And an active organism together with active expansion of precursor fermentation yield adequate amounts for the qualitative determination of the active metabolite. 通过小球上已编码组合过程制备的化合物文库可暴露于细菌,真菌,植物细胞等的溶解物中。 Compound library prepared by the process of the coded composition pellets may be exposed to a lysate of bacterial, fungal, plant cells and the like. 以这种方式,避免在完整细胞中插入被标记复合体的需要,并且球粒上的许多分子中仅相对少量的分子需要进行检测(如在荧光激活结合分析测定中)。 In this manner, the need to avoid the insertion of the labeled complex in intact cells, and a number of molecules on the pellets only required a relatively small amount of molecular detection (e.g., fluorescence activated binding assay).

本发明另一种有用方法包括使用一种微生物培养的产品作为另一种培养物的补料。 Another useful method of the present invention comprises the use of a microorganism culture as another feed product of the culture. 以来自大规模培养物(~1升)的100个不同微生物分离物的收集体进行说明。 Collecting 100 to isolate different microorganisms from large scale culture (~ 1 L) will be described. 通过过滤回收每一种培养物的上清液,并将其分为100个10mL的等份样。 The supernatant was recovered by filtration and each culture, and divided into 100 aliquots of 10mL. 每一等份均以100株中的一株接种并温育。 Each aliquot are inoculated in a 100 and incubated. 由此从100份微生物分离物中产生10,000个样品(代谢物的代谢物)。 Thereby generating 10,000 samples (metabolite metabolites) from 100 microbial isolates parts. 由差异甚大的种可将这种组合代谢的方法延伸为连续代谢:例如将微生物发酵产品与外来植物溶解物一起温育,或将植物组织的提取部分与真菌培养物一起温育。 This combination method may be extended from the metabolism of the great differences in species of sequential metabolism: for example, microbial fermentation products and the lysate incubated with exotic plants, parts of plants or extracts with the tissue incubated with fungal cultures. 这些方法可作用:产生的化学多样性方法的任意一种产品都能用于这些连续代谢产品暴露步骤中。 These methods may act: any product produced by the method can be used for chemical diversity of these metabolites continuous exposure step.

在本发明的另一方面,通过产生组合标记的脂质体混合物来筛选天然产物多样性,每个脂质体优选包囊天然产物化合物文库的仅一种成份或一种简单的混合物。 In another aspect of the present invention, only one component to screen natural products produced by diversity combination mixture of labeled liposomes, preferably encapsulated liposomes each compound libraries or natural product of a simple mixture. 本发明允许同时分析1000个-10,000个化学化合物或天然产物提取物及分析100个色谱分离的馏分,这些馏分由带有阳性信号的天然产物提取物衍生。 The present invention allows the simultaneous analysis of analytes and a 1000 to 10,000 chemical compounds or natural product extract 100 chromatography fractions, these fractions with a positive signal from a natural product-derived extract. 在这种关系中,“同时”的意思是在同一度管中与读出系统的细胞一起分析。 In this context, "simultaneously" means that the analysis cell is read out together in the same system of pipes.

组合标记的脂质体混合物制备如下。 Combination of labeled liposomes mixture was prepared as follows. 对于来自库中的每一种单独的天然产物提取物或化学产品而言,制备分离的脂质体,该脂质体包囊水相中的试测物质,在包囊时特定的脂质体标记物被掺脂质体制剂中。 From the library for each of the individual chemical or natural product extract product, isolated liposome preparation, the liposomes encapsulating an aqueous phase test test substance, in particular when encapsulated in liposomes label is mixed with the liposome formulation. 可将脂质体冻干以便在低温下长期储存,这对收集物及收集位点附近的天然产物样品为储存很有益处,并且将脂质体冻干便于天然产物提取物呈一种适合随后组合实验的形式而长期储存。 It may be lyophilized liposomes for prolonged storage at low temperatures, and the natural product samples collected spaces near the collection point to store very beneficial, and the lyophilized liposomes to facilitate natural product extract was then suitable combination of experiments and long-term storage. 优选脂质体制剂中的类脂对所有样品是相同的并根据类型和组合物进行选择以产生所需大小和完整性的均一薄层的脂质体。 Preferred lipid liposome formulation and the same is selected depending on the type and composition to yield liposomes with the desired size and uniformity of the integrity of the thin layer for all samples. 试剂如海藻糖可在脂质体形成时被包入,以允许冻干及随后通过加水而重建完整脂质体。 Reagent trehalose may be entrapped during liposome formation, followed by lyophilization to allow adding water to full reconstruction liposomes. 在包囊提取物/化学物质的标记脂质体产生/再生时,可使用高压技术,该技术允许包囊的水相体积大于由脂质体包裹的计算出的体积,从而能测试更大体积的测试材料,由此可读出细胞基的更大信号。 Generating labeled liposome encapsulation extract / chemicals / reproducing time, using a high pressure technique, which allows the encapsulated aqueous volume greater than the volume calculated by the liposome, so that it can test a larger volume the test material, whereby a greater signal read out of the cell group.

现存的脂质体技术便于结合有高百分比(>80%)水相(与每种测试物质的使用效果相关)的脂质体的生产。 Existing production of liposome technology to facilitate binding with a high percentage (> 80%) aqueous phase (associated with the use of the effects of each test substance) liposomes. 可用各种“洗涤”方法除去未结合的水相。 By various "washing" methods to remove unbound aqueous phase. 此外,可生产脂质体,其不会泄漏或改变包囊的水相(相关于标记的特异性和没有混合的被包裹水相),并且可生产脂质体,其不会改变插在其类脂单层中的成份(作为标记物插入的糖脂/蛋白质抗原不会被改变)。 Further, to produce liposomes which do not leak or encapsulated aqueous phase change (in relation to the labeled specific and not wrapped mixed aqueous phase), and to produce liposomes, which does not change in its inserted lipid monolayer component (/ protein antigen is not changed as a marker for insertion of glycolipid).

此方法可采用各种标记物,例如,标记物可以是(a)具有激发和发射性质的不同荧光团,这些性质允许每种荧光团在其它种荧光团或其结合物存在时可被检测-荧光团可被选择分配入包囊的水相中或重建脂质体的膜相中,面向外部;(b)不同的稀土元素的金属阳离子,其通过原子吸收光谱可被识别-稀有金属原子将被设计为盐形式分配于重建脂质体的包囊水相中;(c)不同的抗原,根据需要通过其与合适的单克隆抗体和初级/二级荧光探测抗体/荧光团的特异反应来识别这些抗原-在蛋白质,糖蛋白和/或糖脂上的抗原可被选择分配入重建脂质体的膜相中,面向外部;和(d)抗原,荧光团和/或金属离子的结合物,其能大大提高用于筛选的可能信号的数量,标记不同脂质体的附加水平增加的数目可来自不同水平的荧光团/金属离子/抗原的使用,由此可鉴别出信号混合 This method may employ various markers, e.g., markers may be (a) different fluorophores having excitation and emission properties, these properties allow each fluorophore can be detected when the other fluorophore or a combination thereof is present - fluorophores may be selected dispensed into the aqueous phase encapsulated or the reconstruction phase of the liposome membrane, facing outward; (b) a metal cation different rare earth elements, which is determined by atomic absorption spectroscopy can be identified - the rare metal atoms is designed in the form of a salt partitioned encapsulated aqueous phase of the liposomes reconstruction; (c) different antigens as needed through which a suitable monoclonal antibodies specifically reactive primary and / two fluorescence detecting antibody / fluorophore and to recognition of these antigens - the liposome membrane remodeling phase protein, glycoprotein and / or glycolipid antigens may be selected on the allocated outside-oriented; and (d) an antigen, the fluorophore and / or metal ion conjugate which can greatly increase the number of possible signal for the screening, increasing the number of fluorophore labeled liposomes different from the additional levels may be different levels / metal ion / antigen used, whereby the mixing signal discriminated 中每种成份的不同“数量”。 Different in each component of the "quantity."

也可采用一种通用的荧光标记,其为所有脂质体共有,可从那些未与脂质体融合的细胞中快速选择出与脂质体融合的细胞。 May also be a universal fluorescent label, a total of all liposomes, those cells can be selected from the unfused liposomes rapidly out of the cell fusion with liposomes as. 此标记物与任一用于个别脂质体制剂的组合标记不同,并且其与产生脂质体的类脂,信号标记,和药/天然产物的水溶液样品在脂质体产生时进行混合。 This marker is used in combination with any of a liposomal marker different individual, and which is, signal mark, and drug / natural product samples were mixed with an aqueous solution of the lipid to produce liposomes when liposomes produced. 也可采用荧光标记,其在高密度时自动猝灭(即在脂质体膜中),但在脂质体融合和在细胞膜呈现荧光。 It may also be a fluorescent label that automatically quenched (i.e. a liposome membrane), but liposome fusion and the fluorescence in the cell membrane presentation at a high density. 根据脂质体与细胞的融合方式,例如也可结合入一种病毒起点或糖脂的融合蛋白,其将介导脂质体与细胞的紧密粘附(依赖于凝集素类粘附过程,由适当的受体介导,受体是根据需要导入用于读出的细胞系)。 The fusion liposomes to cells embodiment, for example, fusion proteins may also be incorporated into a viral origin or glycolipids, which tightly adhered liposome-mediated cell (depends on the adhesion process lectins from appropriate receptor-mediated, the receptor is required for reading out the introduced cell line). 这类元素不会影响脂质体-脂质体的相互作用(所避免发生的情况),但可提高脂质体-细胞融合的效率。 Such elements do not affect liposome - liposome interaction (happens avoided), but may increase the liposome - efficiency of cell fusion.

该方法可利用细胞读出系统,使用一种在启动子下游含报道基因(如荧光素酶),β-半乳糖苷酶的细胞系,这里启动子在应答外源激素或配体如类固醇,细胞因子,前列腺素,抗体,抗原等等被加入到细胞中时被活化。 The method may utilize cell readout system, the use of a reporter gene downstream of the promoter-containing (e.g., luciferase), [beta] galactosidase half cell lines, where the promoter such as a steroid hormone or in response exogenous ligand, when activated by cytokines, prostaglandins, antibodies, antigens and the like are added to the cells. 活化配体与细胞表面受体或细胞内受体的结合激活一级联信号,其最终导致应答启动子的激活及信号基因的转录。 Activating ligand binding to cell surface receptors or activation of a receptor with an intracellular signal that ultimately results in the activation and gene transcription signals is responsive promoter. 信号蛋白的表达导致从个别的被活化细胞中产生信号,这些细胞可被定量检测。 Protein expression signals results in a signal from an individual cell is activated, these cells can be quantitatively detected. 在寻找化合物时,该化合物作为拮抗剂作用于胞内信号转导级联的任意一部分,可通过加入外源信号激动剂(细胞因子,激素等)对细胞的整个群体进行预处理,并且在脂质体融合之后测量在个体的细胞基础上信号输出降低。 In the search for compounds which are acting as antagonists to any part of the intracellular signal transduction cascade, may be pretreated by the addition of the entire population of cells foreign signal agonist (cytokines, hormones, etc.), and aliphatic plastid measurement signal output decreases after the cell fusion on an individual basis. 在寻找化合物时,该化合物作为激动剂作用于胞内信号转导级联的任一部分,不必向细胞中加入外源信号,并在脂质体融合之后,可测量在个体细胞基础上的信号的出现。 In the search for compounds which are acting as an agonist in any portion of the intracellular signal transduction cascade, not necessary to add exogenous signals to the cells, liposome fusion and then, in a measurable signal on an individual cell basis appear.

标记的脂质体混合物与大量过量的读出细胞混合,过量细胞数目是至关重要的,在脂质体一细胞融合之后仅产生下列产品:(i)不与脂质体融合的细胞;和(ii)与一个脂质体融合的细胞(受体细胞)。 Labeled lipid mixture with a large excess of readout cells are mixed, an excess of the number of cells is critical to produce the following products only after a cell fusion liposomes: (i) not fused to the liposome cells; and (ii) a liposome fusion with the cells (recipient cells). 这一步有效混合是很必要的,可使用连续搅拌或线性流动的细胞悬浮液来达到有效混合,其中将脂质体混合物慢速加入到细胞悬浮液中。 This step is necessary to effective mixing, stirring or using a continuous linear flow of the cell suspension to achieve an effective mixing, wherein the lipid mixture was slowly added to the cell suspension. 用标准方法引发融合,如加入PEG或使用高电压,如果需要,可通过在细胞-脂质体膜中包含入融合剂或配体-受体识别对以增强融合。 Fusion initiation by standard methods, such as high voltage or PEG was added, if necessary, by the cell - into the liposome membrane comprises a fusion or ligand - receptor to enhance the recognition of the fusion. 融合步骤有效地在受体细胞中加入单一脂质体的水相部分。 Fusion step effectively added to the aqueous portion of the liposomes in a single recipient cell. 由此,天然产物提取物水溶液,来自化学库(inventory)的测试化合物或天然产物提取物的层析分离级份现在便能在胞内信号转导途径的任一点起作用。 Thus, natural product extract solution was test compounds from a chemical library (Inventory) or natural product extract parts chromatography stage was now able to any one of the signal transduction pathways that act intracellularly. 融合步骤也可向个别受体细胞中加入特异性标记物,该标记物为特定测试化合物样品提供信号。 Fusion step may also be added to individual specific markers recipient cells, the label provides a signal for the particular test compound sample. 如果那些标记物原来存在于脂质体的类脂膜中,则现在这些标记物分布于受体细胞的外层细胞膜中。 If those markers present in the original lipid membrane liposome, which is now located in the outer cell membrane markers of the recipient cell. 此位置的抗原易接近特异性单克隆抗体的表面(panels)。 Surface (Panels) the accessibility of antigen-specific monoclonal antibodies of this position. 原来存在于特殊脂质体水相中的稀土金属离子现在存在于受体细胞细胞质中。 Originally present in the aqueous phase of the liposomes particular rare earth metal ions now exists in the recipient cell cytoplasm. 融合步骤也可加入共有的识别细胞的脂质体标记物,这些细胞原是受体细胞,来自那些没有参与脂质体融合的过量细胞。 Fusion step may also be added to the total cell identification marker liposome, these cells originally recipient cell from those cells not involved in excess of liposome fusion. 标记物可以是从脂质体膜移向受体细胞膜的荧光团。 The label may be moved from the cell membrane receptor liposome membrane fluorophore.

下一步,细胞、与个别脂质体融合的细胞和任意一种未融合的脂质体的混合物,与外源配体一起温育(如在测试拮抗剂的情况下)或不加任何物质进行温育(如在测试激动剂的情况下)。 Next a mixture of cells, liposome fusion with individual cells and any unfused liposomes (as in the case of the test antagonist) with an exogenous ligand was incubated with or without any substance incubated (e.g., in the case of the test agonist). 使用限定浓度和温育时间的对照化合物来确定这一温育的时间。 Using defined concentrations and incubation time to determine the time of the reference compound incubation.

优选的使用FACS选择所感兴趣的化合物(细胞)。 Preferred compounds of interest using FACS selection (cell). 例如可先使用前面或侧面光散射从任意未融合的脂质体中分选细胞(受体细胞或非受体细胞)。 For example, start with the front or side of any light scattering from the liposomes for sorting the unfused cells (recipient cell or recipient cell). 大的细胞可很快从小脂质体中分离出来。 Large cells may be rapidly separated from small liposomes. 下一步可从那些不是脂质体受体的过量细胞中分选是脂质体受体的细胞,是受体的细胞带有共有的脂质体衍生的荧光标记,而非受体细胞带有的这些标记是非荧光的,当然这一步骤是可选择的,但是如果将其作为预分选步骤进行操作,会得到通常多数细胞的分离,这些细胞与作为受体的少数细胞的随后分析测定是不相关的。 Next to the excess cells that are not liposomes is a cell receptor for sorting receptor liposome, the liposome is derived cell receptor labeled with a fluorescent common, rather than the recipient cells with these non-fluorescent markers, of course, this step is optional, but if it operates as a pre-sorting step, most typically will be isolated cells, then these cells Determination as acceptor few cells are not related. 为确证拮抗剂的特性,可在报道蛋白(如β-半乳糖苷酶或我素酶)发射光的基础上进行分选,从少数负荧光细胞或低荧光细胞分离出多数正荧光细胞(通过早期加入外源性配体来抑制这些正荧光细胞)。 To confirm the characteristics of an antagonist can be made in a reporter protein sorting (e.g. β- galactosidase or luciferase I) based on the emitted light, fluorescent cells isolated from a small number of negative or low positive fluorescent cells showing most fluorescent cells (by early addition of exogenous ligand to inhibit these positive fluorescent cells). 后两种细胞类型由包囊于特定脂质体的化合物的假定拮抗效果产生,这些特定脂质体已与这些个体细胞融合。 After the two cell types is assumed antagonistic effects produced by a liposome encapsulating a particular compound, the particular liposome has fused with the cells of these individuals. 为确证激动剂,可在报道蛋白射光的基础上进行分选,从少数正荧光细胞分离出多数负荧光细胞,后者细胞产生于脂质体衍生的化合物的假定的激动效果。 To confirm agonist, reporter protein may be carried out on the basis of light emitted from the sorting, cells were separated from the majority of negative fluorescence few positive fluorescent cells, cells which assumed agonistic effect on liposome-derived compound.

在某些实验中,根据上述标准可将所有的感光趣的细胞作为一个群体进行分选,并用标准FACS方法收集偶然性细胞作为克隆个体。 In some experiments, all of the photoreceptor may be interesting as a cell population were sorted according to the above criteria, and the individual clones were collected as a contingency cells by standard FACS methods. 可将这些个体细胞作为单一细胞进行分析,分析其所带有的特定标记物,准确确认介导所需效果的特殊脂质体。 The individual cells can be carried out as a single cell analysis, which bears a particular marker, to confirm the accuracy of liposome-mediated specific desired effect. 在其它应用实例中,可分析测定标记物在整个被分选阳性细胞群体中的分布,并且根据实验设计方案及插入来自不同时间/地点/库的样品中的特定标记物,在第一次通过时能确认整个阳性细胞群中标记物类型的多样性。 In another application example, the marker may be analyzed in the whole distribution measuring sorted positive cell population, and according to the experimental design and insert samples from different time / place / library specific markers in the first pass when the entire positive cell population can confirm the type of marker diversity.

通过适于所用的特殊标记物结合物的方法可对收集的单个细胞或细胞群进行分析,用FACS和/或传统的分光光度法可对荧光标记物进行分析。 By methods appropriate to the particular label of the conjugate may analyze individual cells or cell populations collected by FACS and / or conventional spectrophotometrically analyzed on a fluorescent marker. 通过适当标记的单克隆抗体的加入和ELSA,FACS,放射性同位素或发光辅助分析可对抗原标记物进行分析测定。 May be assisted analysis of antigenic markers was analyzed and measured by the addition of ELSA, FACS, or a radioisotope emitting an appropriately labeled monoclonal antibody. 通过原子吸收光谱可分析测定金属离子标记物,对标记物解码后,仅以在第一次通过时产生阳性结果的那些脂质体的混合物重复进行测度,或使用向分离的细胞样品中加入的每一所感兴趣成员的纯脂质体进行重复测试。 Analysis by atomic absorption spectrometry determination of metal ions may be a marker, the marker after decoding, to produce a mixture of only a positive result in the first pass is repeated liposomes those measures, or added to the cells in the isolated sample each member of the pure interest liposomes repeat the test.

本发明的这些及其它方法可以自动化形式进行,以实施本发明的操作,如下。 These and other methods of the invention can be automated fashion, to implement the operation of the present invention, as follows. VII.仪器在某些实施方案中,一些单体(如氨基酸)的偶联步骤会要求相对长的温育时间,由于这个原因及其它原因,使许多单体平行加入的系统是合乎需要的。 VII. In certain embodiments, the instrument, some of the monomers (e.g., amino acids) of the coupling step would require a relatively long incubation time, and for this and other reasons, many systems of the monomers added in parallel is desirable. 本发明涉及自动化仪器,用于产生和筛选被编码的合成分子文库,一种优选的仪器,能同时进行50至100或更多个平行反应,描述于1993所11月2日递交的美国申请No.08/149,675中,这里引为参考文献。 The present invention relates to an automated instrument for generating and screening libraries of synthetic molecules encoded, a preferred apparatus, can be 50 to 100 or more simultaneous parallel reactions, described in 1993, filed on November 2 U.S. Application No in .08 / 149,675, incorporated herein by reference. 这一装置能够在程序控制下,将反应混合物或合成反应的固体载体的料浆分布于各个通道中,进行收集,混合及重分布。 This device can be under program control, the reaction mixture or a solid support synthesis reaction slurry distributed to the respective channels, collecting, mixing and redistribution.

然而,一般情况下生产标记分子合成文库的专门仪器,如肽合成仪,都需要复杂的管道系统,同时还需大批量的单体池/及标记物,且往往伴随各种偶联反应。 However, the production of synthetic libraries of labeled molecules generally specialized equipment, such as a peptide synthesizer requires complex piping systems, it is also required large quantities of the monomer pool / and markers, and often accompanied by a variety of coupling reactions. 由于标记物的分配能力,它将一些简单的指令转化为适当的探针混合物讯号并因此指导混合物质的分配。 Since the distribution capacity of the marker, it is simple instructions into the proper mixture of a probe signal and thus guide the allocation of the mixed material. 由单体合成的产物,按照需要,在指定的混合物中分配。 , As needed, from the product distribution in the specified monomer synthesis mixture. 反应的启动,温度及时间控制功能均由系统提供。 Start the reaction, the temperature and time control functions provided by the system. 经过适当的设计,该产品也可用作多途径肽合成仪,能生产出1~50毫克(粗产品)达100种不同的肽段供分析之用。 With proper design, the product can be used as multi-channel peptide synthesizer capable of producing 1 to 50 mg (crude) of up to 100 different peptide fragments for analysis. 参见专利文献91/17823,再次引用仅供参考。 See Patent Document 91/17823, again incorporated by reference.

典型的该仪器主要由以下几部分组成。 Typically the instrument is mainly composed of the following parts. (1)各种贮存、混和及运送合成产物(如肽段及寡聚核苷酸)的装置:(2)一个封闭的反应室,各种反应物在此不活动的环境中发生各种复杂的反应;(3)一只封闭反应容器板模;(4)引导反应物流向各自反应器的装置;(5)收集和分离微型小球(0.1~100微米)的装置;(6)在每个化学反应完毕后冲洗该反应器中小球的装置。 Means (1) a variety of storage, transport and mixing synthetic products (e.g., oligonucleotides and peptides) in: (2) a closed reaction chamber, the various reactants herein inactive complex environment occurs reaction; (3) a closed reaction vessel plate mold; (4) direct the reaction stream to a respective reactor; means (5) collection and separation micro-beads (0.1 to 100 microns); (6) in each after completion of the chemical reactions means that the reactor is flushed small balls. 反应器的板模可有好几种设计方案。 Formwork reactor may have several designs. 比如,反应器可置一个套环中致使其可按照一个中心轴旋转(相当于一个离心机),也可以将其置于-12×8的板模(96孔微滴定平板格式96-well microtiter plate format)中。 For example, the reactor can be set so that a collar rotatably (corresponding to a centrifuge) in accordance with a central axis, may be placed in the mold plate (96-well microtiter plate format 96-well microtiter -12 × 8 of plate format) in. 这任何一种设计都将使反应物及其产物运输、抽吸(aspirstion)的自动化变得简单易行,而且传递功能(transfer functions)在某些方面也有很高的应用价值。 This design will make any kind of reactant and product transport, aspiration (aspirstion) automation becomes easy, and the transfer function (transfer functions) in some areas also has a high value.

用于粒子的收集及再分配系统也有好几种设计方案。 Systems for the collection and redistribution of particles there are several designs. 例如,小球可悬浮于一适当表面张力及浓度的溶剂中以便于自动吸液装置(pipetting in strument)能将游离小球转化为一个联合的反应器。 For example, pellets can be suspended in a suitable surface tension and the concentration of the solvent to the automatic pipetting device (pipetting in strument) can be converted to a free ball joint reactor. 混合以后,这些小球可通过同样的自动吸液装置被重新分配到反应器中。 After mixing, the beads may be reassigned to the reactor through the same automatic pipetting device. 同样,小球也可通过一特定有阀的反应室将其交联起来。 Similarly, beads may be crosslinked by a particular reaction chamber together with a valve. 阀门打开时溶剂流入,致使小球变成一个交联的容器。 Solvent into the valve opens, causing the ball to become a crosslinked container. 混合以后,再通过与前述流向向反的溶剂流致使小球重新分离。 After mixing, re-isolated by causing the beads to anti-solvent stream and the flow direction.

在另一实施方案中,小球也可通过顶部开口的压缩式(closely spaced)反应器将其交联。 In another embodiment, the beads may be crosslinked by compression (closely spaced) open top of the reactor. 浸泡反应器也可以使小球混合。 Soaking reactor may be mixed pellets. 如果小球带有磁性,那么应用一定的磁场,使小球再通过反应器的底部,可使它们重新得到分离。 If the ball is magnetized, then the application of a certain magnetic field, so that the ball through the bottom of the reactor, they can be separated again. 非磁化的小球可通过真空抽吸(vacuum suction)使小球通过反应器底部而使其重新得到分离。 Non-magnetized beads The beads may be by vacuum suction (vacuum suction) through the bottom of the reactor and it was isolated again. 在其它的实施方案中,就是将小球置平板上,然后在上面覆盖一“切刀”(“cookie-cutter”)形的装置以使其各部分得到分离,下面将有更详描述。 In other embodiments, the pellet is placed on a flat plate, and cover a "blade" ( "cookie-cutter") in the above apparatus shaped portions so as to be isolated, there will be described in more detail.

洗球系统也可有好几种设计方案。 Wash ball system may also have several designs. 小球可通过输液装置(liquid delivery)及抽吸管道系统来加以清洗。 Pellets may be cleaned by an infusion (liquid delivery) and the suction pipe system. 每个反应器都配备有自己的一套管道系统,或者具备一个适合各种反应器的清洗管道系统。 Each reactor is equipped with its own set of piping system, or provided with a pipe cleaning system for a variety of reactor. 在后一种情况下,移液及抽吸管道系统可以安装在一个自动化的臂上以对每个反应器能单独起作用。 In the latter case, the pipetting and aspiration pipe system may be mounted on an automated arm can function to each reactor separately. 可运用离心,磁化小球或一定的磁场等手段将每个反应器中的小球制成球形小体。 Use of the means may be a centrifugal, magnetic beads or the like made of certain magnetic spherical bodies each reactor pellets. 反应器的底壁可镀上一层惰性的膜以使反应物及其产物可用真空吸收器将其清除。 The bottom wall of the reactor may be coated with a layer of inert film to the reactants and products can be used to clear the vacuum absorber. 同样,使用一种允许一个持续流体穿过反应物及清洗液的反应器,即反应器终端设有路配式装置(1uer fittings)及滤膜,也可以使反应内容物得以顺利清除。 Similarly, to allow the use of a fluid through a continuous reactor the reactants and the washing liquid, i.e. the reactor passage is provided with a terminal apparatus of formula (1uer fittings) and the membrane may be smooth reaction contents were removed.

任何一种自动化多功能仪器在使用上都要受到一些很重要的限制。 Any kind of automated multi-functional instrument will be subject to some very important restrictions on use. 因为该体系依靠各个单独的反应室而起作用,每个反应室都必须和反应物运输系统及一“母体罐”(″moth-er pot”)相连。 Since this system relies on various separate reaction chamber functions, must be connected to each reaction chamber and a reactant transport systems and "parent pot" ( "moth-er pot"). 小球被泵到罐中进行混合后又重新分配到各反应室,以便于以后的单体添加得以有条不紊地进行。 Each pellet is pumped into the reaction chamber and then be redistributed to the mixing tank in order to add to the orderly conduct of subsequent monomers. 由于单体或其它一些构建单元数量庞大,且在一个庞大的反应系统中分离小球及反应物都比较困难,故该类仪器在实际应用中受到了局限,使其中同时产生的反应不到100个。 Since monomers or some other construct a large number of cells, and separation of the reactants and the pellet are more difficult in a large reaction system, so that such devices has been limited in practical applications, so that while generating a reaction wherein less than 100 a.

本发明避免了因混和及再分配小球而将其在不同反应室内来回泵的必要,简化了反应物运输物运输手续,并使少量小球的分离准确而易行。 The present invention avoids the need for mixing and redistribution due to their round pellets and the pump chamber in the different reactors, simplifies to transport the reactant transport procedures, and a small pellet of isolated lines apparent accuracy. 最基本的设计主要包括一个平板,平板表面上带有一系列的反应“位点”;该平面或者是水平的,或者带有一系列小孔以形成反应位点。 The basic design includes a plate having a series of reactions "site" on the plate surface; or the plane is horizontal, or with a series of apertures to form a reaction site. 比如,反应表面可设计出256个反应位点,以16×16点阵排列。 For example, the reaction may be devised surface reaction sites 256 to 16 × 16 matrix arrangement. 每一反应位点就是平板表面上的一点或一个小孔,一小批合成的小球将被吸附于此处。 Each reaction site is a point or a small hole on the flat surface, a small group of synthetic beads are adsorbed in here. 吸附力可由磁力、真空过滤(vacuumfiltration)、伴随被动机械分类的重力或其它一些简单手段得到。 By the magnetic attraction force, vacuum filtration (vacuumfiltration), gravity or some other means along with simple passive mechanical classification obtained. 首先将稀释的小球悬浮液均匀地在平板表面涂成一薄层,然后同一定的吸附力将小球集中到每个反应位点。 First, the diluted suspension of beads evenly into a thin layer on the plate surface, and with constant suction force to concentrate pellets each reaction site.

当小球被集中到一定反应位点以后,应用隔离工艺将每个反应位点制成一个隔离的反应室(暂时性的),如图29所示。 When the ball is concentrated to a certain reaction site after application of the isolation process each reaction site made of a reaction chamber isolated from the (temporary), shown in Figure 29. 通过一种变通手段,隔离装置可永久性地吸附于平板表面,以形成浅的反应小孔。 By means of a flexible means, isolation means may be permanently adsorbed on the plate surface, the reaction to form a shallow hole. 反应物被运送到每个反应室,小球释放到由反应物而形成的悬液中,反应就会进行。 Reactants are delivered to each reaction chamber, the ball is released into the suspension formed by the reactants, the reaction will be carried out. 条件理想时,小球还可被重新吸附到平板表面并将反应物清除。 When ideal conditions, pellets may also be re-adsorbed to the surface of the plate and the reaction was clear. 当所有偶联反应循环步骤完成后,反应室隔离体系也随之清除,小球被收集到反应位点上方的小池里保存。 When all the coupling reaction cycle step is completed, the reaction chamber isolation system also will be clear, pellets were collected into a small pool above the reaction site storage site.

小球的混和是由在反应池里诱导产生对流而得以完成的,然后小球被重新吸附到平板表面以进行下一轮新的偶联反应。 The pellets are produced by mixing induced convection in the reaction pool has been accomplished, and pellets were re-adsorbed onto a flat surface for the new coupling reaction. 随后的步骤,包括冲洗步骤是通过类似方式完成的。 Subsequent steps including the step of rinsing is done in a similar manner. 反应物的添加及清除是通过自动吸液及管道系统联合来实现的。 Add and remove reactants and by an automatic pipetting system piping joint achieved. 特定反应物的添加(如:单体)可用自动多功能吸移管管理器(multi pipettors)来完成。 Add specific reactants (eg: monomer) available automatic pipettor multifunction device (multi pipettors) to complete. 普通反应物的添加及清除可由一固定管道系统来完成。 Was added and the reaction may be ordinary clear a fixed piping system to complete. 该系统在每个反应室无需阀门。 The system does not need a valve in each reaction chamber. 在反应室隔离装置安装之前或清除之后,一些常规工序如清洗工序可以直接在小球上进行(on the beads en masse)。 Before and after the reaction chamber in isolation or removal device is mounted, some conventional processes such washing step may be performed (on the beads en masse) directly on the ball.

大批量单体及其它结构单元的使用给编码工作带来了额外的负担。 The use of large quantities of monomers and other structural units to work encoding bring an additional burden. 在一寡聚核苷酸标记物编码过程中,一基本的1000单体序列需要一个5碱基序列来标记每个反应步骤;超过1024单体序列就需要6碱基序列十为之编码。 In an oligonucleotide tag encoding process, a single basic sequence requires a 1000 nucleotide sequence 5 marks each reaction step; over 1024 monomer sequence requires ten 6-base sequence encoding whom. 为减少合成仪管道系统的复杂性(即减少特定反应添加步骤),本发明提供了一种特定的编码策略。 To reduce the complexity of the synthesizer duct system (i.e., a particular reaction step of adding reduction), the present invention provides a specific coding strategy. 为说明此方法,现以16×16反应板为例加以说明。 To illustrate this method, 16 × 16 is to be described as an example the reaction plate. 这种排布可使256个不同的反应同时进行。 This arrangement allows 256 different reactions simultaneously. 应用多碱基偶联为每个反应单独编码是一项十分艰巨的任务。 Multi-base coupling reaction for each separate coding is a very difficult task.

这种点阵排布具16行、16栏,每个点都有一个唯一的地理位置。 This lattice arrangement with 16 rows and 16 columns, each point has a unique geographical location. 每行位点可标记为一组,所有16可被特征性地用2碱基密码子(“亚密码子”)加以编码。 Each row sites may be labeled as a group, all 16 may be characteristically be encoded with 2 base codons ( "alkylene codon"). 一条形板或槽形块可用作2碱基添加的基板,以形成16个反应室(注意,偶联的碱基是作为单体的磷酰胺化产物(phosphoramidites),而不是作为二聚体)。 A plate-shaped or channel-shaped base block 2 may be used as the substrate was added to form a reaction chamber 16 (note, is coupled as a monomer nucleotide phosphoramidite product (of the phosphoramidites), rather than as a dimer ). 参见专利文献No.WO93/09668,再次引用仅供参考。 See Patent Document No.WO93 / 09668, again incorporated by reference. 这种反应位点定位形式与其它的类似;例如,可用光学方法标记小球,参见美国专利No.5,143,854,其中的条形覆盖法(striped masking process),这里引用仅供参考。 And other similar forms such reactive sites positioning; for example, be optically labeled beads, see, U.S. Patent No.5,143,854, wherein the strip-shaped covering method (striped masking process), herein incorporated by reference. 如果使用平板或槽形块,那么平板必须降至与网格合成表面同一水平面上,以使合成分子文库过程中,使每个反应得以分开。 If plate or channel block, the plate must be reduced to the same level with the surface of the synthetic mesh, to the synthetic molecular libraries process, each reaction is separately.

在标记反应过程中,小球不能释放进去。 In the labeling reaction, the ball can not be released into it. 然而,它们的空间分离一直要维持到下一小步完成为止。 However, their spatial separation has to be maintained until the next small step is completed. 当行的每个反应位点被亚密码子标记后,将板上升且旋转90°,然后降低以形成覆盖栏的条带。 After the reaction sites each row is labeled sub-codon, the plate rises and rotates 90 °, and then lowered to form a strip covering bar. 16栏又重新被2碱基亚密码子标记,这样,256个反应位点都被4碱基“超密码子”(“supercodon/)特征性地标记上。通过识别反应位点位置,超密码子就能给出每个反应室所要加的单体。 16 column again labeled 2 Nokia base codon, so that the reaction sites 256 are marked on the base 4 "super codon" ( "supercodon /) characteristically. By identifying the position of a reaction site, super password each reaction chamber can give sub desired additional monomers.

VIII.平行偶联合成反应仪(Apparatus for Parallel ouplingSynthesis Reactions)总体来说,本发明为合成多种多样的物质提供了一个固体底物(solid substrate)。 VIII. Synthesis parallel coupling reaction apparatus (Apparatus for Parallel ouplingSynthesis Reactions) In general, the present invention provides a solid substrate (solid substrate) for the synthesis of a wide variety of substances. 例如,该仪器使用了上面所述的小球。 For example, the apparatus described above using the pellets. 下面就以肽合成为例介绍一下该仪器的基本情况。 Here's an example to introduce the basic peptide synthesis situation of the instrument.

首先它将使底物多元化(plurality),用“S”表示,合成反应将可以在多元化底物上进行。 First, it would diversify the substrate (Plurality), with "S" represents the synthesis reaction may be carried out on the diversification of the substrate. 在发生偶联反应的联系分子“L”的帮助力,使底物能有选择地提供。 Information molecules in the coupling reaction of "L" assistance force, so that the substrate can be selectively provided. 底物被分配后与各种单体起反应,比如与“A”及“B”单体反应形成以下的底物形式:SA 和 SB然后,底物又重新组合、混和并再次分配。 After the substrate is assigned with various monomers react, such as the "A" and "B" monomer to form a substrate formed of the following: SA and SB then, again substrate composition, mixed and redistributed. 经过混合及分配步骤后,就形成两种或更多的底物形式,每种形式中既包括SA又包括SB。 After mixing and dispensing steps, two or more formed substrates forms, each form comprising both SA and SB.

其余的偶联反应便可照此进行。 The rest of the coupling reaction can proceed accordingly. 例如,上述的聚合产物可再与单体C及D反应以形式以下的产品形式:1.SAC SA-D2.SBC SBD通过上面简单的例子就可看出该合成技术可在短时间内得到大量不同的底物形式。 For example, the above-described polymerization product can be further reacted with the monomer C and D products in the form of the following form: 1.SAC SA-D2.SBC SBD can be seen from the above simple example of this synthesis may be obtained in a short time a large number of in the form of different substrates. 通过对合成这类分子多种收集物的周密设计和(或)通过对该底物标记物提供平行合成方法,如上所述,这些底物将会有极高的应用前景。 By careful design of the synthesis of such molecules of collection and (or) by providing a method of parallel synthesis for the substrate markers, as described above, these substrates will have a high prospect. 在一特别的实施方案中,该发明提供了一些能有效生产各类用途底物的设备和方法。 In a particular embodiment, the invention provides some methods and apparatus capable of efficiently producing all kinds of uses of the substrate.

A.概说(General)图1所示的是一能合成多种收集物分子的设备。 A. General said apparatus is capable of synthesizing a plurality of collections of molecules represented by (General) FIG. 该设备包括一个母体混合器200,通过一普通项式多阀箱体(topcommon manifold)212及215,221-229管道与一多元化反应器201-209相偶联。 The apparatus 200 includes, through a conventional multi-term valve housing (topcommon manifold) 201-209 215,221-229 phase coupling 212 and a conduit with a parent diversification reactor mixer. 212箱体又与221-229及215管相偶联。 221-229 and 212 and the housing 215 and the phase coupling. 反应器201-209也通过111-119阀及241-299管选择性地与添加单体的反应物供应池231-239相偶联。 The reactor also passed 201-209 111-119 241-299 tube and the valve is selectively adding monomer phase reactant supply reservoir coupled 231-239. 压力传递系统(pressurized dilivery systen PDS)265分别通过管260及256与母体混和器200及反应器201-209相偶联。 Pressure delivery system (pressurized dilivery systen PDS) 265 and 256, respectively, the tube 260 to the parent and the mixer 200 through the phase coupling reactor 201-209.

当小球悬液从母体混合器200转移至反应管201-209时,合成反应即开始。 When the transfer from the mother pellet suspension to the reaction tube 200 blender 201-209, the synthesis reaction starts. 当129阀打开(所有其它阀关闭)时,小球悬液从母体混合器200通过215管进入顶式箱体212。 When the valve 129 is opened (all other valves closed), the ball 200 enters the mixer from the mother suspension-top box 212 through 215. 然后小球悬液通过221-229管在201-209反应器中进行分配。 Beads suspension was then partitioned 201-209 221-229 tube reactor by. 从单体库231-239选出的反应物通过各自的管241-249进入各自反应器201-209。 231-239 monomer selected from the library by a respective reactants into the respective reactor tubes 201-209241-249. 这样,在有小球的201-209反应器中,偶联反应便发生。 In this way, the reactor at 201-209 small ball, the coupling reaction will occur.

图1所示的压力传递系统265通过管260与母体混合器200相偶联。 Pressure 265 shown in FIG transfer tube 260 by the mixer 200 to the parent phase conjugation system. 当阀门10及通风阀90打开时,该系统通过管260将受压的反应物从系统运到母体混合器。 When the valve 10 and vent valve 90 is opened, the system is pressurized through line 260. The reaction was transported from the mixer to the parent system.

压力传递系统265也通过管256,分离阀100,低位箱体阀101-109,低位管271-279,注射阀111-119以及管241-249与反应器201-209相偶联。 Pressure delivery system 265 through the tube 256 also, the separation valve 100, lower valve housing 101-109, 271-279 lower tube, an injection valve and a tube 111-119 241-249 201-209 reactor phase coupling. 为了将反应物从PDS265选择性地运到反应器201-209,受压的反应物通过打开的100阀时入管256及一低位多阀箱体214。 To the reaction was selectively transported to PDS265 201-209 reactor, the reaction was pressurized through the opening 100 of the valve into the tube 256 and a multi-valve casing 214 low. 然后,受压的反应物在压力条件下通过选择性打开的101-109阀,针对性地上升到管271-279。 Then, the reaction was pressurized through the valve 101-109 is selectively open under pressure, targeted to rise to 271-279 tube. 然后反应物再通过选择性打开241-249管进入各自的反应器201-209。 The reaction was then re-opens into the respective tubes 241-249 of the reactor by selectively 201-209.

例如,为使反应物从一给定的单体池231运送至反应器201,一定量受压溶液被从PDS265被压向管256,然后进入低位多阀箱体214,进而进入管271。 For example, the reaction was transported from a given pool of monomer 231 to the reactor 201, an amount of the solution is under pressure from the tube 256 is pressed PDS265, then into multiple lower valve housing 214, inlet pipe 271 and further. 在一个适当的时刻,一定量的单体反应物从单体池231注射到正在流向管271的溶液流中。 At an appropriate moment, a certain amount of monomeric reactants 231 is injected from the pool to the monomer solution stream is flowing to the tube 271. 注射单体过后,从反应池231流出的混合液通过241管流向反应器201以参加下面的偶联反应。 After injection of the monomer, the reaction vessel 231 from flowing mixture 241 flows through the reactor 201 to participate in the following coupling reaction.

为了用单体池406-412中的单体选择性地标记反应器201-209中的小球,经过打开的阀门4-7,一个受压的来自库406-412标记单体反应物溶液流进入-PDS265的普通型多阀箱体255。 For use of the monomer pool monomer 406-412 201-209 selective marker in the reactor pellets, after opening valves 4-7, a pressure of 406-412 marker monomer reactant solution flows from the library into the common multi -PDS265 valve housing 255. 然后,受压标记物单体及一适当的压力溶剂流通过打开的100阀及256管进入低位多阀箱体214。 Then, a monomer and a compression marker suitable solvent stream into the lower pressure by opening valve 100 and valve housing 214 over 256. 受压的单体标记物及其适当的溶剂流上升到各自的271-279管,然后通过各自打开的101-109阀进入反应器201-209,在此将发生小球标记反应。 Pressurized monomer markers and suitable solvent stream rises to the respective tubes 271-279, 201-209 and then enter the reactor through the respective valve open 101-109, herein labeled beads will react.

经过理想的单体和/或标记物添加反应后,反应器201-209的小球悬液可回收到母体混合器200供重新聚合和混和。 After the desired monomer and / or marker is added after the reactor, the reactor is recycled pellet suspension to the parent 201-209 200 mixer and mixed for re-polymerization. 为使小球悬液从反应器201-209回收到母体混和器200,可通过打开的122及101-109阀,从管250向小球悬液中吹入氩气以使其增压。 The beads suspension 201-209 is recovered from the reactor to the parent mixer 200, through open valve 122 and 101-109, an argon gas blown from the pipe 250 to the suspension to make pellets boost. 100及110阀关闭,固此受压压小球悬液便流向管221-229,然后进入普通型多阀箱体212,再通过打开的129阀进入215管,最终进入母体混和器200。 Valves 100 and 110 closed, this compression pressure solid pellet suspension flow tubes 221-229 will then enter the common multi-valve housing 212, valve 129 and through opening 215 into the final mixer 200 into the mother. 在200混和器中,小球悬液重新混和,然后再重新分配到反应器201-209,以为更进一步合成其它分子作好准备。 In mixer 200, the mixed pellet re-suspension, and then redistributed to the reactor 201-209, that further prepare synthetic molecules other.

在另一种设计里,201-209的每个反应器之产及与母体混和器200之间,可安装上一多功能三通阀门(plurality ofthree-way vessel)。 In another design, the reactor 201-209 of each product of mixing between parent and 200, may be mounted on a multi-functional three-way valve (plurality ofthree-way vessel). 该阀门最好安装在普通顶式多阀箱体212中。 The valve is preferably mounted on top of the ordinary multi-valve housing 212. 通过这种方式,一些反应器可与母体混合器200分开。 In this way, some of the reaction may be separated from the mixer 200 to the parent. 这对为进一步合成而重新分配小球悬液将特别有用。 This will be particularly useful for the synthesis of further redistribution of the ball suspension. 例如,小球最初被分配到反应器201-209用于合成反应,然后回收到母体混和器200中,在此重新分配过程中,仅一部分3通道阀门打开,比如这样,仅仅反应器201、205及209能够进入小球悬液以供进一步合成之用。 For example, pellets initially assigned to 201-209 for synthesis reactor, and then recovered to the parent mixer 200, this reallocation process, only a portion of the valve opening passage 3, so that for example, only the reactor 201, 205 and 209 can enter the beads suspension for further synthesis.

图1也显示了非同心搅拌器(nonconcentric agitators)280及285,分别与一顶式反应器托架290及一底式反应器托架295相偶联。 Figure 1 also shows a non-concentric agitator (nonconcentric agitators) 280 and 285, respectively, and a reactor 290 and a bottom bracket reactor coupled with a bracket 295. 顶式反应器托架290是不可动的,但底式295可随非同心搅拌器与涡形马达(vortexing motor)300联合,从而使底式反应器托架295及底端反应器201-209中产生搅拌力。 Reactor top bracket 290 is not movable, but may be combined with the bottom of Formula 295 and a non-concentric volute stirrer motor (vortexing motor) 300, so that the reactor bottom bracket and a bottom end of the reactor 295 201-209 stirring force is generated. 由于托架之间的管道十分灵活,故搅拌力使反应器201-209中的反应物产生涡流,并因此促进合成反应的进行。 Because the pipeline between the bracket is flexible, the stirring force so that the reactor was 201-209 eddy current is generated in the reaction, and thus facilitate the synthesis reaction.

顶部普通多阀箱体212在一端与215管相连,这样便提供了物质在母体混和器200及多阀箱体212之间运输的管道。 Common multi-top box 212 is connected to one end of the valve 215 and, thus providing a precursor material between the mixer 200 and the multi-valve housing 212 of the transport pipe. 在另一端,多阀箱体212与126相连。 At the other end, and the multi-valve 126 is connected to the housing 212. 管216通过121阀向箱体212中通入高压氩气,同时也使箱体212通过120阀能够向其内部通气。 Tube 216 into the high pressure argon to the housing 212 through valve 121, while also capable of housing 212 through valve 120 vent the interior thereof.

两只功能强大的传感器90S及99S被安装在母体混和器200的外表面,以探测其中的液面高度。 Powerful features two sensors 90S and 99S are mounted on the outer surface of the mixing matrix 200 to detect the liquid level therein. 如果流体存在于伟感器的测量范围(detection envelope)之内,传感器将打开。 Wei if the fluid present in the sensor measuring range (detection envelope), the sensor opens. 反之,传感器将关闭。 Conversely, the sensor will turn off.

传感器101S-119S为光学传感器,主要对半透明管中流体的存在与否进行监测。 101S-119S sensor is an optical sensor, the presence of mainly semi-transparent fluid in the pipe or not monitored. 当一股流体存在于管中,传感器将打开。 When one fluid present in the tube, the sensor will open. 反之,传感器将关闭。 Conversely, the sensor will turn off.

同样地,一只声学传感器120S也监测流体的存在。 Similarly, an acoustic sensor 120S also monitors the presence of the fluid. 流体,包括小球悬液在内,流经管道时将被其监测到并将传感器打开。 Fluids, including suspensions, including beads, which flows through the conduit to be monitored and a sensor opening. 相反,当装有声学传感器的管道里无流体存在,传感器120将自动关闭。 In contrast, when the pipe's acoustic sensor with no fluid is present, the sensor 120 will automatically shut down. 由于光学传感器在有些条件下不能可靠地区分半透明空的聚四氟乙烯管和含有小球悬液的同样管道,声学传感器的应用则弥补了这一不足。 Since the optical sensor can not be reliably distinguished under some conditions translucent Teflon tube and the same air duct pellets suspension, comprising the application of acoustic sensors to compensate for this deficiency. 此外,虽然传感器120S使用了声学传感器,但任何其它能够区分出空管及含有流体或小球悬液管的传感器均可使用。 Further, although the use of sensors 120S acoustic sensor, but any other sensor capable of distinguishing an empty bobbin and suspension fluid containing tubes or beads may be used. 因为声学传感器较之于其它类型传感器,如光学传感器要贵得多,故每台合成仪中仅120S一只声学传感器。 Because the acoustic sensor compared to other types of sensors, such as an optical sensor is much more expensive, so that each synthesizer only one acoustic sensor 120S.

反应器库(bank)的设计使母体混和器与各反应器之间只需一个或更少的阀门。 Reactor bank (Bank) is designed so that mixing between parent each reactor with only one or fewer valves. 这种设计有其独到的优越性。 This design has its unique advantages. 因为阀门的机械打开和关闭常会造成对脆弱小球及合成多聚体的损害,这种设计大大降低了此过程的损坏程度。 Because of the mechanical valve is opened and closed often cause damage to the fragile little ball and synthetic polymer, this design greatly reduces the extent of the damage this process. 同样,如果小球体积过大,它们常被阻滞于阀门周围致使系统运转受阻。 Similarly, if the ball too large, they are often arrested at around the valve causing the operation of the system is blocked. 此外,众多阀门的来回关开将会产热使体系温度升高,这对一些温度敏感性的聚合分子将会产生不良的副作用。 Further, the plurality of valves will round off on the heat generating temperature of the system increases, which will have adverse effects for some temperature sensitive polymeric molecules. 因此,减少小球悬液通过的阀门数是个十分有利的举措。 Therefore, reducing the number of suspensions by the ball valve is a very beneficial move. 如果129阀不包括在内,可利用增压技术来防止流体在母体混和器与反应器库(reaction vessel bank)之间的流动。 If not included, the valve 129 may be utilized to prevent the flow of pressurized fluid technology between the parent library mixer reactor (reaction vessel bank) of.

如前所述,上例中所说的阀门当有在工作状态时都是关闭的。 As previously described, the valve of said embodiment in working condition when there are closed. 没有一个特定的打开指令,阀门总是处于不工作的关闭状态。 It is not a specific instruction to open the valve in a closed state does not always work.

B.机械文件从机械角度,该自动化合成仪可大致分为三个系统:反应器库、母体反应器及压力传递系统。 B. Mechanical file Mechanically, the automated synthesizer can be roughly divided into three systems: the reactor bank, and a pressure reactor precursor delivery system. 如上所述,偶联合成反应在反应器库的各反应器内进行。 As described above, the coupling reaction is carried out in the respective synthesis reactors of the reactor bank. 母体反应器负责来自各反应器小球的容纳、收集及混和工作。 The parent is responsible for containing the reactor, collecting and mixing work from each of the reactors ball. 压力传递系统负责适当溶剂和/或适当反应物溶剂浓度溶液的合理运送,将它们在合成反应的一定阶段分送至母体反应器或各个反应器库。 Solvent delivery system for the proper pressure and / or transport of the appropriate reactants reasonable solvent concentration of the solution, they were distributed to each reactor or reactor parent library at some stage of the synthesis reaction. 此外,整个体系是在封闭状态下工作的。 In addition, the entire system works in a closed state. 必要时的增压往往是通过通入惰性气体如氩气来得以实现的。 When supercharging is often necessary by passing through an inert gas such as argon gas to be achieved. 氩气由于容易得到及其化学惰性,故为一种理想的增压气体。 Argon chemically inert and easy to obtain, it is ideal as a pressurized gas.

为了叙述方便,现以一特定的自动化合成仪为例来加以说明。 For convenience, now to be described in a specific Example of an automated synthesizer. 该例将涉及在小球上合成一套多肽。 This embodiment would involve the synthesis of a polypeptide on the beads. 小球被标记以易识别,接着是每个氨基酸与四个碱基单体:A、T、C及G的偶联反应。 Pellets were marked with recognizable, each amino acid monomer followed by four bases: coupling reaction A, T, C and G,.

然而必须认识到自动合成仪既不仅仅局限于上述特例中所描述的特定多聚物的合成,也不仅仅局限于标记多聚物的合成。 However, it must be appreciated both automated synthesizer synthesizing the specific polymer is not limited to the described specific example is also not limited to labeled synthetic polymers. 以下描述中以多肽合成及以上述核苷酸标记小球反应为例,该例中一个反应器库(reaction vessel bank)含有9个反应器。 In the following description, the above peptide synthesis reaction of labeled nucleotides and pellets, for example, in this embodiment a library reactor (reaction vessel bank) contains nine reactors. 但这只是一个特例,反应罐中反应器的数目并没有固定的上下限。 But this is only a special case, the number of reaction tank reactor and no fixed upper and lower limits. 实际上,合成仪的模块化构造(modular con-structien)使反应罐的添加变得简便易行。 In fact, a modular construction of the synthesizer (modular con-structien) was added to make the reaction tank becomes easy. 此外,许多其它分子及标记物也可在小球上进行合成,或者反应体系中无须标记物的存在。 In addition, many markers and other molecules can also be synthesized on the beads, or the existence of the reaction system without tag.

1.压力传递系统前述的压力传递系统265是经特殊设计来完成多肽的合成,其可再细分为:多肽合成池、小球标记池以及运送阀。 1. Pressure pressure delivery system 265 is the aforementioned delivery system specially designed to complete the synthesis of polypeptides, which can be subdivided into: pool peptide synthesis, a small pool, and delivery valve ball marker.

a.多肽合成池该例中除需要9种氨基酸单体作为合成多肽的原料外,其它几种“常规”(common)试剂也在合成中得以应用。 a. Peptide Synthesis Example except the pool requires nine amino acids of the polypeptide monomer as the starting material, the several other "conventional" (Common) synthesis reagents also be applied. 例如,在一典型多肽合成反应中,主要用到下列试剂:表2功能 化学成分脱保护(Deprotection) 含10%哌啶的DMF溶液激活(Activation) 0.2M HBTU及0.6M DIEA溶于3∶1的DMF/DCM混合液帽封(Capping) 乙酸酐的THF溶液n-甲基味唑的THF溶液清洗液 DMFTHFb.标记小球库在该例中,小球被选择性地标记。 For example, in a typical peptide synthesis reaction, mainly uses the following reagents: Table 2 Function deprotected chemical composition (Deprotection) DMF solution of activated (Activation) containing 10% piperidine was dissolved in 0.6M DIEA and 0.2M HBTU 3:1 the DMF / DCM mixture closure cap (capping) of acetic anhydride in THF n- methyl-imidazol-cleaning solution in THF DMFTHFb. tag library pellets this embodiment, the pellets were selectively labeled. 在另一例肽合成反应中,小球被四种核苷酸A、T、C和G标记上。 In another embodiment the peptide synthesis reaction, the pellets were the four nucleotides A, T, C and G tag. 除了四种苷酸以外,在标记物合成中还得用到以下溶剂及反应物。 In addition to four kinds of nucleotide, the label used in the synthesis had the following reactants and solvent.

表3功能 化学组成脱保护 三氯醋酸的DCM液氧化 碘、三甲基吡啶,水及MeCN激活 0.5M四唑MeCN液帽封 乙酸酐THF液N-甲基咪唑THF液清洗 NeCN这些核苷酸反应物装于足够大体积和数量的小池中以使自动合成仪完成标记物的合成。 Function Table 3 Chemical composition deprotected with trichloroacetic acid solution in DCM oxide iodide, trimethyl pyridine, water and MeCN 0.5M tetrazole activation MeCN cap seal anhydride THF solution N- methylimidazole THF solution was washed NeCN these nucleotides the reaction was mounted in a sufficiently large volume and the number of cuvette that the automated synthesizer to complete the synthesis of the marker.

如图2所示一代表性的400池容纳表3中的MeCN。 The receiving pool 3 MeCN 400 representatively illustrated in FIG. 2 in a table. 表2、表3两个池都连有2根导管。 Table 2, Table 3 has two pools are connected two conduits. 如图2所示,管450内装有高压氩气,以将小池中内容物压向452管。 2, the tube 450 built with high pressure argon, to press the contents of the cuvette 452. 有些设计中,反应池总是处于高压状态。 In some designs, always at high pressure reaction cell. 而另一些设计中,仅在需要池中物质时,氩气管通一些特定的可控阀门向其中吹气升压。 While other designs, the pool only when needed substance, argon line through certain controllable valve boost blowing thereto.

c.运送阀图3详述了一个典型3-通(3-port)螺线管阀。 C. Figure 3 details the delivery valve of a typical 3-way (3-port) solenoid valve. 如,新泽西General Valve Corp.of Fairfield生产的2-110-900型阀门。 E.g., NJ General Valve Corp.of Fairfield 2-110-900 production type valve. 此外,图3所示的3通阀门,比如阀门4从412池中运送反应物。 Further, as shown in FIG. 3, 3-way valves, such as valve 4 from the reaction tank 412 was forwarded. 图1的PDS265也用是的三通阀门。 PDS265 FIG. 1 is also used in the three-way valve. 该例中所用的三通阀门包括第一通道454及第二通道456,还有一个贯穿其本身的通道458,与一通道454及二通道456都有物质交流,且可使流体在一和二通道自由流动。 The three-way valve used in the embodiment includes a first channel 454 and second channel 456, and through a channel 458 itself, the substance has a channel to communicate with two channels 456 and 454, and the fluid can in one and two channel flow freely. 如要形成普通型多阀箱体462,那么,此方说二通道456就必须和另一三通道阀的第一通道454相偶联。 To form a multi-common valve housing 462, then, this side channel, said first and second channel 456 must be another three-channel valve 454 phase coupling. 其它三通道阀门还有图1所示的阀门5。 The other three channels as well as valves the valve 5 shown in FIG. 其内部的一只螺线管通过线190对控制信号。 A solenoid inside which a control signal line 190 through the pair.

作出应答,并选择性地打开第三通道460,使其与458通道发生物质交换。 Respond, and selectively opening the third passage 460, passage 458 so as to exchange with the substance. 图1中阀4的第三通道460与412小池相偶联。 In FIG. 4, valve passage 460 and third 412 phase coupling cuvette.

为了控制好反应物从412小池流向多阀箱体462,载有高压反应物的线管464与阀门4的第三通道460相联。 In order to control the flow of reactants from the multiple cuvette 412 of the valve housing 462, carrying a high pressure reactant conduit 464 and valve 460 of the third channel is associated. 在适当的时候,螺线管打开,致使第三通道460与458通道发生物质交换,籍以使受压的3通道460内的反应物注入4阀的458通道并进而进入普通型多阀箱体462。 At the appropriate time, the solenoid is opened, so that the third channel 458 and the channel 460 exchange material occurs, so that the pressurized membership 3 reactant channels 460 within the injection passage 458 and thus the valve 4 into the common multi-valve housing 462.

图4所示一典型开关型2-通道螺线管阀10。 Figure 4 shows a typical type 2-channel switching solenoid valve 10. 比如新译西General Valve Corp.of Fairfoield生产的2-17-900型阀门止适合于此。 For example, a new translation of the West General Valve Corp.of Fairfoield production 2-17-900 only suitable for this type of valve. 如图4所示,阀门10包括468和470二个通道。 4, the valve 10 includes two channels 468 and 470. 阀门10也有一螺线形管道贯穿其中,使其与第一能道468及第二通道470发生信息交流,从而使液体或气体能在两个通道之间流动。 Valve 10 also has a spiral conduit therethrough, so that the exchange of information occurs between the first and the second channel 468 can track 470, so that the liquid or gas can flow between the two channels. 阀门10内的螺线管,通过472线对控制信号作出应答,籍以使第一通道468选择性与第二通道470发生交流。 The solenoid valve 10, respond, through a membership to a control signal line 472 to the first passageway 468 and second passageway 470 selectively exchange occurs. 当阀门10的一个通道连上一个带有高压气体或液体的管道时,阀门10便可用作控制流体从该通道流向其中其它通道的开关。 When a channel on the valve 10 connected with a high pressure gas or liquid conduit, the valve 10 will serve as a control fluid to switch to other channels from among the channels.

图5更为详尽地描绘了一个压力传递系统265,其中有一方面与本发明十分一致。 FIG. 5 depicts a more detailed pressure delivery system 265, which is consistent with an aspect of the present invention. 图5表示出了一个多阀箱体0-23号24个阀门。 Figure 5 shows more than a valve housing No. 0-2324 valves. 把2通道及三通道阀门的第一和第二通道绞连(daisy-chained)以形成普通型多阀箱体让反应物流过。 The three-channel 2-channel valve and the first and second twisted passage connected (daisy-chained) to form a common multi-valve housing so that flow through the reactor. 三通道阀门1-7,9-12以及14-22,比方说在结构上与图4中的阀门10比较类似。 Three-channel valve 1-7,9-12 and 14-22, for example in the structure of the valve 10 in FIG. 4 relatively similar. 普通型多阀箱体以下几部分构成,具贯穿管道的三通道阀、开关(on/off)阀以及相邻阀门间的偶联管。 Common multi-valve housing composed of the following parts, having a three-channel valve through the conduit, the pipe coupling between the switch (on / off) valve and an adjacent valve. 正如前所述,三通道阀门的第三通道受阀门内的一螺线管控制。 As noted earlier, the third passage by a three-channel valve of the solenoid control valve. 三通道阀门的第三通道与反应物或溶剂池的导管相连,从而使反应物或溶剂能够在PDS265中来回传送。 The third three-channel valve passage is connected to the conduit pool reactants or solvents, or a solvent so that the reaction can be sent back and forth in PDS265. 同样,第三通道还可用作一个出口,比如,它可将反应物运送到反应器组中的阀门100。 Similarly, a third outlet passage may also be used, for example, it may be transported to the reactant valve 100 of the reactor group. 两通道阀门主要用于隔离及通入氩气。 Two-channel valve is mainly used to isolate and purged with argon.

如图5所示,24个阀门被安装在三个分离的反应器组中以节省空间。 5, the valve 24 is mounted on the reactor three separate groups to save space. 图5中的压力运输系统表示出一导管480将阀门左边的反应器组与中部的反应器组相联。 FIG. 5 shows a transport system pressure conduit 480 to the reactor and the middle set of reaction group linked to the left of the valve. 表4列出了运输系统中所用的阀门,并对一些典型的阀门类型及受阀门控制的反应地加以说明和解释。 Table 4 lists the valves used in the transport system, and some typical types of valves and valves controlled by the reaction to be described and explained.

表4阀 型号 池 池内含物0 开/关 -) 氩1 FWO60 400 MeCN2 FWO60 402 1%三氯乙酸的DCM溶液3 FWO60 404 四唑4 FWO30 406 C5 FWO30 408 T6 FWO30 410 G7 FWO30 412 A8 开/关9 FWO30 414 废物10 FWO30 416 母体反应器底部11 FWO30 100 RV库12 FWO30 420 废物13 开/关14 FWO60 422 顶端母体15 FWO60 424 碘、可力丁、水、乙腈16 FWO60 426 乙酸酐的THF溶液17 FWO60 428 N-甲基咪唑的THF溶液18 FWO60 430 派啶19 FWO60 432 HBTU20 FWO60 434 DIEA21 FWO60 436 乙腈22 FWO60 458 DMF23 开/关 氩例如,阀22被表明是一种FWO60阀,即具有60/1000英寸通行管道的快速冲洗(FWO)三通道阀。 TABLE 4 Valve Model 0 pond contents on / off -) Ar 1 FWO60 400 MeCN2 FWO60 402 1% trichloroacetic acid in DCM tetrazol 3 FWO60 404 4 FWO30 406 C5 FWO30 408 T6 FWO30 410 G7 FWO30 412 A8 / off 9 FWO30 414 bottom waste 10 FWO30 416 precursor reactor 11 FWO30 100 RV library 12 FWO30 420 waste 13 on / off 14 FWO60 422 to the top of the parent 15 FWO60 424 iodine, collidine, water, acetonitrile 16 FWO60 426 acetic anhydride in THF 17 FWO60 428 N- methylimidazole in THF 18 FWO60 430 send piperidine 19 FWO60 432 HBTU20 FWO60 434 DIEA21 FWO60 436 22 FWO60 458 DMF23 acetonitrile / off e.g. argon, valve 22 is indicated that a FWO60 valve, i.e. having 60/1000 inch pipeline passage of rapid processing (FWO) three-channel valve. 而且阀22控制着压力池438中的一种反应物,如表4所示,该池含有DMF。 And valve 22 controls the pressure of one of the reactants pool 438, as shown in Table 4, the cell contained DMF. 再如,阀23是一种开关阀,它控制着从氩镤应源(未给出)到PDS265通用箱体中压缩氩的流量。 Again, valve 23 is a switching valve, which controls should protactinium from the argon source (not shown) in the housing to PDS265 general flow of compressed argon.

图5表示与阀0相连的供从一端给PDS265通用多阀箱体加压的管486。 Figure 5 shows a valve for 0 PDS265 connected from one end to the common multi-tube pressure valve housing 486. 另一管486是与阀23相连并且从另一端用氩给PDS265通用多阀箱体加压。 Another pipe 486 is connected to valve 23 and to a common multiple of the valve housing PDS265 pressurized with argon from the other end.

作为说明,在一个多肽合成降解循环中的加压投掷系统265的操作被描述如下。 By way of illustration, a pressurized polypeptide synthesis and degradation cycle throwing operation system 265 is described below. 对于多肽的降解,10%哌啶的DMF溶液被运至反应器(RV)库中的反应器中。 For the degradation of polypeptides, DMF solution of 10% piperidine library is transported to a reaction reactor (RV) of. 表4表明阀18允许来自池430的哌啶流。 Table 4 shows the valve 18 allows flow from the pool piperidin-430. 因此,阀18需要有出口以允许来自压力池430的哌啶流至PDS265通用多阀箱体中。 Thus, the need for the valve 18 to allow the outlet from the pressure tank 430 piperidin flows to PDS265 common multiple valve housing. 为了迫使溶液进入RV库阀11,关闭阀8而打开阀13以迫使加压哌啶进入开放的RV库阀11。 RV library in order to force the solution into the valve 11, closing valve 8 to open the valve 13 to urge the pressurized piperidine RV library into the open valve 11.

如图5和表4所示,RV库阀11和母体反应器阀10被安置在阀串的中心位置。 5 and Table 4, RV library valve 11 and valve reactor precursor is placed in the center position of the valve train 10. 这种安置有利于减少多阀箱体内这些阀与某一特定试剂阀间部位的长度。 This arrangement helps to reduce the length of the multi-valve cabinets valves between a particular agent and the valve site. 因此,需要较小量的试剂来填充该多阀箱体部位。 Thus, a smaller amount of reagent required to fill the multi-valve housing portion. 隔离阀8和B可被关闭以防止试剂从多阀箱体的一端喷入RV库阀11或母体反应器阀10或者不必要地进入通用多阀箱体的一部位。 B and isolation valve 8 may be closed to prevent the agent from the one end of the valve housing is injected into the valve 11 or the parent RV library reactor valve 10 or unnecessary portion into a common multi-valve housing.

表4也表明阀4-7和9-12是具有大小为30/1000英寸通行管道的3通道阀。 Table 4 also shows the valve 4-7 and 9-12 having a size of 30/1000 inch valve passage conduit 3 channels. 相对而言,多阀箱体的其余阀具有大小为60/1000英寸的通行管道。 In contrast, the remaining multi-valve housing having a valve size of 60/1000 inch pipe passage. 管道通行交叉部位的减少更减少了多阀箱体各自部位的体积。 Reduction conduit passage intersecting portion further reduces the volume of the respective parts of the multi-valve housing. 因而,需要用来填充多阀箱体的试剂更少。 Accordingly, it is necessary to fill multiple valve housing less reagent.

例如,核苷酸A、T、C、G相对很贵。 For example, a nucleotide A, T, C, G is relatively expensive. 因此很值得让所用试剂量保持在所需的最小量。 So it's worth getting the amount of reagents required to maintain a minimum amount. 核苷酸阀4-7被安置在靠近RV库出口阀11的地方以保持核苷酸阀和RV库出口阀11间的距离很短因而所需试剂量低。 Nucleotide valve 4-7 is disposed in close proximity to the outlet valve RV library nucleotides 11 to maintain the valve and the outlet valve RV library very short distance 11 and thus lower the required amount of the reagent. 沿着从任何核苷酸阀至RV库出口阀11的通路上的多阀箱体的交叉部位也保持很小以便进一步减小存在于多阀箱体中的核苷酸试剂量。 Any nucleotide along the valve from multiple valve housing portion to the cross passage on the outlet valve RV library 11 is also kept small to further reduce the amount of agent present in a plurality of nucleotides in the valve housing. 实际上,图5的管480和多阀箱体中核苷酸阀和隔离阀8之间的部位具有被减小了的30/1000英寸的交叉部位。 In fact, the portion between the tube 8 in FIG. 5 of the valve housing 480 and a plurality of nucleotides and isolation valves having a reduced cross-site 30/1000 inch.

C.反应器库图6表示了根据本发明一个方面的一个简化反应器组500。 C. reactor bank 6 shows a simplified set of reactor 500 according to an aspect of the present invention. 为了便于说明,溶液流经的管道已被部分地删去了。 For convenience of explanation, the solution flowing through the conduit has been partially deleted. 反应器库500包括一个顶部托架(bracket)502、两个侧面托架504和506和两个底座508和510。 Reactor bank 500 includes a top bracket (bracket) 502, two sides 504 and 506 and two brackets 508 and 510 of the base. 顶部反应器托架290连在侧面托架504和506上。 The reactor top bracket 290 attached on the side of the bracket 504 and 506. 一个涡流马达300安在顶部反应器托架290上以便通过传动带521给反应器201-209复合体提供动力。 An eddy current in a motor 300 on the top of the reactor via a bracket 290 to power transmission belt 521 to a composite reactor 201-209. 反应器201-209的底部安在反应器托架295的底部上。 201-209 security bottom of the reactor in the reactor bottom portion 295 of the bracket. 托架502、504、506、290和底座508和510可由任何合适的材料制成。 502,504,506,290 and the bracket 510 and the base 508 may be made of any suitable material. 考虑到机械制造、强度和轻重量,在本发明中用铝来制造上述所提的托架。 Considering the manufacture, strength and light weight, aluminum for manufacturing the above mentioned carrier in the present invention.

底部反应器托架295通过轴套520中的两个非同心轴280固定在顶部反应器托架290上。 Reactor bottom bracket 295 attached to the top of the reactor through the sleeve 290 on the carriage 520 in the two non-concentric shaft 280. 非同心轴280通过顶部反应器托架290上的孔(未标明)可旋转地被安装好。 Non-concentric shaft hole 280 (not indicated) is rotatably installed on the bracket 290 through the top of the reactor. 通过传动带521将非同心轴280和涡流马达300相偶联。 Belt 521 by the non-concentric shaft 280 and motor 300 are coupled vortex. 如后面将要讨论的,非同心轴280将涡流马达300提供的旋转力转移成驱动力以使底部反应器托架295以环型形式而运动。 As will be discussed later, the non-concentric shaft 280 of the motor 300 provides a rotational force of the swirl to transfer the driving force to move the bottom of the reactor in a ring form and movement of the carriage 295. 该环形运动对反应器201-209中的内含物产生涡流效应。 The circular movement of the eddy current effects in the 201-209 reactor contents. 由于反应器201-209中的每一个都在各自的底端与底部反应器托架295相连,因此所有反应器都被同时而一致地驱动。 Since each of the reactor in their respective bottom end is connected to the reactor bottom bracket of 295201-209, so all of the reactors are simultaneously driven in unison.

图6也显示了底部托架522。 FIG 6 also shows the bottom bracket 522. 底部托加522是连在侧面托架504和506上,并且也可由任何合适材料制成,包括铝。 Astorga bottom 522 is attached to the side surface of the bracket 504 and 506, and may also be made of any suitable material, including aluminum. 氨基酸池231-239安装着底部托架522下。 Amino acids 231-239 pool 522 is mounted at the bottom bracket. 氨基酸池231-239中的氨基酸试剂被用作合成特定实施方案中各套多肽的基本成分。 Pool 231-239 amino acid reagent is used as the synthesis of specific embodiments of the basic components of the respective sets polypeptide. 为合成多肽本发明考虑使用九种不同的氨基酸单体。 It is a synthetic polypeptide of the present invention contemplates the use of nine different amino acid monomers.

图6也显示了安在侧面托架504和506上的隔离阀托架526。 FIG 6 also shows safety on the side of the bracket 504 and the bracket 506 of the isolation valve 526. 隔离阀托架526包括一个代安置较低多阀箱体阀101-109的管道530。 The isolation valve 526 includes a bracket disposed on behalf of multiple lower valve housing of the valve conduit 530101-109. 如图6所示,在本发明中每个低多阀箱体阀101-109都固定在管道530中。 6, in the present invention, each of the multiple low valves 101-109 are fixed to the valve housing 530 in the pipeline. 然而,低多阀箱体阀101-109也可使用市场上可得到的固定金属器具或其它固定方法来被固定在隔离阀526上。 However, the low multiple valves 101-109 of the valve housing may also be fixed on the isolation valve 526 using the fixation hardware or other fixing methods available on the market. 较低多阀箱体阀101-109是三通道螺线管阀并且是与以前讨论的与图3有联系的3通道阀相同。 Lower valve housing multi-channel valve is a three solenoid valves 101-109 and is the same as FIG. 3 associated with the valve passage 3 discussed previously. 如反应器组500中所使用的,低多阀箱体阀101-109中的通行管道偶联在一起以形成一个通用低多阀箱体214,来自加压投掷系统265中的溶液通过它而流动。 The reactor group 500 is used, the low pass pipe coupling multiple valve housing valve 101-109 together to form a common low multi-valve housing 214, the system 265 was thrown from a pressurized by it and flow.

三个2通道阀100、110和122也在图6中显示了。 2 three valve passages 100, 110 and 122 are also shown in FIG. 6. 九个阀101-109控制着溶液从低多阀箱体214到九个反应器201-209的流动。 Nine valves 101-109 control the flow of solution from the lower valve housing 214-9 plurality reactor of 201-209. 在通用低多阀箱体214的第一末端的隔离阀110开口于一废物线(在图6中未显示)。 In general the low end of the multi-valve housing a first isolation valve 110 opening 214 in a waste line (not shown in FIG. 6). 在通用低多阀箱体214的第二末端的隔离阀100选择性地禁止或允许溶液从PDS265流向箱体214的其余部位。 In general low multiple valve housing second end 214 of the isolation valve 100 selectively allow or prohibit the flow of solution from the rest of the housing 214 PDS265. 一个可供选择的隔离阀122供应局部氩压力以协助溶液运到或运自反应器组500的各部分。 An alternative isolation valve 122 serves to assist the partial pressure of argon to a solution transported or shipped from the portions of the reactor group 500.

在另一实施方案中,这十一个隔离阀100~110和122以一个11阀组的形式提供,正如由利福尼亚州Foster City的ABI所提供的模型P/N601374那样。 In another embodiment, the eleven isolation valves 100 to 110 and 122 to provide a form of valve 11, as a model P Li Funi Foster City, Georgia is provided by ABI / N601374 above. 该阀组已预组装了因而简化组建程序。 The valve block has been pre-assembled thus simplifying the formation process. 该11阀组的十一个阀的功能实际上跟上面所描述的一样。 The eleven functional valve 11 with the valve actually as described above.

由合适材料如铝构成的一个喷射阀托架532被固定在侧面托架504和506上。 The injection valve is made of a suitable material such as aluminum is fixed to a bracket 532 on the side of the bracket 504 and 506. 喷射阀群111-119通过喷射阀托架532上的孔而被安装上。 Injectors 111-119 are mounted on a cluster through a hole in the bracket 532 injection valve. 图6表示共有9个喷射阀111-119来控制氨基酸来自九个氨基酸池231-239的喷射。 6 shows a total of 9 to control the injection valve 111-119 amino acids from the nine amino acids 231-239 of cell injection. 喷射阀111-119是3通道螺线管阀并且同早在图3中所述的3通道阀是一样的。 111-119 passage injection valve is a solenoid valve 3 and the same early in the passage 3, the valve 3 is the same as FIG. 各喷射阀的第一部分与反应器201-209相偶联。 A first portion of the reactor injection valves 201-209 of phase coupling. 而第二部分与低多阀箱体101-109的第三部分相偶联。 And a second valve housing portion and the lower portion of the third plurality of phase conjugation 101-109. 该偶联是用合适大小的管来实现的,如本实施方案中采用的1/16英寸特氟隆管。 The coupling of an appropriate size tube is achieved, according to the present embodiment as 1/16 inch Teflon tubing employed. 如前已明白的,各喷射阀111-119的通行管道允许溶液在反应器201-209和低多阀箱体阀101-109之间的自由流动。 As previously appreciated, each injector passage conduits 111-119 201-209 allows the free flow of the solution in the reactor and lower multi-valve housing between the valve 101-109. 各喷射阀111-119的第三部分与氨基酸池231-239相连以便选择性地禁止或允许氨基酸池231-239相连以便选择性地禁止或允许氨基酸被射入在低多阀箱体阀101-109和反应器201-209间的溶液流。 The third portion of each injection valves 111-119 and the 231-239 amino acid pool is connected to selectively permit or prohibit 231-239 amino acid pool is connected to selectively prevent or allow multiple amino acid to be incident on a low valve 101 of the valve housing solution flow between the reactor 109 and 201-209.

图6也显示了一个顶部通用多阀箱体212。 FIG 6 also shows a general multi-valve housing top 212. 该箱体包括九个多阀箱体通道542以使该箱全与九个反应器201-209相连。 The cabinet comprises more than nine passages 542 of the valve housing so that the whole tank is connected to the nine reactor 201-209. 在本实施方案中,1/8英寸弹性特氟隆管被用来使箱体通道542与反应器201-209的顶端相偶联。 In the present embodiment, 1/8-resilient Teflon tubing was used to make the housing passage 542 to the top of the reactor with 201-209 conjugated. 顶部通用多阀箱体212也含有一个第一末端通道544以使它与母体反应器(未显示)相连,其中来自各个反应器201-209的小球已经混合在一起了。 General Multi top of the valve housing 212 also contains a first end of the channel 544 so that it is attached to the parent reactor (not shown), wherein each reactor is from 201-209 beads have been mixed together. 第二末端通道546把顶部通用箱体212与一个3通道压力/通气阀(未显示)相连。 546 universal box top of the second end of the channel 212 is connected to a 3-channel pressure / vent valves (not shown). 该压力/通气阀和第二末端通道546提供了另一条管路,通过该管路压力氩、溶液和试剂等可被供应到顶部箱体212中。 The pressure / vent valve and a second end of the channel 546 provides another line, it may be supplied to the top of the case 212 through the line pressure of argon, a solution of the reagent and the like. 另一方面,该压力/通气阀和第二末端通道546提供了另一条管路,通过该管路压力氩、溶液等可从箱体212排放到合适的池中。 On the other hand, the pressure / vent valve and a second end of the channel 546 provides another conduit, through which the line pressure of argon, solution and the like can be discharged from the tank 212 to a suitable tank.

图6A显示了对池231-239的另一种安排。 Figure 6A shows another pool of 231-239 arrangements. 它不是提供单一池组,如池231-239,而是提供,组群,如231a-239b、231b-239b等。 It is not providing a single battery pack, such as the 231-239 pool, but to provide, groups, such as 231a-239b, 231b-239b and the like. 这些安在一个可旋转传送带1000中。 These security in a carousel 1000. 它们都开口于传送带1000的顶面1002以便使池中的试剂能从顶面1002来估算。 They are open to a top surface of the conveyor belt 1000 to 1002 of the reagent from the top surface of the pool 1002 is estimated. 传送带1000安在压力器(未显示)内以使每个池都在压力器内受到相同压力。 1000 Ann pressure belt (not shown) within each pool is subjected to the same pressure in the pressure filter. 为了把池中试剂转移至反应201-209,与管241-249相连系的一管群被安在压力器内。 To the pool was transferred to the reaction reagent 201-209, 241-249 is connected to the tube system is a group of security in the pressure device. 压力器中的管被放置在一选择池组的池内,如池231a-239a。 The pressure in the tube reactor is placed in a pool of selected cell pack, such as pool 231a-239a. 通过将管平移到池中或者把传送带1000平移至管道中可把管安置在库中。 Translated to the tube by the pool or to the conveyor belt 1000 can be translated to the conduit tube disposed in the library. 传送带1000可旋转以使这些管与所选择的池组一致。 Belt 1000 is rotated so that the tubes may be the same pool to the selected group. 压力器内的压力会使池和管241-249间存在一个压力梯度。 Pressure will pool and 241-249 in the pressure pipe between a pressure gradient exists. 当这些管被安在池中或选择阀111-119被打开时,压力梯度驱使池中的试剂到管241-249中去便运至反应器201-209中,这正如前所述。 When these tubes are safety valves 111-119 in the pool is selected or opened, the pressure gradient driving the tube into the reagent cell 241-249 will be transported to the reactor to 201-209, which indicated earlier. 因此通过允许从各种不同试剂中选择试剂,例如,允许使用各合成步骤中的各套基本原料,传送带1000给合成器提供了灵活性。 Thus reagent selected from a variety of reagents by allowing, for example, allow the use of various sets of basic raw materials of each synthesis step, the conveyor belt 1000 provides flexibility to the synthesizer.

图7更详细地显示了在氨基酸池231-239、喷射阀111-119、低多阀箱体阀101-109和反应器201-209之间的相互连接。 Figure 7 shows in more detail the pool of amino acids 231-239, 111-119 injector, low multi-valve housing between the valve interconnecting the reactor and 201-209101-109. 低多阀箱体阀,例如阀101(它是一种三通道阀),是与通用低多阀箱体214相连的,以使溶液在低多阀箱体阀101的第一通道和第二通道之间自由流动。 Lower valve housing multiple valves, such as a valve 101 (which is a three-port valve), and a plurality of low common valve housing 214 connected to the solution at low multi-channel valve of the first valve casing 101 and the second flow freely between channels. 低箱体阀101的第三通道通过管271与3通道喷射阀111的第一通道或第二通道相连。 Lower valve housing 101 through the third pipe passage 271 and the injection valve 3 channel or first channel 111 is connected to the second channel. 而第一能道和第二通道中的另一通道通过管241与反应器201的一端相连。 And the other channels of the first channel and the second channel can be connected via a pipe 241 and one end 201 of the reactor. 反应器201的另一端通过管221与顶部通用多阀箱体212(未在图7中显示)中的多阀箱体通道542相连。 Multi-channel 542 is connected to the other end of the valve housing 201 with the top of the reactor Generalized Multi-valve casing 212 (not shown in FIG. 7) through the tube 221. 管271、241和221都是由化学耐性材料如特氟隆制成。 The tubes 271,241 and 221 are made of a Teflon material chemical resistance. 实际上,本实施方案中采用了具有许多交叉部分的半透明PTFE和FET特氟隆管,因为它具有低反应性和半透明特氟隆材料的光学特征。 Indeed, the present embodiment uses a FET having a plurality of translucent PTFE and Teflon tube cross section, because it has low reactivity and optical characteristics of a translucent Teflon material.

氨基酸池231是利用惰性气体如氩经管道562来加压的。 231 is the amino acid pool with an inert gas such as argon through the conduit 562 to pressurize. 氨基酸池231中的加压氨基酸溶液通过管道560进入了通道喷射阀111的第三通道。 Amino acid solution is the pressurized tank 231 through line 560 into the passage 111 of the injection valve of the third passage. 在接收到一适当的指令后,喷射阀111被打开以允许加压氨基酸溶液进入到喷射阀111的通行管道中去图7也显示两个光学传感器111S和101S光学传感器材111S和101S探测半透明特氟隆管271和241内的液体的存在与否。 Upon receiving an appropriate command, the injection valve 111 is opened to allow the pressurized injection valve into the amino acid solution passage pipe 111 to Figure 7 also shows two optical sensors 111S and 101S and 101S 111S optical sensor probe translucent material Teflon tubing 271 and the liquid present in the 241 or not. 如图7所示,光传感器111S位于喷射阀111之下而光传感器101S位于反应器201之下。 As shown in FIG 7, the optical sensor 111S is located below the injector 111 and the optical sensor 201 101S is located below the reactor. 来自光传感器111S和101S的数据被送至一控制计算机(图7中未显示)以用一控制合成反应的各阶段。 111S and 101S from the optical sensor data is sent to a control computer (not shown in FIG. 7) to control each stage by a synthetic reaction.

图8更详细地显示图1中的非同心搅拌器280。 Figure 8 shows in a non-concentric agitator 280 in FIG. 1 in more detail. 非同心搅拌器280包括与圆柱开钩爪566相偶联的两圆柱形轴564和568。 Agitator 280 includes a non-concentric with the cylindrical opening finger 566 coupled to two phase 564 and a cylindrical shaft 568. 轴564与圆柱形钩爪566的辐射轴纵向校直并且在其两平面之一相偶联。 The cylindrical finger shaft 564 longitudinal axis 566 of the linear radiation correction and phase coupling in one of its two planes. 轴568在另一平面相偶联。 In another shaft 568 coupled plane. 并且偏置于圆柱形钩爪的辐射轴。 And the bias to the radiation axis of a cylindrical claw. 在一实施方案中,轴564和568、圆柱形钩爪566由同一架全属刨台制成。 In one embodiment, the shaft 564 and 568, by a cylindrical finger 566 with a plane table made of full metal.

当圆柱轴564在一固定旋转支持物如滚轴、圆柱轴568(它偏置于圆柱轴564的轴心作环形运动。操作上可偶联多于一个的非同心搅拌器280,例如通过带一轮结构允许一组非同心搅拌器280,例如通过带一轮结构允许一组非同心搅拌器280一起运动。在本实施方案中,两个非同心搅拌器280的轴568与单一托架相连以便在轴564旋转时以环形形式移动托架。而且,轴568和涡流马达300被设计来以每分钟约1500转在环形轨道内移动各反应器的底部。为了防止小球的损坏,本实施方案中的环形轨道最好限于半径约为3.5mm。 When the cylinder axis 564 at a fixed rotational support such as roller, the cylindrical axis 568 (which is biased to the cylindrical axis of the shaft 564 for circular motion. Operatively coupled to more than one non-concentric agitator 280, for example by a belt a set of non-concentric structure allows a stirrer 280, for example, allows a group with a non-concentric configuration by a stirrer 280 move together. in this embodiment, the shaft 568 is connected to a single carrier two non-concentric agitator 280 in order to move the shaft 564 is rotated to form an annular carrier. Further, the shaft 568 and the motor 300 is designed to swirl about 1500 revolutions per minute track movement within the annular bottom of each reactor. in order to prevent damage to the pellets, the present embodiment circular orbit embodiment is preferably limited to a radius of about 3.5mm.

图9更详细地显示了反应器组500的上面部分,它包括图6中的反应器201-209和涡流马达300。 Figure 9 shows in more detail the upper part of the reactor group 500, which includes a reactor in FIG. 6 201-209 300 and eddy current motors. 与图6中所讨论的,反应器组500包括与顶部托架290相连的反应器组201-209。 As discussed in FIG. 6, the reactor group 500 includes a top bracket 290 connected to the reactor group 201-209. 顶部托架290有供反应器201-209相连的一组孔洞630。 A top bracket 290 has a hole 630 for the set of connected reactors 201-209. 来自该孔(未显示)上的一特氟隆管道在孔630与各反应器201-209的上末端相连,其连接方式允许液体在特氟隆管和反应管201-209之间自由流动。 From the aperture (not shown) on a Teflon pipe 630 and the hole at the end of each reactor 201-209 are connected, the connection tube and allow the liquid in a Teflon reaction tube flow freely between 201-209.

反应器201-209的低末端与低反应器托架295相连。 201-209 reactor lower end of the bracket 295 and is connected to the reactor low. 图10A和10B更详细地显示了图6中的低反应器托架295的底面视图。 10A and 10B show in more detail a view of the bottom surface of the reactor lower bracket 295 in FIG. 6. 图10A是低反应器托架295-部分的放大底面视图。 FIG. 10A is a portion of the low reactivity bracket 295- enlarged view of the bottom surface.

图10B显示在低反应器托架295的一群管道650。 10B shows the carriage in a group of low reactor pipe of 650,295. 一个弹性而半透明特氟隆管(从图10B中省略以便于演示)从各反应器201-209(也从图10B中省去)的低末端伸出并安在管道650中。 A resilient and translucent Teflon tubing (omitted from FIG. 10B for presentation) 201-209 from each reactor (also omitted from FIG. 10B) and lower projecting end security in the conduit 650. 供安放光传感器的沟652与各管道650相连。 The groove for mounting the optical sensor 652 is connected to each of the ducts 650. 沟652在图10A中被清楚显示。 Groove 652 is clearly shown in FIG. 10A.

图10B显示了将光传感器固定在低反应器托架295上的一群安装孔654。 10B shows the optical sensor is fixed on a group of mounting holes 295 of the reactor 654 lower bracket. 图10B也显示了一群选择孔656以减轻重量。 Figure 10B also shows a group selected hole 656 to reduce weight. 如前所讨论的,低反应器托架295随着非同心搅拌器的环形运动而涡流反应器中的内含物。 As previously discussed, the reactor lower bracket 295 moving with non-concentric annular vortex stirrer, and the reactor contents. 供选择的孔656也可通过低反应器托架295制成以减轻托架的质量,进而减少移动托架所需的动力。 Alternative bracket 295 aperture 656 may be formed by low reactor to reduce the quality of the carrier, thereby reducing the power required to move the carriage.

在低反应器托架295的每一端附近的通行孔658将一个非同心搅拌器280连接到低反应器托架295上。 Low reactor passage hole 658 of the bracket 295 near each end to a non-concentric agitator 280 is connected to the lower bracket 295 of the reactor. 反应器201-209的低末端随着低反应托架295的环形运动而运动,它们是从弹性特氟隆管241-249伸出来安在低反应器托架295的通行管道650上。 201-209 lower end of the reactor with the low reactivity of the carriage 295 is moved circular movement, which are extended from the elastic 241-249 Teflon tube reactor at low Bryan bracket of passage conduits 650,295. 当低反应器托架295以环形运动时,反应器组中的所有反应器201-209中的内含物都以相应方法作涡流运动。 When the low reactor circular motion to carriage 295, all the reactors of the reactor contents set 201-209 are made in a swirling motion in a corresponding method.

图9也显示了供安装非同心搅拌器280的可选择性的非同心搅拌器套520。 Figure 9 also shows the installation of non-concentric with a stirrer 280 for selectively sleeve 520 of non-concentric agitator. 该搅拌器套围着在空圆柱套内的非同心轴以防止在非同心轴运动时对使用者的可能伤害和对设备的损伤。 The stirrer was set around the shaft in a non-concentric hollow cylindrical sleeve to prevent user injury and possible damage to equipment at the time of non-concentric axis.

本实施方案使用级型马达(加尼福尼亚Oriental MoforU.SA Corp.of Torrance提供的PX245-01AA模型)和级型马达控制器(加尼福尼亚Semix Corp.of Fremont提供的RD122模型)以给非同心搅拌器提供旋转力。 The present embodiment uses a motor-stage type (PX245-01AA Oriental MoforU.SA Corp.of Torrance model areas like California provided) motor controller and a stage type (RD122 model Semix Corp.of Fremont provided in areas like California) to provide rotational force to the non-concentric agitator. 如图9所示,三个滑轮670、672和674与涡流马达300和两个驱动带521和676一起操作以便在非同心搅拌器套520内使两个非同心搅拌器280一起旋转。 9, three pulleys 670, 672 and 674 and the motor 300 and the eddy current two drive belts 521 and 676 operate together so that the two non-concentric agitator stirrer was set in the non-concentric 520,280 rotate together. 尽管本实施方案使用了级型马达和组型马达控制器,旋转力可以由任何其它型号的马达供应,这包括其它电动或气动马达。 Although the present embodiment uses a set of type-stage type motor and a motor controller, the rotational force of the motor can be supplied from any other model, including other electric or pneumatic. 而且,由涡漩马达300提供的动力可以通过任何合适的传递系统,包括链和轮齿、滑轮和带、齿轮等,传递到非同心搅拌器280去。 Further, the power provided by the swirl motor 300 by any suitable delivery system, including teeth and chain, belt and pulleys, gears, etc., is transmitted to the agitator 280 to the non-concentric.

图11A显示了用于探测半透明特氟隆管内液体存在与否的一个典型光传感器680。 FIG 11A shows a typical optical sensor Teflon tube 680 for detecting the presence or absence of liquid translucent. 光传感器680是与本实施方案中的光传感器101S-119S相同的。 Optical sensor 680 is an optical sensor 101S-119S in the present embodiment is the same as the embodiment. 光传感器680(易利路易州Omron,Inc.of Schaumburg提供的EE-SX671模型)包括供悬挂光传递器和聚光器的两个分叉末端682和684。 An optical sensor 680 (EE-SX671 model state Yi Li Luyi Omron, Inc.of Schaumburg provided) for suspending comprises two bifurcated end of the light transmitter and a condenser 682, and 684. 光传感器680也具有一个686室以供安放合适的电路将光感数据传递到主控计算机中并且也为了将光感器680安于托架上。 The optical sensor 680 also has a chamber 686 suitable for mounting light sensing circuit to pass data to the host computer and also to content with the light sensor 680 on the carriage.

图11B更详细地显示了光传感器680的分叉末端682和684。 FIG 11B shows in more detail the split ends 682 and 684 of light sensor 680. 安装在叉末端682内表面的是一个大体为长方形的传递器685,以供将光传递到箭头688方向的大体为长方形的聚光器(图11B中未显示)中。 Mounted in the fork end surface 682 is a generally rectangular transmitter 685, for transmitting light to the direction of the arrow 688 is generally rectangular concentrator (FIG. 11B not shown). 聚光器安在分叉末端684的内表面并且同样是大体为长方形。 An condenser bifurcated end 684 and the inner surface is likewise generally rectangular. 为了探测大致半透明特氟隆管内液体的存在,特氟隆管被安在两分叉末端682和684之间的缝隙中。 In order to detect the presence of a substantially translucent liquid within Teflon tubing, Teflon tubing 682 is safe and the gap 684 between the two split ends. 当在大致半透明特氟隆隆管内存在液体时,聚光器被引发,预示着探测到特氟隆管内有一种液体。 When the long TEFLON tube substantially translucent liquid exists in the condenser is triggered, indicating that there is a detected liquid Teflon tubing.

在实践中,已发现当是空的时,大致半透明特氟隆材料不能引起光传感器680引发。 In practice, it has been found that when the blank is substantially translucent Teflon material does not cause the optical sensor 680 initiator. 为了很好地使用通用光传感器来感受半透明管内液体的存在,便用了一种新型光学校准挡光物。 To make good use of common optical sensor to feel the presence of a translucent tube for the liquid, then a new type of optical alignment with the light barrier. 图11C更详细地显示了光学校准挡光物690。 Figure 11C shows in more detail the alignment of the optical light blocking material 690. 它是由足以阻挡任何由传递器685发出的不透光材料制成。 It is sufficient to block any light transmissive material emitted by the transmitter 685 is made. 它包括第一表面696的两个挡光壁692和694,它们是为了使挡光物690摩擦地附在光感器680的分叉末端之一上,并且使光学校准挡光物690固定在分叉末端上。 It comprises a first light blocking surface 696 of the two walls 692 and 694, which are provided so that a light barrier 690 is frictionally attached to the bifurcated one end of the light sensor 680, and the optical alignment light barrier 690 fixed to fork on the end. 在一个实施方案中,挡光壁692和694被设计为使聚光器附着图11B的分叉末端684上。 In one embodiment, the light blocking walls 692 and 694 are designed such that the condenser FIG adhered on split ends 684 11B.

光学校准挡光物690也含有建在第二挡光表面700的一个管道698。 Optical alignment light blocking material 690 also contains a built in the light-blocking surface 700 of the second conduit 698. 第二挡光表面700是背对上述第一表面696的表面。 The second light blocking surface 700 is a surface opposite to the first surface 696. 管道698的轴心线垂直于挡壁692和694。 The conduit 698 is perpendicular to the axis of the retaining wall 692 and 694. 管道698的大小恰好使其紧夹住特氟隆管。 Size of the pipe 698 so that it clamps down exactly Teflon tubing. 因此,特氟隆管是固建在管道698内的,并且当光校准挡光物700被安在分叉端682和684间的缝隙时它是垂直于上述传递器带685的。 Thus, solid Teflon tubing is built in the pipe 698, and when the optical calibration safety light barrier 700 is slit at 682 and 684 of the bifurcated end which is perpendicular to the transfer belt 685.

图11D显示了位于管道698中心线的一个孔702。 FIG 11D shows a hole 698 located in the center line of the conduit 702. 孔702允许少量光沿着第一表面696和第二表面700间的孔穿过光校准阻光物690。 Hole 702 allowing a small amount of light of the collimated light passes through the light blocking material 690 along the first surface 696 and a second bore surface 700. 当大致半透明特氟隆重管如管241-249或271-279被牢固于管道698内时,孔702的轴心穿过特氟管的中心。 When the weight substantially translucent Teflon tube The tube is securely 241-249 or 271-279 in the conduit 698, axial hole 702 through the center of Teflon tube. 孔702的形状和大小是一个管的光学特征函数。 The shape and size of the holes 702 is a function of optical characteristics of the tube.

当光校准阻光物690以图11A所示方式安装在分叉末端682和684之间时,来自分叉末端682内的传递器685的光穿过一个大致透明管704。 When the light was collimated light barrier 690 in the manner shown in FIG. 11A is mounted between the bifurcated end of 682 and 684, transmitted from the bifurcated end 682 within the light passes through a substantially transparent tube 685 is 704. 在穿过管704后大部分光被光校准挡光器阻挡。 After most of the tube 704 by the light passing through the light barrier blocking calibrated. 穿过管704的一些光到达孔702(在图11A中被挡住)并且沿着孔702的孔眼到达分叉末端684内的聚光器。 Some of the light through the tube 704 reaches the hole 702 (hidden in FIG. 11A) and along the aperture hole 702 reaches the condenser 684 in the bifurcated end.

既然孔702的轴心穿过半透明管704的中心,穿过管心的光到达分叉端684内的聚光器部位。 Since the axial center hole 702 through the center of the translucent tube 704 through the center of the light pipe reaches the condenser 684 in the bifurcated end portion. 既然通过空管704的光被发散,就没有足够的光到达聚光器而引发感受器。 Since air is dissipated through the light pipe 704, there is not enough light reaches the condenser caused receptors. 当半透明特氟隆重管704内存在液体时,穿过满管的光被其中的液体聚光。 When the liquid in the memory 704, the light passes through the full bobbin which is translucent Teflon heavy liquid collecting tube. 被聚集的光进入孔702从分叉端682的方向引发分叉端684内的聚光器。 Light is collected initiator access hole 702 in the condenser 684 from the direction of the bifurcated end of the bifurcated end 682. 当液体不存在时,聚光效应甚微。 When liquid is present, the condensing effect is minimal. 因此,很少的光进入孔702。 Therefore, little light into the hole 702. 实际上,当特氟隆管704中没有液体时,就没有足够的光穿过孔702来引发分叉端684内的聚光器。 In fact, when there is no liquid in the Teflon tube 704, there is not enough light through the aperture 702 is initiated within the bifurcated end of the condenser 684.

如前所述,光校准挡光物690的大小恰好是紧夹住特氟隆管704。 As described above, the size of the optical calibration light barrier 690 is exactly 704 that clamps Teflon tubing. 当光校准挡光物690装在分叉端682和684之间时,如图11A所示,特氟隆管被光传感器680和挡光物690牢牢夹紧。 When the light-collimating light barrier 690 disposed between bifurcated end 682 and 684, as shown in FIG. 11A, a Teflon tube 680 and the light sensor is a light barrier 690 firmly in place. 通过将光感器680固定在一个托架上,特氟隆管704也随着固定在托架上。 By the light sensor 680 is fixed on a carriage 704 along with the Teflon tubing is fixed on the bracket. 以同样方式,本实施方案反应器201-209的底部伸出来的特氟隆管241-249也被固定在底部反应器托架295上。 In the same manner, the present embodiment of the reactor bottom outstretched 201-209 241-249 Teflon tubing is also fixed to the bottom bracket 295 of the reactor.

如图12所示,本实施方案中的反应器201之类的反应器由一段FEP特氟隆管710构成。 12, this embodiment of the reactor 201. The reactor or the like FEP Teflon tubing section 710 from the configuration. 管710的外径为1/4英寸,内径为0.19英寸。 OD tubing 710 1/4 inches, an inner diameter of 0.19 inches. 如后面更详细的描述,管710的直径为了应用起见也可做的更大,在此两种或更多试剂可同时在反应器201中混合。 As described in more detail later, the diameter of the tube 710 is greater for the sake of the application can do this in two or more agents can be mixed in the reactor 201 at the same time. 管710封闭性地与弹性特氟隆管271相偶联。 Tube 710 closed with a Teflon tube 271 elastically coupled. 弹性特氟隆管271被固定在反应器组的低反应器托架295上。 Low elasticity Teflon reactor tube 271 is fixed to the bracket of the reactor group 295. 当低反应器托架295作环形运动时,该弹性特氟隆管271的底部随着底部反应器托架作所述的环形运动以使管710内的内含物涡流运动。 When the carriage 295 is low reactor for endless movement, the bottom portion 271 of the elastic tube with a teflon reactor bottom bracket for circular motion to allow the contents of the swirling motion within the tube 710.

在这个实施方案中,管271的外径为1/8英寸而内径为1/16英寸。 In this embodiment, the outer diameter of the tube 271 is an inner diameter of 1/8 inch and 1/16 inch. 图12显示了一个管道连接器,它由第一偶合器714、第二连接器716和第三耦合器718组成以使不同交叉部分的管紧密地连在一起。 Figure 12 shows a pipe connection, which consists of a first coupling 714, the second connector 716 and the third coupler 718 so that the tube composed of a different cross-section closely together. 前述管连接器可从Norton,Inc.of Akron购得。 The pipe connector is commercially available from Norton, Inc.of Akron. 在管710的底部有一个滤器或过滤物质1102(图12中被挡住)以供防止管710内的巷质进入弹性管712。 In the bottom of the tube 710 has a filter material or filter 1102 (hidden in FIG. 12) for preventing the mass in the tube lane 710 into the elastic tube 712. 例如,该滤器可以是2微米钛滤器。 For example, the filter may be a 2 micron titanium filter.

在管710的另一端,一个第四耦合器720、一个第五连接器721和一个第六耦合器722紧密地将管710连接到管221上。 At the other end of the tube 710, a fourth coupler 720, a fifth and a sixth connector 721 is tightly coupled to the tube 722 to the tube 710 is connected 221. 耦合器720和722以及连接器721是必需的,因为本实施方案中管221有来自管710的不同交叉部位。 Couplers 720 and 722 and the connector 721 is required, since the present embodiment, the tube 221 from different parts of the cross tube 710. 管221连接到顶部多阀箱体212的一个多阀箱体通道542上(图6中未显示)。 Pipe 221 is connected to the top of the valve housing 212 of the multi a multi-channel valve housing (not shown in FIG. 6) 542.

一个弹性O-环724被安压托架290内的一个洞726内(在图12中以截面显示)。 An elastomeric O- ring 724 is pressure safety bracket 726 within a hole 290 (shown in cross section in FIG. 12).

O-环724弹性地夹住耦合器722,因而弹性地将反应器201牢固在托架290上。 O- ring 724 resiliently grip coupler 722 and thus resiliently reactor 201 on the carriage 290 securely. 当反应器201的底端被驱动时,O-环作为一个支点而使反应器201的顶端相当不动以增加涡漩效应。 When the bottom end of the reactor 201 is driven, O- ring as a fulcrum the top of reactor 201 to increase quite movable swirl effect.

如图12A和12B所示,在一特定实施方案中的反应器201可选择性地备有一个控制套1100以控制反应器201-209的温度。 Shown in FIGS. 12A and 12B, in a particular embodiment the reactor 201 in the embodiment may be selectively provided with a control sleeve 1100 to control the reactor temperature is 201-209. 在该实施方案中的温度控制套1100包括一个接头配件1104以将反应器201连接到221线和241线(未显示)。 In this embodiment, the temperature control set 1100 includes a fitting 1104 to the joint 201 is connected to the reactor 221 and the line 241 lines (not shown).

进/出通道1106、1108是为了将热导液体供应到反应器201中去。 In / out channel 1106 to the thermal conductivity of the liquid supplied to the reactor to 201. 该热导液体可被用来给反应器201加热或冷却。 The thermal conductivity of the liquid may be used to heat or cool the reactor 201. 在这个实例中,反应器201是由一个特氟隆管1110形成,最好其外径为1/4英寸,壁厚1/32英寸。 In this example, the reactor 201 is formed from a Teflon tube 1110, preferably having an outer diameter of 1/4 inch, a wall thickness of 1/32 inch. 空间上相离于管1110的是一个外管1112,它最好由特氟隆构成并具有外径3/4英寸、壁厚1/32英寸。 1110 spatially from the tube is an outer tube 1112, which is preferably composed of Teflon and having an outer diameter of 3/4 inch, a wall thickness of 1/32 inch. 外管1112围住接头配件1104而形成一个环形空间1114以供接收热导液体。 The outer tube surrounds fittings 1112 1104 1114 to form an annular space for receiving the thermal conductivity of the liquid. O-环1116、1118位于外管1112和接头配件1104之间以形成一液体密闭区。 O- rings 1116, 1118 and 1112 located on the outer pipe fittings between 1104 to form a liquid tight zone. 以这种方式,热导液体可通过通道1106和1108之一被导入环形空间1114以反应器201加热或冷却。 In this manner, the thermal conductivity of the liquid reactor 201 to 1114 may be heated or cooled by passages 1106 and 1108 is introduced into one of the annular space. 通道1106或1108也允许热导液体通过环形空间1114而持续循环。 Channel 1106 also allows a thermal conductivity of 1108 or continuously circulating the liquid through the annular space 1114.

反应器1120具有一个整体形式温度控制套1112的另一个实施方案在图12C和12D中显示了。 The reactor 1120 has an overall form another embodiment of a temperature control jacket 1112 shows in FIG. 12C and 12D. 除了反应器1120最好由玻璃构成外,反应器1120的功能大体与反应器201的相同。 In addition to 1120 is preferably constituted by an outer glass, the reactor 201 is substantially the same functionality as the reactor 1120 reactor. 温度控制套1122也最好由玻璃构成。 Temperature control sleeve 1122 is also preferably made of glass. 这样的结构允许反应器1120和温度控制套1122作为一个整合单位而形成。 Such a configuration allows the reactor 1120 and a temperature control jacket 1122 is formed as one integral unit. 反应器1120包括过滤器1124和连接1126、1128以连接到221线和241线(未显示)上。 The reactor 1120 includes a filter 1124 and 1126, 1128 are connected to the lines 221 and 241 connected to lines (not shown). 进/出通道1130、1132是为了使热导液体在套1122和反应器1120间的环形空间内循环。 Entrance / exit passage 1130 is provided to enable the thermal conductivity of the liquid circulating in the reactor jacket 1122 and 1120 of the annular space. 软管倒钩1136、1138是为了便于将进/出通道1130、1132连到合适的液体源上。 1136, 1138 hose barb to facilitate the entrance / exit passage 1130 connected to a suitable source of the liquid. 以这种方式,热导液体能在环形空间1134内循环以给反应器1120加热或冷却。 In this manner, the thermal conductivity of the liquid can be recycled to the reactor 1120 to be heated or cooled in the annular space 1134.

D.母体反应器图13更详细地显示了本发明实施方案的母体反应器。 FIG parent D. The reactor 13 shown in more detail the reactor of the parent embodiment of the present invention. 例如,母体反应器200的体积约为30毫升。 For example, the volume of the mother reactor 200 is approximately 30 ml. 管260与母体反应器200的底端紧密相连以将试剂或氩运到或运自投掷系统PDS 265(图13未显示)。 Parent pipe 260 and bottom 200 of the reactor are closely linked to the agent or argon or transported to the transport system from the throwing PDS 265 (not shown in FIG. 13). 一滤器过滤物746被安在母体反应器200底部附近以防止基质进入管260。 Filter 746 is a filter material near the bottom of the parent safety of the reactor inlet tube 200 to prevent the substrate 260. 在母体反应器200的顶端装有可移动盖743以供加入或除去材料。 A cover movably mounted to the top 743 in the reactor 200 precursor for addition or removal of material. 穿过盖743的管215在母体反应器200和反应器组的顶部通用多阀箱体212(图13中未显示)之间转移材料。 743 through the cover 215 at the top of the tube reactor 200 and the reactor precursor group of generic multi-valve housing 212 (not shown in FIG. 13) between the transfer material. 管215从盖743穿出。 Pipe 215 from piercing the lid 743. 一条可供选择的冲洗管路281连接到溶剂源以将加压溶剂运到母体反应器5的内壁冲洗该内壁。 Alternatively a flush conduit 281 is connected to the inner wall of the pressurized solvent to the solvent source to the parent transported flushing the reactor inner wall 5.

图13显示了两个电容传感器905和995(ElectromaticControl Corp.of Hoffman Estates,Illinois的模型18-08),它们安在母体反应器200的外面附近。 Figure 13 shows the two capacitive sensors 905 and 995 (ElectromaticControl Corp.of Hoffman Estates, Illinois model 18-08) which are in the vicinity of the outside of the parent Ann reactor 200. 电容传感器905或995探测其附近液体的存在情况并将传感数据分别通过线756和785传递到一控制计算机(未显示)。 Capacitive sensor 905 or 995 which detect the presence of liquid near the sensor data and are transmitted to a control computer (not shown) via lines 756 and 785. 电容传感器995用于探测向母体中加入的试剂。 The capacitive sensor 995 for detecting reagents were added to the precursor. 电容传感器905用于探测重新分布的球粒悬浮液的。 The capacitive sensor 905 for detecting the redistribution pellet suspension. 根据小球量和所用的反应容器数量可调节两种传感器。 The number of beads and the amount of the reaction vessel can be adjusted with the two sensors. 电容传感器的数据被软件使用,以控制合成过程的各个循环。 Capacitive sensor data is software used to control the synthesis process of each cycle.

E.控制系统1.控制计算机自动合成仪使用控制计算机由传感器获得数据,并在合成过程各循环步骤中控制阀门和涡流马达。 E. A control system controlling an automated synthesizer using a computer to control a computer to obtain data from the sensors, and the swirl control valve and the step motor at each cycle of the synthesis. 当与自动合成仪连接使用时,任何计算机,包括普遍公知的微机,小型机,工作站,大型机等等,可以用来处理传感器数据并发布命令控制阀门和涡流马达。 When connected to the automated synthesizer using any computer, including generally known microcomputer, minicomputer, workstation, mainframe, etc., it can be used to process sensor data and issue commands swirl control valve and the motor.

而且,来自合成仪传感器的传感器数据可以通过任何商售的使用一般数据显示方法的数据显示装置获得。 Further, sensor data from sensors synthesizer may use any generally commercially available display data obtaining method of the display device. 同样,用商售输入/输出控制器,响应合适的计算机命令,阀门和涡流马达也可被控制。 Similarly, using a commercially available input / output controller, suitable computer in response to the command, the eddy current motors and valves can also be controlled.

在一实施方案中,IBM-兼容微机(也认为是个人计算机或PC)被用作控制计算机(Model Gateway 200。4DX2-50V,by Gateway 200Inc.of No.Sioux City,South Dakota)。 In one embodiment, IBM-compatible computer (or a personal computer is also considered PC) is used as a control computer (Model Gateway 200.4DX2-50V, by Gateway 200Inc.of No.Sioux City, South Dakota). 在PC中,有允许加入各种扩充板的复杂扩充槽。 In the PC, for allowing expansion board added various complex expansion slot. 这些插线板与PC总线源接着头并让PC与卡上的电路系统联通以完成电子功能。 The patch panel head and then the PC bus and let the source circuitry on the PC card to perform electronic functions Unicom. 一些扩充板也允许PC与外部设备和电路系统联通。 Some expansion board also allows the PC to external devices and circuitry Unicom. 例如,一普遍公知的为调制解调器板的插线板插入PC上的扩充槽,并让PC与另一个具有调制解调器的计算机联通。 For example, insertion of a generally known expansion slot on the PC board of the patch panel of the modem, and so that another computer has PC and modem link. 用个人计算机使用扩充板是一般技术知识。 Use expansion board with a personal computer is general technical knowledge.

在一实施方案中,自动合成仪与PC通过多通道数字I/O插线板联通(Model PCTIO120-P by Industrial ComputerSource of San Diego,California)。 In one embodiment, an automated synthesizer multi-channel digital I / O board Plug link (Model PCTIO120-P by Industrial ComputerSource of San Diego, California) by the PC. PCDIO120-插板的说明详细描述于Product Manual No.00431-050-20A,其也可由In-dustrial Computer Source得到。 DESCRIPTION PCDIO120- interposer described in detail in Product Manual No.00431-050-20A, which can also be obtained In-dustrial Computer Source.

每一PCDIO120P提供120个以24通道一组的缓冲输入/输出(I/O)通道。 Each PCDIO120P 120 provided to a set of 24-channel buffer input / output (I / O) channels. 每个24道组被程序辅助设备接头(PPI)8255A芯片控制。 Each group 24 is a control program 8255A chip accessory connector (PPI). 这些通道可选择地以8通道为一组用以输入或输出。 Alternatively, these channels to 8 channel group for input or output.

图14显示了控制硬件部分的简化图解。 Figure 14 shows a simplified illustration of part of the control hardware. 具有复杂扩充槽的计算机760,如PC与显示监视器762和键盘764连接。 Computer expansion slot 760 has a complex, such as a PC and a display monitor 762 and a keyboard 764 are connected. 将PCDP120-PI/O板766插到一扩充槽中使PC与五个控制器线路7688联通。 The PCDP120-PI / O board 766 is inserted into a PC expansion slot so that the controller circuit 7688 with five Unicom. 每一控制器线路760通过导体通道与I/O板766联通。 Each link controller 760 via a conductor line channel I / O board 766. 在本实施方案中,导体通道770包括一具有服务为于24个通道的能力的50-导体带。 In the present embodiment, the channel 770 includes a conductor having a service capability of the channel 24 with conductor 50.

2.控制器线路I/O板766的输出信号,典型地为15mA源电流和24mA接收器电流,不足以操动电磁阀。 2. The controller output signal lines I / O board 766, typically 15mA 24mA source current and a current receiver, insufficient solenoid actuator. 所以,控制器电路768将来自I/O板766的输出信号转化为动力信号,其具有足够的动力实际操动电磁阀。 Therefore, the controller circuit 768 from the I / O board 766 of the output signal into a power signal having sufficient power actual actuator solenoid. 控制器线路具有接收来自I/O板766的24个输出信号的能力,并且顺序输出24个动力信号771来控制合成仪的各种装置。 The controller circuit 24 has a receiving capability of an output signal from the I / O board 766, and sequentially outputs the power signal 24 to control the various devices 771 synthesizer. 每个控制器线路768的24条动力线771见图14。 Each controller 24 power line 771 line 768 shown in Figure 14.

每个控制器线路768也提供一个中心物理存储单元,在那里每一传感器多至24条传感线的传感数据被集中。 Each controller provides a central line 768 physical storage unit, where each sensor in the multi-sensor data to the sense line 24 are concentrated. 来自多至24个不同传感器的数据可以通过每个控制线路768上的传感器端口772被接收。 Data from up to 24 different sensors may be received by the sensor port 772 on each control line 768.

因此,每一控制线路768能服务于I/O板766上多至48I/O通道,24个输入和24个输出。 Thus, each control circuit 768 can service up to the 766 48I / O channel I / O board 24 inputs and 24 outputs. 图15显示了根据本发明一个方面的控制器线路768的示意图。 Figure 15 shows a schematic diagram of a controller circuit 768 in accordance with one aspect of the present invention. 50-插接首部781将控制器线路768与I/O板766(未在图15中显示)连接。 50 plug header 781 is connected to the controller circuit 768 I / O board 766 (not shown in FIG. 15). 首部781是一输入首部,并与传感器端口772通过总线780连接。 The first portion 781 is a first input unit, and connected to the sensor port 772 via bus 780. 传感器端口包括首部或用来连接控制器线路768和多至24个输入设置如传感器的接头。 Sensor port includes a header portion or linker used to connect the controller 768 and a plurality of lines 24 to the sensor input is provided. 总线780上的每个导体将信号由一个传感器传到输入首部781。 Each conductor on the bus 780 a signal transmitted by the sensor input a header portion 781. 图15也显示了一个可选择的用来将来自传感器端口772的LED导体信号传送到一可选择的发光二极管(LEDs)786的LED总线784。 FIG 15 also shows an optional LED conductors for transmitting a signal from the sensor port 772 to a selectable light emitting diodes (LEDs) 784,786 of the bus LED. 可选择LED总线784的每个导体将一个信号传递到库786中的一个LED。 Alternatively each LED bus conductor 784 will pass a signal to a LED 786 in the library.

图15也显示了另一个用来连接控制器线路766和I/O板766(未在图15中显示)的另一50-插接首部788。 Figure 15 also shows another circuit used to connect the controller 766 and I / O board 766 (not shown in FIG. 15) of the plug header 50 of another 788. 首部788是一用来接收来自I/O板766输出信号的输出首部。 788 is a header for receiving an output signal 766 output from the I / O board header. 总线790将多至8个来自首部788的输出信号载送到一个八进制反相器74LS240芯片792上。 The bus 790 to eight output signal from the header portion 788 is carried to a 74LS240 octal inverter 792 chips. 八进制反相器74LS240芯片792由Texas Instiuments,Inc.,Dallas,Texas制造。 Octal inverter 74LS240 chip 792 by the Texas Instiuments, Inc., Dallas, Texas manufacturing. 总线794将反转的缓冲输出信号由芯片792运载到一个八进制锁存器趋动器796上。 The buffered output signal bus 794 will be reversed by the chip carrying 792-1 chemotactic octal latch 796 on the actuator. 锁存器趋动器芯片796是Model MIC59p50,由Micrel,Inc.,San Jose,California制造。 Latch 796 is a chip-driven Model MIC59p50, the Micrel, Inc., San Jose, California manufactured. 来自每个锁存趋动器芯片796的输出信号与一输出端口798通过总线800连接。 An output signal from each of the latch-driven chip 796 is connected to an output port 798 through the bus 800. 一个可选择的LED决线802将来自芯片796的LED导体信号传递到一可选择的发光二极管(LEDs)804库。 LED transmitting a signal conductor must selectable LED chip 796 from the line 802 to an optional light emitting diode (LEDs) 804 library. 可选择LED总线中的每个导体802可载运一个信号至库804中的一个LED。 Each conductor 802 may be selectively LED buses carry a signal to a LED 804 in the library.

3.阀用在本发明实例中的电磁阀如,例如阀4-7,10,14,90-91,100-121和129通常是闭合的,除非命令其打开,因此合成仪中系统预置状态是闭合的。 3. In the example of the present invention with the valve in the solenoid valve, such as, e.g. 4-7,10,14,90-91,100-121 valve 129 is normally closed and, unless the order to open, thus synthesizer System Preferences closed state. 因为当合成仪处系统预置状态时没有材料允许流动,因此保障了安全性。 Because no material allowed to flow, and therefore guarantees the security of the system when the synthesizer preset state. 当开动时,阀使用了动力并热化。 When actuated, the valve used and the heating power. 除动力系统明显损耗外,热阀可以不利地影响化学物质通过其出入口。 In addition to significant loss of the power system, the thermal valve may adversely affect the chemical through which the doorway. 因为与保持一个已打开的阀门不闭合相比,打开一个电磁阀一般需要更大的动力,一种触击继动器,例如由Crydom,Inc.,Long Beach,California提供的DID20型,被用来操动阀门。 Since the holding open a valve is not closed as compared to open a solenoid valve generally requires more power a relay struck, for example DID20 type provided by Crydom, Inc., Long Beach, California, is used to the valve actuator. 一个触击继动器如DID20,在特定时间内,典型地为100毫秒,给阀门提供+12伏来打开关闭的阀门。 A strike such as a relay DID20, within a specific time, typically 100 ms, +12 volts to provide the valve opening-closing valve. 触击继动器提供+12伏的时间片段可特定地通过软件控制,并且触击通过一I/O通道而起作用。 Strike relay provides +12 volt time segment may be specified by software control, and a striking work by I / O channels. 此后,触击继动器提供一降低的电压,典型地为额定电压的一半或在本发明的实例中大约为6伏,来保持电磁阀的打开状态。 Thereafter, the strike relay provides a reduced voltage, typically a half of the rated voltage or example of the invention is approximately 6 volts to the solenoid valve remains open. 从而需要较少动力来操动阀并产生较少量的热。 Thereby requiring less power to the valve actuator, and a smaller amount of heat is generated.

本发明提供了四种分别的动力供给。 The present invention provides a power supply of the four kinds, respectively. 第一动力供给输出+5伏以趋动77L芯片,如那些控制器线路768上的芯片。 Supplying a first power-driven to output +5 volts 77L chips, such as those on the controller circuit chip 768. 第二动力供给输出+32伏用于步进马达。 +32 volt output of the second power supply for the stepping motor. 第三动力供给提供+12伏打开电磁阀。 The third power supply provides +12 volts electromagnetic valve is opened. 可选择第四种动力供给也提供了+12伏用于传感器。 Alternatively fourth power supply also provides a + 12V for a sensor. 分开的给阀提供动力的第四动力供给保证当阀门打开和关闭所产生的任何噪音不干扰传感器工作。 Separate fourth power supply providing power to the valve to ensure that when the valve is opened and closed any noise generated by the sensor does not interfere with the work.

在其系统预置状态,所有阀门都闭合。 System Preferences in its state, all valves are closed. 作为附加的安全措施。 As an additional safety measure. 合成仪进一步提供了一固态触击监视器,当控制计算机出现故障时会关闭所有阀门。 The synthesizer further provides a solid strike monitor, when the control computer failure will close all valves. 固态监视器如Brentek Inter-national提供的SM-WDT5型(由Industrial Computer Sourceof San Diego,Califonia获得),被安装在控制计算机和阀门动力供给之间。 The solid-state monitor provided Brentek Inter-national SM-WDT5 type (manufactured by Industrial Computer Sourceof San Diego, Califonia obtained), is mounted between the control valve and the computer power supply. 软件产生的脉冲由控制计算机传递到一个I/O通道上的触击监视器。 Software generated pulse is transmitted by the control computer to strike on a monitor I / O channel. 当脉中消失,例如CPU错误,锁上键或锁下键错误,触击监视器关闭阀的动力供给,从而关闭所有阀门。 When the pulse disappears, such as a CPU error, lock the lock key or key error, touch monitor power supply shut-off valve to close all valves.

F.控制软件现将参照图16-24的程序框图详细讨论控制软件。 F. control software control software will now be discussed in detail with reference to the block diagram of FIG. 16-24. 这些程序框图说明了由控制计算机为完成合成方法的相关相而发布的命令。 The block diagram illustrates the command from the control computer to complete the synthesis methods associated with the release. 为简化下面的讨论,假设在所有相关时间,压力输送系统的阀门接到了控制计算机的正确的命令,将所需试剂送到了反应器库阀门。 To simplify the following discussion, assume that all relevant times, the pressure of the delivery system to correct the command valve control computer, the required reagents to the reactor bank valve.

图16是说明控制计算机为排泄反应器201-209内含物而发布的程序的组合的程序框图。 FIG combination block diagram of a control computer 16 is the drain of the reactor contents and 201-209 of the published procedures. 在排泄前,反应容器201-209含有液体或小球悬浮液。 Before excretion, the reaction vessel containing 201-209 pellet suspension or liquid. 与顶部共同管道212连接的氩气供气阀121接到开启命令并开启,用氩气给顶部共同管道212加压。 Argon gas conduit 212 connected to the common supply valve 121 to the top and open on command, to the top of the pipe joint 212 pressurized with argon. 同时,选择的了一端口阀门101-109开启使液体由反应器201-201流入较低管道214。 At the same time, a selected valve ports 101-109 201-201 inflow opening of the liquid conduit 214 from the reactor low. 只有选择的201-209阀门打开,因为在一些偶联反应中不是使用所有反应器201-209。 201-209 only selected valve opens, because not all reactors 201-209 In some coupling reactions. 如果一个反应器在一个反应阶段是空的,则没必要排泄其内含物。 If a reactor is in a reaction stage is empty, it is not necessary to drain its contents. 底部管道214中的排泄阀开启让液体流出。 Bottom of the pipe 214 so that the drain valve is opened the liquid flows. 压力不同的结果使氩气压力驱使液体由反应器201-209,通过较低管道214,由排泄阀110流出。 Different pressures resulting in pressure driven liquid argon, 214, 201-209 effluent from the reactor via the lower drain valve 110 by a conduit. 当传感器110S关闭,表明没有剩余的液体要排泄,阀门仍保持开启2-5秒以确保所有的液体由反应器库排出。 When the sensor 110S closed indicating no excess liquid to drain, the valve remains open 2-5 seconds to ensure that all liquid discharged from the reactor bank.

图17表示计算机控制清除较低管214发布的命令的组合。 17 shows a computer-controlled command to clear the lower tube 214 of the release composition. 在起始步骤,在较低管道214存留有材料。 In the initial step, the lower conduit 214 has material remaining. 此后,分离阀100开启让加压氩气由PDS265进入较低管道214。 Thereafter, the separation valve 100 opened so that the pressurized argon from the conduit 214 into lower PDS265. 同时,排泄阀110开启将材料从较低管道214排出。 Meanwhile, the drain valve 110 is turned on to discharge the material from the lower duct 214. 材料由较低管道214排出直到没有材料剩余。 Material from the lower discharge duct 214 until no material remained. 当光学传感器检测到排泄管中无材料时,阀门又回到其系统预置状态。 When the optical sensor detects when no material to the drain pipe, and the valve returns to its preset state system.

图18说明一套控制计算机混合母体容器200内含物发布的命令。 18 illustrates a parent control computer commands the mixing container 200 the contents of the publication. 这一系统将氩气由底部31入母体容器200来混合内含物。 This system will argon content was mixed into the parent vessel from the bottom 31 to 200. 氩气气泡当从母体容容底部升至母体容器中内含物的表面时,搅拌了母体容器200的内含物。 When the argon bubbles rise to the parent when the parent vessel from the bottom surface of the workpiece contained in a leisurely, stirring the contents of the container 200 of the parent. 没有被表达命令保持开启的所有阀都处于其系统预置状态。 All valves are not kept open commands are expressed in their preset state of the system. 阀门100开启使加压氩气由PDS265进入到母体容器的底部来搅拌其中内含物。 Opening the valve 100 the pressurized argon to the stirred contents PDS265 wherein the bottom into the parent vessel. 阀90也开启从母体容器中排出氩气。 Exhaust valve 90 is also open from the mother vessel with argon. 大约15-30秒后,阀100和90回到其系统预置状态。 After about 15-30 seconds, and 90 valves 100 to return to its preset state system.

图19A和19B表述了控制计算机把小球悬浮液由母体容器分配给各个反应器编写的命令程序。 19A and 19B expresses the control computer the pellet suspension was dispensed from the container to the parent command program written in each reactor. 该程序隐含了用小球悬浮液填充各反应器的测量体积的技术。 The filling procedure implies measuring the volume of each reactor a suspension with a pellet technology. 使用这一程序,反应器的装填几乎没有流速依赖性。 Using this procedure, the reactor is charged almost no velocity dependence. 因此将看到,不管小球悬浮液将进入的反应器和管道端口之间的距离的远近,小球悬浮液被均匀地分布在各反应器中。 Will thus be seen, regardless of the distance between the bead suspension into the reactor and the conduit port distance, pellet suspension was uniformly distributed in each reactor. 分配周期开始时,反应容器库只含有氩气,且母体容器在步骤852含有小球悬浮液。 When the dispensing cycle begins, the reaction vessel contains only argon library, and at step 852 the parent vessel containing pellet suspension.

在步骤854,在再分配阶段之前,反应容器库部分地装有DMF来代替反应容器库中存在的氩气。 In step 854, before the redistribution stage, a reaction vessel equipped with a library part instead of DMF the reaction vessel with argon present in the library. 分离阀100开启使加压DMF由加压输送系统进入较低管道。 Separation valve 100 opened by the pressurized DMF pumped into the lower duct system. 阀101-109开启向反应器上送DMF。 101-109 DMF feed valve open to the reactor. 阀120开启从顶部共同通道排出氩气。 Open the valve 120 is discharged from the top of the argon common channel. 预程序时间周期(每阶段大约3秒)后,阀100,101-109和120回到,优选顺序地,其系统预置状态。 After the preprogrammed period of time (about 3 seconds per stage), 100,101-109 and 120 back to the valve, preferably sequentially, the system preset state. 然后反应器201-209被混合去除气泡。 The reactor was then removed air bubbles are mixed 201-209. 阀100-109又开启大约3秒钟。 100-109 and valve open for about 3 seconds. 在预程序时间期满时,通向反应器的每一管241-249中的DMF含量应接近顶部共同管道212。 When preprogrammed time expires, DMF 241-249 content of each tube to the reactor should be close to the top of the pipe 212 together.

然后在步骤858,反应器库与母体容顺200装入DMF。 Then in step 858, the parent library reactor container 200 was charged cis DMF. 阀100开启使加压DMF由PDS265进入反应器库。 Open valve 100 from the pressurized DMF PDS265 library into the reactor. 阀101-109开启继续向反应器库加入DMF。 101-109 continue to open the valve DMF was added to the reactor bank. 阀129开启使流过反应器库的DMF进入母体反应器200。 Flowing through the valve 129 open reactor bank DMF reactor 200 into the mother. 阀90也开启从母体反应器200排出被替代的氩气。 Also open the discharge valve 90 is replaced by argon 200 from the mother reactor. 继续填入直至传感器99S检测出其DMF在其检测范围内时,母体容器200中液体含量升至上限容量传感器99S的水平。 Continues to fill until level sensor 99S which detects the DMF is within detection range, the liquid content of the precursor container 200 raised to the upper limit of the 99S capacitance sensor. 然后反应器库完成填入。 The reactor is then filled in to complete the library. 母体容器200填充物多至大约第二容量传感器99S水平。 The parent container 200 was filled up to about a second capacitance sensor 99S levels. 然后在步骤860,阀90,100,101-109又回到其闭合态。 Then in step 860, the valve back to its closed state 90,100,101-109.

下一步,在862步骤,顶部共同通道被清除DMF。 Next, at step 862, the top of the common channel is cleared DMF. 阀121开启,用加压氩气给顶部共同管道加压。 Open valve 121, to the top of the pipe joint is pressurized with a pressurized argon. 阀129开启让DMF由顶部共同管道进入母体体反应器。 DMF opening valve 129 so that the common pipe into the top of the reactor body precursor. 阀90开启由母体容器排出被替代的氩气。 Opening the discharge valve 90 from a parent container is replaced with argon. 从而DMF从顶部共同管道转移至母体容器中。 DMF thus transferred from the top of the pipe joint to the parent vessel. 母体容器被设计具有足够体积容纳没有流过的另加的DMF。 The parent container is designed with sufficient volume to accommodate additional in DMF is not flowing. 例如大约5秒的预程序时间周期后,阀90,121和129在步骤864又回到其系统预置状态。 For example, after a preprogrammed time period of about 5 seconds, the valves 90,121 and 129 at step 864 the system returned to its preset state. 预程序时间周期是可变的,但一定等于或超过清除顶部共同通道DMF所需的时间。 Preprogrammed period of time is variable, but must equal or exceed the time required for common channel DMF top clearance.

如果在进入小球悬浮液之前反应容器中没有装入DMF,即如果反应器是空的,则不球悬浮液的分配将是不均匀的。 If the reaction vessel is not charged with DMF before entering the pellet suspension, i.e., if the reactor is empty, no suspension will distribute the ball is not uniform. 如果反应器是空的,则离小球悬浮液由母体容器送入而通过的管通端口最近的反应器首先被装入。 If the reactor is empty, the beads from the suspension into the container from the parent tube passing through the port nearest the reactor is first charged. 如果允许几个容器过量装入,则可能对一些反应器没有剩下的小球悬浮液。 If excess charged allows several containers, some of the reactor may no remaining pellet suspension.

本发明使用了一种新的控制反应器接收小球悬浮液体积的方法。 The present invention uses a new method of controlling reactor receives pellet suspension volume. 首先,在步骤866,一小柱氩气被引入使反应器与顶部共同管道连接的每个管的顶部。 First, at step 866, an argon gas is introduced into the cartridge so that the top of each reactor tube is connected to a common pipe at the top. 为产生氩气气泡,阀121开启让加压氩气进入顶部共同管道。 Argon bubbles to generate, so that the valve opening 121 into the top of argon pressure common pipe. 选择的阀101-109顺序打开让一些DMF由反应器排至较低管道。 101-109 sequentially selected to open the valve for some DMF discharge from the reactor to a lower conduit. 阀110开启让被替代的DMF由底部管道排出。 Valve 110 is opened so that alternate in DMF withdrawn from the bottom duct. 大约0.3秒后,一小柱氩气出现在将反应器与上部共同通道连接的管的顶部。 After about 0.3 seconds, the argon column a small tube at the top of the reactor together with the upper channel connection. 然后在步骤868阀101-110和121又回到它们系统预置状态。 Then, in step 868, and valves 121 and 101-110 return to their preset state of the system.

在步骤869,在制备中,母体容器中的小球悬浮液被混合以在反应器间分布。 In step 869, in preparation, the parent container pellets are mixed to the suspension between reactor profile. 与此步骤相联系的命令与图18相关的讨论相似。 Command associated with this step is similar to the discussion related to FIG 18. 阀10和90在步骤870又回到其系统预置状态。 Valves 10 and 90 in step 870 the system returned to its preset state. 这一步骤保证均匀小球悬浮液被均匀地分布在所选择的反应器间。 This step ensures a uniform pellet suspension was evenly distributed among the selected reactor.

然后小球悬浮液的一部分被引入顶部共同管道,步骤871。 Pellet suspension then is introduced into the top portion of the common conduit, step 871. 这一步骤根据预程序时间周期定时,以使进入顶部共同管道中的小球悬浮液代替顶部共同管道中的氩气的大部分而不流过管道端口。 This step according to a preprogrammed time period of the timing, so that the top of the common pipe into the pellet suspension argon replace most common pipe and not through the top of the pipe ends. 这个端口与最后一个反应器试管,例如与传感器109S连接的反应管连接。 The port tube with the last reactor, for example the reaction tube connected to the sensor 109S is connected. 已发现大约0.5秒的预程序周期是令人满意的。 About 0.5 seconds has been found that the pre-program cycle is satisfactory. 为将这部分小球悬浮液送入顶部共同管道,阀91开启用氩气给母体容器加压。 This part of the ball joint into the top of the suspension pipe, valve 91 is opened to the parent vessel was pressurized with argon. 阀129开启使小球悬浮液流进顶部共同管道。 Ball valve 129 open so that the top of the suspension flow into a common conduit. 阀120开启排出存留在顶部共同管道中的氩气。 Exhaust valve 120 remain open in a common argon top of the pipe. 先前讨论的预程序时间周期期满后,阀91和120在872步又回到其系统预置状态。 After expiration of the pre-programmed time periods previously discussed, the valve 91 and the system 120 returns to its preset state and at step 872. 更重要的是,阀129继续开启以防止小球和聚合物由于阀的关闭动作而受损伤。 More importantly, the valve 129 continues to open to prevent the polymer pellets and due to the closing operation of the valve being damaged. 在本发明实施方案中,2-端口阀129继续接到控制计算机命令信号以上述方式保持开启。 In an embodiment of the present invention, the 2-port valve 129 continues to command signals to a control computer in the above manner remains open. 但是129可以是一个活门阀,其一收到控制计算机命令脉冲就套接在开和闭状态之间。 However, a shutter valve 129 may be, one of the command pulse received on the control computer is sleeved between open and closed states. 如果阀129是一活门阀且已经打开,则控制计算机不需要发布命令保持活门阀129开启。 If the valve is a shutter valve 129 has been opened and the control computer need not issue a command shutter valve 129 remains open.

图19B是图19A的继续。 FIG. 19B is a continuation of Figure 19A. 当存在于顶部共同管道212中的部分氩气被替代后,剩余小球悬浮液在874步被转移到顶部共同管道212。 When present in the top part of an argon gas common pipe 212 is replaced, the remaining pellet suspension was transferred to the top of the pipe joint 874 in step 212. 阀91开启用氩气给母体容器200加压。 Valve 91 is opened to the parent container 200 pressurized with argon. 保持开启的阀129使加压小球悬浮液进入顶部共同管道212。 The valve 129 remains open so that the pressurized suspension enters the top of the ball joint pipe 212. 选择性阀101-109开启使反应器200中的DMF流出至较低管道214。 101-109 selectively opening the valve in the reactor effluent to a lower conduit 200 DMF 214. 阀110开启从较低管道214排泄DMF。 Open valve 110 from the lower duct 214 of excretion DMF. 部分由于小球悬浮液的抗性,DMF相对慢地顺着选择性反应管201-209退下。 Since the resistance of the ball portion of the suspension, DMF relatively slowly withdraw along the selectivity of the reaction tubes 201-209. 来自使选择反应器与顶部共同管道212连接的选择管221-229的替代DMF被小球悬浮液替代。 Selecting from the selection reactor common pipe 212 connected to the top of DMF 221-229 is replaced by an alternative pellet suspension. 悬浮起泡的DMF柱向较低管道214缓慢向下前进。 It was suspended in DMF post foaming proceeds slowly down to the lower conduit 214.

较早引入起泡的柱也可有利地作为一个体积标记,即提供了一条控制计算机测定什么时候每个反应器已接收了足够量小球悬浮液的途径。 Introduced earlier bubble column may advantageously be used as a volume marker, i.e., the computer provides a way to control when the measurement of each reactor received a sufficient amount of the pellet suspension. 由于聚四氟乙烯的表面性质和DMF与聚四氟乙烯之间的高接触角,发泡柱被截留在DMF柱和悬浮液柱之间。 Due to the high contact angle between the surface properties of polytetrafluoroethylene and polytetrafluoroethylene and DMF, it is trapped between the foam columns and columns DMF suspension liquid column. 如上文所讨论的,在本发明实施方案中使用的光学传感器可检测基本透明的聚四氟乙烯中液体柱的存在或不存在。 As discussed above, the optical sensor used in the embodiment of the present invention may be detected in the presence of substantially transparent polytetrafluoroethylene or absence of the liquid column. 由于DMF被向下前进的大量小球悬浮取代,气泡向下移动到反应管和将反应器与较低管道连接的管。 Since DMF is advanced downwardly substituted pellet resuspended large bubbles move downwardly to the reaction tube and the tube connecting the reactor and the lower duct. 此时用光学检测器101S-109S检测氩气气泡向下运动,检测器瞬间关闭。 At this time, an optical detector detecting 101S-109S argon bubbles downward motion detector is momentarily turned off. 传感器数据,如上所讨论的,传给控制计算机,控制计算机立即发出命令信号关闭各个阀101-109。 Sensor data, as discussed above, transmitted to the control computer, the control computer issues a command signal to close the respective valves 101-109 immediately. 所有阀101-109关闭后,小球悬浮液转移物在878步再开始并继续该步骤直至声学传感器120S检测到在连接母体容器和顶部共同管道的管中没有液体。 After all of the valves 101-109 closed, the suspension was transferred pellet was re-started at step 878 and Continue until acoustic sensor 120S detects no liquid in the container and connected to the top of a common parent pipe tube. 在步骤882,除129外的所有阀回到其系统预置状态。 In step 882, all valves except the system 129 returns to its outside preset state. 正如所讨论的,阀129持续打开以避免损伤小球和阀129。 As discussed, the valve 129 continuously open and to prevent damage to the ball valve 129. 此合刻成仪在其反应器中含有悬浮液。 This combined instrument carved in its suspension containing reactor. 并且可能有一些小球悬浮液残留在母体容器中。 And possibly some remaining pellet was suspended in the mother vessel. 为保证所有小球都转移到了反应器中,要使用至少一个淋洗循环的淋洗过程。 To assure that all pellets are transferred to the reactor, to be used during at least a rinsing cycle rinse.

该淋洗循环自890步用DMF喷洒母体容器内壁以疏松粘附在壁上的小球悬浮液残余物。 The spray rinse loop from step 890 with the inner wall of the parent vessel DMF to stick to the wall of the loose pellet suspension residue. 阀14开启喷洒DMF从上至下洗壁。 Valve 14 opens wash spray from top to bottom wall DMF. 阀90开启从母体容器中排出替代氩气。 Valve 90 open from the parent vessel substitute argon discharge. 继续用DMF喷洒内壁直到母体容器DMF水平升至较低容量传感器90S的水平,并将其关闭。 Continue spraying with DMF until the inner wall of the parent vessel DMF is raised to the level of the lower capacity sensor 90S, and close it. 控制计算机接收到传感器数据并立即发出命令在892步让除阀129以外所有阀回到其系统预置状态。 The control computer receives sensor data 892 and issue a command immediately step all valves except valve 129 is returned to its preset state system. 然后以上述讨论的方式让氩气气泡搅拌母体容器内含物。 Then in the manner discussed above so that argon bubbles parent vessel contents were stirred.

或者通过再装入DMF并混合以疏松粘附于母体容器内壁或玻璃料上的小球悬浮液残余物。 Or by DMF and mixed to reload loosely adhered to the inner wall of the parent vessel or frit glass beads suspension residue. 首先,在步骤884用新鲜DMF再装填母体容器。 First, at step 884 refilled with fresh DMF parent container. 通过开启阀10让DMF从加压输送系统进入母体容器完成这一步骤。 By opening the valve 10 into the parent vessel DMF allow completion of this step from the pressurized delivery systems. 阀90也开启让被替代的氩气由母体容器排出。 Valve 90 is also open so that gas discharged is replaced by a parent container argon. 当母体容器中DMF液面升至顶部容量传感器99S水平,顶部容量传感器99S接通,控制计算机接收到传感器数据立即发布命令在886步让除129外的所有阀回到其系统预置状态。 When the parent vessel was raised to the top level of DMF capacity level sensor 99S, a top volume sensor 99S is turned on, the control computer receives sensor data immediately issue a command in step 886 so that all the valves except the system 129 returns to its outside preset state. 用上面讨论过的方式,通过将氩气气泡导入母体容器在步骤888混合母体容器内含物。 With the embodiment discussed above, by introducing argon gas bubbles parent vessel contents mixed at step 888 the parent vessel.

然后通过开启阀91用氩气对母体容器加压并将混合物通过阀129转移到顶部共同管道,在步骤894将DMF和小球悬浮液的混合物转移到反应器中,其中阀129在整个淋洗过程中保持打开状态。 91 is then pressurized with argon gas by opening the valve on the mother vessel and the top of the mixture was transferred to a common pipe via a valve 129, at step 894 the pellet mixture of DMF and the suspension was transferred into the reactor, wherein the valve 129 in the entire Leaching the process remains open. 选择阀101-109开启让液体由反应器流至较低管道。 101-109 selector valve opened to let the liquid flow from the reactor to a lower conduit. 阀110开启将DMF从底部管道排出。 Open the valve 110 is discharged from the bottom of the pipe DMF. 反应器底部的玻璃料排出了反应器中的所有小球。 Glass frit at the bottom of the reactor effluent in the reactor of all the pellets. 最后母体容器被排空。 Finally, the parent container is emptied. 当连接母体容器和顶部总共同管道的管中没有液体时,传感器120关闭。 When the common pipe connects the main container and the parent is no liquid in the top of the tube, the sensor 120 is closed. 传感器数据传给控制计算机显示在母体容器中没有剩下的液体要转移。 Sensor data to the control computer in the parent container does not show the rest of the liquid to be transferred. 控制计算机继续开启阀915-10秒给顶部共同管道加压使任何存留的混合物进入反应器。 Computer control to continue to open the valve 915-10 seconds and pressure to the top of the pipe joint of any of the remaining mixture into the reactor. 5-10秒后除129外所有的阀回到系统预置状态。 5-10 seconds after the addition of all of the valves 129 back to the system outside the preset state. 完成了一个淋洗循环。 He completed a rinse cycle.

如上所讨论,可进行多次淋洗循环以保证基本上所有的小球悬浮液由母体容器转移至反应器。 As discussed above, it may be multiple rinse cycles to ensure that substantially all of the beads suspension were transferred from the parent vessel to the reactor. 发现2至3次淋洗循环是令人满意的。 It found that 2-3 times a rinse cycle is satisfactory.

当完成所有的淋洗循环时,包括129的所有阀在896步回到系统预置状态。 When all of the rinse cycle, all of the valves 129 includes a step 896 back to the system in a precharged state. 注意阀129在整个小球悬浮液再分配过程中,包括淋洗过程,是保持打开的,以减少对球和聚合物的损伤。 Note that the ball valve 129 in suspension throughout the process of redistribution, including rinsing process, it is kept open, and the ball in order to reduce damage to the polymer. 用早先讨论的方法在步骤898从反应器库排出所有液体。 All the liquid is discharged from the reactor bank of the method discussed earlier in step 898.

从图19A-19B的步骤,可以看出使用氩气气泡可以使小球悬浮液均匀分布在反应器间,而不考虑母体容器与反应器间各流路的流速。 From FIGS. 19A-19B of the step, it can be seen that the argon bubbles can pellet suspension was evenly distributed between the reactors, regardless of the flow rate of each flow path between the reactor vessel and the precursor. 只要氩气气泡在小球悬浮液和DMF试剂之间保持稳定,就提供了一个存在记号,使传感器测定反应器的情况。 As long as argon bubbles remain stable between beads and suspension agents DMF, it provides the presence of a mark, so that the sensor measuring the reactor. 如上所提到的保持稳定氩气气泡的能力是由于DMF的良好物理性质,即DMF与聚四氟乙烯的高接触角。 Argon bubbles stabilized as mentioned above, the ability to maintain good physical properties is due to the DMF, DMF i.e., high contact angle with polytetrafluoroethylene.

但是当向反应器传送小球时也可能希望使用不是DMF的液体。 However, when ball transfer into the reactor may also be desirable to use liquid instead of DMF. 这可能会产生问题,尤其是当取代的液体不具有能产生稳定氩气气泡的物理性质。 This may cause problems, especially when the liquid does not have a substituent capable of generating physically stable argon bubbles. 例如乙腈(MeCN)用作DNA合成的溶剂,就不能产生稳定的气泡。 Such as acetonitrile (MeCN) used as a solvent for DNA synthesis, it can not produce stable bubble. 因此需要不同的方法来分配小球到反应器中。 Requiring different methods for allocating the ball into the reactor.

图19C-19D说明了当不能产生稳定氩气气泡时,控制计算机混合母体容器内含物而发布的可选择的一套命令。 FIGS. 19C-19D illustrate when not produce a stable argon bubbles, the control computer commands a selectable hybrid precursor and release of the container contents.

当只使用少数反应器,例如至多4个至5个时,该技术特别有用。 When only a small number of reactors, e.g. at most 4 to 5, the technique is particularly useful.

步骤854a~860a与图19中的步骤854~860相似,只是少数阀101-109开启,即只有对应于选择反应器的那些阀打开。 Step 854a ~ 860a similar to step 860 of FIG. 19 854 ~, 101-109 only a few valve open, i.e. only those corresponding to the selection valve open reactor. 选择容器部分地装有溶剂,如MeCN,以替代在再分配阶段之前存在于反应器库中的氩气。 Selecting a container partly filled with a solvent, such as MeCN, in place prior to redistribution phase is present in the argon gas in the reactor bank. 此后,选择反应器和母体反应器中装入MeCN。 Thereafter, a reactor and the reactor was charged with the parent MeCN. 当母体反应器200中MeCN的量升至第二容量传感器99S水平时,停止MeCN流,表明装填已完成,完成后,选择阀回到其闭合状态。 When the amount of precursor in the reactor 200 was increased to the second volume MeCN level sensor 99S, the flow stopped MeCN indicating loading has been completed, after the completion of the selector valve returns to its closed state.

接着,步骤872a,在制备中混合母体容器中的小球悬浮液以向选择反应器分配。 Next, in step 872a, the pellet suspension was prepared in the mixing vessel precursor assigned to the selector reactor. 在步骤874a,开启阀91,129和选择阀101-109小球悬浮液进入顶部共同管道并分布到选择反应器中。 In step 874a, the valve opening selector valves 101-109 and 91,129 beads suspension was distributed into the top and to select the common pipe reactor. 这些阀持续打开直到传感器120S检测连接母体容器和顶部共同管道的管中不存在液体。 The valve remains open until the sensor 120S detects the absence of a liquid container connected to the mother and the top tube common pipe. 由于没产生氩气气泡,不使用传感器101S-109S。 Since no bubbles generate argon, without using a sensor 101S-109S. 因为只有少数反应器接收了小球,因此小球到达每一反应需时间大约相等,因而通常保证了每一反应器的等同分配。 Reactor because only a few received pellets, beads thus reaches approximately equal time required for each reaction, and thus typically guarantees equally allocated in each reactor.

在步骤890a,应用淋洗过程,已在图19A-19A讨论过,以保证所有小球被转送到反应器。 In step 890a, the application sparging, has been discussed in FIGS. 19A-19A, to assure that all pellets were transferred to the reactor. 淋洗过程完成后,包括129的所有阀在896a步回到系统预置状态。 After the rinsing process is completed, all valves 896a includes step 129 in the preset state back to the system. 在步骤898a,反应器库排出了所有液体。 In step 898a, the reactor bank all liquid discharged.

图20是控制计算机为用输送系统的所需的试剂装入反应器而发布命令顺序的程序框图。 FIG 20 is a block diagram of a control computer charged into the reactor with the desired reagent delivery system and the procedure of issuing commands. 参照图20讨论的步骤也大略在图21A-21D中作了说明。 Referring to FIG. 20 step also discussed in roughly 21A-21D in FIG been described. 这一方法假设较低管道中只装有惰性氩气(步骤900)。 This method assumes that only the lower conduit with an inert argon gas (step 900). 图21A图示了具有空管道的反应器库的相关部分。 FIG 21A illustrates a relevant portion of the pipe reactor having an empty library. 较低管道首先在步骤902装入一试剂。 Lower conduit 902 is first loaded at step a reagent. 阀100开启让试剂进入较低管道,阀110开启以较低管道排出被取代的氩气。 Open valve 100 so that the reagent into the lower duct, duct valve 110 is opened to discharge the low substituted argon. 当试剂被传感器110S检测到时,所有阀在903步回到系统预置状态。 When the reagent is the detection sensor 110S, all valves in the system back to the step 903 the preset state.

在一些例子中,传感器101S-109S在正当装载连接反应器和较低管道的管时,可能会被偶然地或过早地关闭。 In some examples, the sensor 101S-109S proper loading tube when connected to the reactor and the lower conduit, may be accidentally or prematurely closed. 例如,传感器可能在试剂实际到达之前被试剂的散滴驱动。 For example, the sensor may be driven bulk reagent dropwise until actual arrival reagent. 这样会导致没有足够量的试剂存在于向反应器输送的管中。 This will not cause a sufficient amount of agent present in a delivery tube to the reactor.

为了减少或排除与过早传感器驱动相关的问题,控制计算机在904步为预装载管而可任意选择地命令将阀101-109开启一定时间。 In order to reduce or eliminate the problems associated with premature sensor drive, in step 904 the control computer is pre-loaded tube may be arbitrarily selected to command the valve open a predetermined time 101-109. 一般在被传感器101S-109S检测之前限定时间以使装载管达大约75%。 It is generally defined time before detection sensor 101S-109S to the loading tube by approximately 75%. 正如所述,该步骤不依赖传感器101S-109S的使用。 As described, this step does not depend on the sensors 101S-109S. 因此预装载保证即使传感器过早被驱动,管中也会至少存在足够量试剂来注入反应器以适当地进行混合。 Thus even if the sensor is preloaded to ensure that premature driven, the tube will be at least a sufficient amount of reagent to be injected into the reactor suitably mixed. 在步骤905,在制备中装载管的预定时间期满后,阀回到其系统预置状态。 In step 905, after a predetermined time expires prepared in loading tube, a valve system is returned to its preset state.

在步骤906,连接反应器与较低管道的管用试剂装载至传感器水平。 At step 906, the connection pipe reactor and the low reagent conduit to the sensor load level. 装载过程可以平行进行以节省时间或顺序进行。 Loading procedure may be performed in parallel or sequentially in order to save time. 阀100开启使加压氩气由加压输送系统进入较低管道。 Valve 100 is opened by the pressurized argon pumped into lower conduit system. 选择阀顺序或平行开启,使试剂进入连接反应器和较低管道的管中。 Sequential or parallel selector valve opened, the reagent into the reactor and the lower connecting conduit tube. 当管中试剂含量达到光传感器101S-109S上限时,光传感器接通,控制计算机瞬间关闭相关的阀101-109。 When the tube reaches the upper limit of the content of the reagent photosensor 101S-109S, the optical sensor is turned on, the control computer associated valve momentarily turned 101-109. 当所有传感器101S-109S灯全接通时,所有阀在步骤908全都回到系统预置状态。 When all the sensors 101S-109S lights full on, all the valves back to the system at step 908 all the preset state. 图21B大意说明装载过程完毕后的结果。 FIG 21B illustrates the results after the effect of the loading process is completed.

然后用上文讨论的方法在步骤910清除底部管道。 The method discussed above and then step 910 clears the bottom of the pipe. 图21C说明了具有清洁管道和一柱试剂的反应器的相关部分,其中试剂是在顶部传感器例如101S和阀例如101之间的管部分中。 FIG 21C illustrates a relevant part of the cleaning conduit and the reactor having a column agent, wherein the agent is for example in the top of the sensor 101S, and a valve such as a tube portion 101 between. 底部管道清除后,所有阀在912步又回到系统预置状态。 After the bottom of the pipe purge, all valves in step 912 the system returned to the preset state. 管中的试剂通过玻璃料涌入反应器。 Reagent tube reactor through a glass frit influx. 为使试剂进入反应器,氩气阀122开启使氩气由加压输送系统进入加压较低管道。 Is a reagent into the reactor, argon valve 122 is opened by the pressurized Argon gas delivery system into a low pressure conduit. 排泄阀120也打开。 Drain valve 120 is opened.

在步骤913,开动涡旋马达共同搅拌反应器库一预测时间。 In step 913, the motor actuation vortex stirred reactor bank a common prediction time. 该时间周期足以使反应器内含物混合完全。 The period of time sufficient to complete the reactor contents were mixed. 发现一般大约4秒是合适的,但也可根据反应类型而变化。 It found that about 4 seconds is generally suitable, but it may vary depending on the reaction type.

在上述混合周期期间,在步骤914,将阀101-109开启一预程序时间周期以使少量试剂流和主反应器。 During the period of the mixing, at step 914, a valve open 101-109 preprogrammed period of time to allow a small amount of reagent flow and the primary reactor. 通常在合成过程中重复装载并排空反应器。 Typically repeated loading and emptying of the reactor during the synthesis. 每次反应器排放,其中小球就变干并聚集在一起形成“球块”。 Each time the reactor vent, wherein the ball becomes dry and together form a "block the ball." 步骤914使反应器中的内含物液体化并溶解球块。 Step 914 in the reactor liquid contents of dissolved and block the ball. 因为涡旋运动在小球在或接近反应器底物时是最有效的,所以只需注入少量液体。 Since vortex motion in the ball near the bottom of the reactor or was the most effective, so only a small amount of liquid is injected. 另一方面,小球块可能漂浮在反应器顶部,需要更多的时间将其溶解。 On the other hand, the ball block could float to the top of the reactor, which requires more time to dissolve. 在步骤915,在预定时间期满后,阀回到系统预置状态。 In step 915, after a predetermined time expires, the preset state of the valve back to the system.

在步骤916,通过开启阀101-109,使剩余试剂部分倒入反应器。 In step 916, by opening valves 101-109, the remaining portion of the reagent was poured into the reactor. 装载各容器直到相应传感器101S-109S检测没有液体存在,控制计算机关闭阀。 Each container is loaded until 101S-109S respective sensor detects no liquid is present, the control computer closes the valve. 当所有传感器101S-109S关闭时,所有反应器都装载了。 When all the sensors 101S-109S closed, all the reactors are loaded. 为提高混合效果,在装载的同时搅拌容器库。 To improve the mixing effect, while stirring container magazine loading. 如上所指出的,通过一次开启全部的阀101-109或顺序开启,可设计反应器使平行装载容器或顺序装载。 As noted above, once opened by opening all the valves 101-109 of the sequence or, the reaction can be designed or allows parallel loading container loading sequence.

或者,可不依赖传感器101S-109S装载反应器。 Alternatively, the sensor may not be dependent on the loading of the reactor 101S-109S. 例如,可以在足以装载容器到所需含量的预程序时间周期开启阀101-109。 For example, the container may be sufficient to load the pre-programmed time period required to open the valve content 101-109. 已发现0.1~1秒的时间周期是令人满意的。 It has been found from 0.1 to 1 second period of time is satisfactory. 但是,这一时间周期可根据所用容器数目而变化,即使用容器数目越多,所需时间越长。 However, this time period may vary depending on the number of containers used, the number of containers i.e., the use of the more, the longer the time required. 图21D显示了装载后反应器的简图。 FIG 21D shows a schematic of the reactor after loading.

注意,进入反应器的容器的体积也可通过改进阀101-109和传感器101S-109S之间管的长度和直径而容易地改变。 Note that the volume of the container into the reactor can be easily changed by modifying the length and diameter of the pipe between the valves 101-109 and the sensor 101S-109S. 这种改变通过用一具有不同长度或横截面规格的管取代连接反应器和注入阀,例如,阀111的管而容易地实现。 This is changed by using a different length or cross-sectional size of the tube connecting the reactor and the substitution injection valve, for example, the valve tube 111 is easily realized.

在某些例子中也可有利地增加反应器自身的直径。 In certain instances it may also advantageously increase the diameter of the reactor itself. 例如,也许希望同时混合小球和两种或多种试剂。 For example, maybe the ball and hope at the same time mixing two or more agents. 这样的混合可以在图21A-21D将第一种试剂引入反应器的步骤后进行。 Such mixing step for a first reagent may be introduced into the reactor in the FIGS. 21A-21D. 然后第二种试剂可以通过重复图21B-21D的步骤引到反应器中。 Then the second agent may be introduced into the reactor by repeating the steps of FIGS. 21B-21D. 但是这样做时,由于在引入第二种试剂之前有氩气留在管241-249和271-279中,因此将清除小球悬浮液和第二试剂间的氩气气泡。 However, when doing so, since the second agent prior to introduction of argon gas in the tube to remain in the 241-249 and 271-279, and therefore clears the argon bubbles between the pellet suspension and the second reagent. 为了将氩气气泡从反应器中排出,可将反应器内直径作大来降低反应器中小球悬浮液的高度,从而使氩气气泡容易从小球悬浮液中逸出。 To an argon gas bubble discharge from the reactor, the reactor diameter can be made large to reduce the height of the reactor suspension of small balls, so that argon bubbles easily escape from the pellet suspension. 去除氩气泡后,通过如上所述进行涡旋,将小球悬浮液和第二试剂一起混合。 After removing the argon bubbles, vortexed as described above, the pellet suspension were mixed together and the second reagent.

图22是控制计算机为向反应器中加注活化的氨基酸试剂而发布的命令的顺序流程图。 FIG 22 is a flowchart illustrating a control sequence for the computer filling activated amino acid reagent into the reactor and release commands. 图23A-23C大略说明氨基酸加注过程的相当步骤。 FIGS 23A-23C illustrate relatively rough filling step acid process. 又假设初始时较低管道是清洁的,即只含有氩气。 Another initially assumed the lower pipe is clean, i.e. contains only argon. 图23A是初始时反应器,较低管道,阀,传感器及相关管的示意图。 23A is a schematic view of a reactor, the lower piping, valves, sensors and associated initial tubes.

容器231-239另一选择性安置见图6A。 231-239 container further selective placement Figure 6A.

为使运动带动多组容器的旋转圆盘传送带具体化,在步骤917旋转圆盘传送带有选择容器组的管列成一排。 In order to drive the plurality of sets of motion of the rotating carousel container embodied, at step 917 the rotating carousel with a tube fitted to a selected row of groups of containers. 一旦选择了合适的容器,就在918步用氨基酸活化剂如0.2MHBTV和0.6M DIEA的DMF与DCM比为3∶1的溶液注入管道。 Once the appropriate containers selected, at step 918 the amino acid activating agent such as DMF and DCM and 0.6M DIEA ratio with 0.2MHBTV 3:1 solution for injection pipe. 用与图20相关的讨论过的方法装载管道,即开启阀100和110直到传感器110S接通。 The method discussed in FIG. 20 related to the loading pipe, i.e., opening valves 100 and 110 is turned on until the sensor 110S. 此后所有阀在920步全都闭合。 After all the valves are all closed in step 920.

然后活化剂进入连接反应器和较低管道的管中。 Then the activator into the reactor and a lower tube connector pipe. 阀100和120开启让加压活化剂在922步进入较低管道。 Opening valves 100 and 120 allow pressurized activator conduit 922 into lower step. 阀101-109的选择阀开启直到较低充传感器111S-119S显示存在有液体。 101-109 selector valve valve opening until the lower filling sensor 111S-119S showed the presence of a liquid. 在步骤924,所有阀又关闭。 In step 924, and all valves closed. 图23B图示了这一初始注入步骤后反应器库相关部分中液体的存在。 FIG 23B illustrates the existence of the relevant portion of the reactor bank implantation step after the initial liquid.

为完成注入,在926步,阀100和120又开启让加压活化剂进入较低管道。 To complete the injection, at step 926, and 120 and opening valve 100 so that pressurized activator into lower conduit. 阀101-109中的选择阀平行开启让加压活化剂柱在上述管中上升。 Selector valve in parallel with the valve open so that the pressurized 101-109 activator column rises in the tube. 同时,阀111-119中的相关阀开启将氨基酸注入加压活化剂的上升柱并与活化剂混合。 At the same time, the valve opening associated valves 111-119 in the amino acid injection pressure rise in the column and mixed with the activator is an activator. 图23C图示了这一注入步骤。 FIG 23C illustrates the implantation step.

当每一传感器101S-109S检测到有液体存在时,控制计算机关闭与这个传感器相关的阀101-109。 When each sensor 101S-109S detects a presence of a liquid, closing valves 101-109 control computer relevant to this sensor. 当所有的阀在928步关闭时,反应器库的所有阀回到系统预置状态。 When all the valves closed in step 928, all valves of the reactor back to the system library preset state. 氨基酸和活化剂的混合物优选允许停留在上述管中大约2分钟以保证在步骤930适当的活化。 A mixture of amino acid and an activator is preferably allowed to stay in the tube for about 2 minutes to ensure proper activation step 930.

此后,用早些时候讨论的与图17相关的方法在步骤932清除底部管道。 Thereafter, the method associated with FIG. 17 discussed earlier with step 932 clears the bottom of the pipe. 然后阀101-109和上传感器101S-109S之间的混合物柱以讨论过的与图20相关的方法在步骤913-916进入反应管。 FIG 20 is related to a method and a valve between the posts 101-109 and the mixture of the sensor 101S-109S discussed in into the reaction vessel at step 913-916.

图24显示控制计算机为将反应器中的小球悬浮液转移回母体容器而发布的命令的顺序。 Figure 24 shows the control computer to the reactor pellet suspension was transferred back to the parent vessel and the release of the order of commands. 开始时,假设反应器是排空的,旦较低管道充有氩气。 Initially, the reactor is emptied assumed, lower denier pipe filled with argon. 首先反应器在936步以讨论过的与图20相关的方法注入溶剂。 FIG 20 is related to a method in the first reactor in step 936 discussed solvent injection. 此后所有阀回到系统预置状态。 After all of the valves back to the preset state of the system.

接着在步骤938涡旋小球和试剂的混合物得一悬浮液。 Then the mixture was vortexed at step 938 to obtain pellets and a suspension agent. 或者在反应器注入时涡旋小球混合物。 Or pellets vortexed mixture was injected in the reactor. 这种方法提高了成悬浮液的速度并有助于防止小球在反应器底聚结。 This method increases the speed of the suspension and to help prevent the pellets coalesce bottom of the reactor. 反应管内含物在步骤940被转移到母体容器中。 The reaction tube contents are transferred to a step 940 in the parent vessel. 阀122打开用氩气给较低管道加压。 Valve 122 is opened to the low pressure pipe with argon. 阀129开启让小球悬浮液由反应器库流流入母体容器。 Opening the ball valve 129 so that small libraries suspension from the reactor flows into the parent vessel. 阀101-109中的选择阀开启,优选顺序开启,让氩气推动每一反应器内含物向上到达顶部共同通道并进入母体容器。 101-109 valve opening selector valve, preferably sequentially turned on, so that each push argon to the top of the reactor contents and upwardly into the common channel parent container. 因此反应器内含物被依次转移到母体容器中。 Accordingly reactor contents are sequentially transferred to the parent vessel. 尽管上述转移也可通过同时开启阀101-109平行进行,但顺序转移使保持反应器库中的高氩气压,因此是优选的。 Although the above-described transfer can also be performed in parallel simultaneously opening valves 101-109, but the order to maintain a high argon pressure so that transfer of the reactor library, which is preferable. 而且每一阀101-109优选保持开启大约4秒以保证基本上所有给定反应器内含物被转移到了母体容器。 And each valve 101-109 is preferably kept open for about 4 seconds to ensure that substantially all of a given reactor contents were transferred to the parent container. 在这一过程中,阀90,122和129保持开启状态。 In this process, the valve 129 remains open and 90,122.

所有反应器内含物被转至母体容器后,淋洗反应器也进行另一转移过程。 After all the reactor contents are transferred to the mother vessel, the reactor was rinsed also another transfer process. 为淋洗反应器,从用步骤936含DMF反应器的再注入开始,重复以上步骤。 The reactor is rinsed, starting from the refilling of the reactor with a DMF containing 936 step, repeat the above steps. 如步骤937和941所示,在一系列循环中,阀129保持开启状态以防止损伤球和阀129。 As shown in step 937 and 941, in a series of cycles, the valve 129 remains open and to prevent damage to the ball valve 129. 母体容器优选具有至少三次转移量的体积。 Parent container has a volume of preferably at least three times the amount of the transfer. 再组合末期,母体容器最好通过开启阀110和91排放,直到母体容器中液体含量达到底部较低容量传感器90S并关闭该传感器。 Recombination end, preferably the parent vessel 91 by opening valve 110 and discharge, the parent vessel until the liquid content reaches the bottom of the lower volume sensor 90S and close the sensor. 已发现三个淋洗循环是令人满意的。 Three rinse cycles have been found to be satisfactory.

此后,包括129的所有阀在942步回到其系统预置状态。 Thereafter, all valves including 129 in step 942 the system returns to its preset state. 用讨论过的与图20相关的方法在步骤944搅拌母体容器内含物以混合来自各容器的小球。 By the method associated with FIG. 20, discussed in the parent vessel 944 contents were stirred to mix pellets from each vessel in step. 如果要从母体容器中移出小球,优选通过开启阀10和91在946步排泄母体容器直到母体容器中液体含量达到底部较低容量传感器90S并关闭该传感器。 If the container is removed from the matrix pellets, preferably by opening the valve 10 and the drain 91 at step 946 the parent vessel until the liquid content of the parent container reaches the bottom of the lower volume sensor 90S and close the sensor. 在步骤948,所有阀依次回到其系统预置状态。 In step 948, all the valves are sequentially system back to its preset state. 然后可以从母体容器中移出含小球的混合物使用。 The mixture containing pellets may then be removed from the parent vessel used.

或者,用与图21A-21B相关的方式将小球再分配到反应器,再分配后,所有阀回到其未发生态。 Alternatively, a related embodiment of FIGS. 21A-21B redistribution pellets to the reactor, after redistribution, all of which are not sent back to the valve ecological.

G.总的软件图表图25是本文附录I包括的源码程序框图。 G. Total FIG 25 is a graph software Appendix I herein comprises a block diagram of the source program. 模块950代表使用者。 Module 950 on behalf of the user. 命令解释程序952接到使用者正文命令。 Command interpreter 952 to the body of the user command. 或者,使用者可以使用菜单系统953输入命令启动合成仪。 Alternatively, the user may use the menu system 953 to enter commands to start synthesizer. 菜单系统接受的集合或者转化成命令解释程序952可使用的格式,或直接在支援例行程序模块962调用支援程序。 Or menu system receiving a set of command interpreter 952 is converted into the format which can be used, directly or in the support block 962 calls the routine support program. 命令解释程序952也分析正文进入或使用者输入的命令。 Analysis also enter text or commands entered by the user command interpreter 952. 此后,分析命令调用或执行支援程序模块962的支援程序。 Since then, call or execute a command analysis support program module support program 962. 并且分析命令被显示格式954格式化并在显示762上显示。 And the command is analyzed 954 to format and display format on the display 762 display.

模块958含有大量宏指令文件。 958 module contains a lot of macro files. 一个宏指令文件定义,例如实际进行合成或构建库的步骤顺序。 A macro definitions file, such as the actual sequence of steps for constructing a synthetic or library. 在其最基础水平,一个宏指令文件含有,例如依次含有控制阀和阅读传感器情报的分散命令体系的宏指令。 In its most basic level, a macro file containing, for example, comprising successively and macro control valve sensor information reading command of the dispersion system. 宏指令可以使用其它基础宏指令以完成高水平功能,如排放反应器201-209。 Other macros can be used to perform high level macros basis functions, such as reactor discharge 201-209.

接到来自宏指令文件958命令解释程序952的宏指令进入合成仪库控制960,合成仪库控制器960调用宏指令进行实际合成。 958 received a command from the macro file interpreter 952 into the macro library control synthesizer 960, synthesizer macro library controller 960 calls the actual synthesis. 例如,一个宏指令可以规定合成开始前一定要建立的最初全局改变。 For example, a macro can be defined initial global change before the start of the synthesis must be established.

图25说明运行各种支援亚程序的支援例行程序模块962。 Figure 25 illustrates the operation of the various sub-programs of support routines support module 962. 一个这样的亚程序是自动装填,它是自动装填反应器直到传感器接通的亚程序。 Such a sub-program is automatically loaded, it is automatically loaded into the reactor until the sensor sub-program is turned on. 支援程序962接到来自命令解释程序952或菜单系统953的输入。 Support program from the command interpreter 962 to the input 952 or 953 of the menu system. 功能选择表964含有功能等指示字。 Function selection function table 964 contains a pointer. 图26给出了功能选择表964的输入和输出及其与命令解释程序952和支援例行程序模块962的关系。 Figure 26 shows the input and output function selection table 964 and the command interpreter routines 952 and support module 962 of the relationship.

也有掌握全局变化和全局设置的初始文件966。 There wasnt change and global settings of the original file 966. 初始文件966掌握,例如,代表给开启一闭合阀提供触击电压时间量的数值,等等。 The initial master file 966, for example, the representative values ​​is provided opening a valve closing time of the strike voltage amount, and the like. 查表文件968与合成仪命令文库控制器960一起执行以,例如,让单体进入合适的选择反应器。 Look-up table file 968 and library controller 960 commands the synthesizer performed together, e.g., the monomers into a suitable choice of the reactor. 查表文件968可以含有,例如,各反应器名单,其相应标记单体,合成所需肽所必须单体的名单。 Look-up table file 968 may contain, for example, each reactor list, which lists the corresponding marker monomers required for the synthesis of peptide monomers necessary.

图25也给出了记录文件970。 FIG 25 also shows the log file 970. 记录文件970接受来自命令解释程序952和合成仪文库控制器960的输入。 Log file 970 and synthesizer 952 receives an input from the command interpreter library controller 960. 记录文件970含有诊断目的的操作数据。 Log file 970 containing operating data for diagnostic purposes. 记录文件970的表目含有,例如,与所调用宏指令相关的情报。 970 log file entries contain, for example, related to the macro call intelligence.

相联文件972含有各反应器及其相连阀和传感器的名子。 Associated file 972 containing each reactor and its associated Hinako valves and sensors. 相联文件972与合成仪文库控制器960和支援程序962一起操作以简化记录各反应器及其相连阀和传感器地址的任务。 File 972 associated with the operation of synthesizers 960 and library controller 962 to simplify support program recording each reactor and its associated task address valves and sensors.

数字命令由支援程序962输出进行平行驱动器974。 Parallel digital command by the support program 974 driver 962 output. 平行驱动器974可以是例如PCDIO 120-PI/O板766。 Parallel drive 974 may be, for example PCDIO 120-PI / O board 766. 平行驱动器974通过其I/O通道输出阀控制信号976驱动网络管阀。 974 which is parallel to the drive I / O channel output signal 976 drives the valve control valve through the network. 阀控制信号,如所讨论的,被控制器电路768进一步处理。 Valve control signal, as discussed, the controller circuit 768 for further processing. 而且平行驱动器974输出步进马达控制器信号978来控制涡旋步进马达。 And parallel to drive the stepping motor controller 974 outputs a signal 978 to the stepper motor-controlled scroll. 来自合成仪光传感器。 Synthesizer from the light sensor. 超声传感器,容量传感器的传感器输入980也被平行驱动器974接收,通过传感器检查亚程序982由支援程序962处理。 Sensor ultrasonic sensor, capacitance sensor 980 is also a parallel input driver 974 receives, through the sensor check processing by the sub-program 982 962 support program.

图27说明了控制阀所需的数据结构。 Figure 27 illustrates the data structures required for the control valve. 阀索引VINDEX数组986接收一阀数目984为输入,并对VALVES数组988提供了指示字。 VINDEX valve array 986 receives the index number of a valve 984 as input, and VALVES array 988 provides pointers. 每一VALVES数组988的单元含有一数字字节990的指示字。 Each array 988 VALVES unit containing a digital pointer 990 bytes. 每一数字字节990含有阀数字情报8个二进制位。 990 bytes of each digital valve comprising 8 bits of digital information. 含有给定阀阀数据情报的二进制位通过其数据字节990的地址,和代表数据字节990中二进制位的相对位置的移动值,是可存取的。 Valve containing a given binary bit data information by which 990 bytes of address data, and a bit shift value representing the relative position of the data byte 990, are accessible. 数据字节990的每个二进制位可以受控来合适地接通或关闭一个阀。 990 bytes of data each bit can be suitably controlled turn on or off a valve. 每个二进制位的阀数据情报通过输出端口992控制相应的阀。 Each bit of the control valve corresponding to the data information through the output port 992 of the valve.

图28说明接收来自输入端口994传感器情报必需的数据结构。 28 illustrates the data structure received from a sensor information input port 994 necessary. 代表每个传感器二进制态的情报储存在数据字节996的一个二进制位中,为存取数据字节996中的情报,使用了传感器号数存取SENSOR数组998。 Each sensor representative of the binary state information is stored in a bit data byte 996, byte 996 is accessing data in the information, the access number using a number of sensor array 998 SENSOR. SENSOR数组998每一成员含有一个合适数据字节的指示字。 Each member of the array 998 comprises a suitable SENSOR data byte pointer. 每个数据字节996含有传感器数据情报的8个二进制位。 Each data byte 996 contains eight bits of information of the sensor data. 含有给定阀阀数据情报的二进制位通过其数据字节996的地址和代表数据字节996中二进制位相对位置的移动值,是可存取的。 Valve containing a given binary bit data information by the bit position of its movement value relative byte address data represent data bytes and 996 996, are accessible.

H.窗口接口控制软件可以包括窗口型接口和工作空间。 H. window interface control software may include a window-type interface and a working space. 通常,窗口接口是一矩形图形使用者接口(GVI),其提供一个或多个在屏幕上显示的窗口。 Typically, the interface window is a rectangular graphical user interfaces (the GVI), which provides one or more windows displayed on the screen. 辅助窗口对象如所希望的可以各种大小和格式(例如砖状或格状)显示。 Auxiliary window object as desired can be of various sizes and formats (e.g. brick-shaped or lattice-shaped) display. 在窗口顶部是一个有大量使用者命令选择的选项单条形,每一条形可以调用辅助亚菜单和软件工具为应用目的而使用。 In the top of the window is a large number of users have the option of a single strip command selection, each sub-menu bar can be called auxiliary software tools and applications for the purpose of use. 窗口也包括用于显示和操纵屏幕对象的空区。 Also includes a window for displaying and manipulating screen objects empty spaces. 这一空区是使用者与存储在控制计算机系统存储器中的数据对象交换信息的工作空间或视口。 This area is empty and the user data object is stored in the control computer system memory workspace information exchange or viewport.

窗口接口包括屏幕光标和用来选择和或者调用所感光趣屏幕对象的指示字。 Window interface includes a cursor and screen or to select and invoke screen objects of interest the photosensitive pointer. 响应使用者指示装置如鼠标的运动信号,光标在屏幕上漂移(即自由移动)到所需屏幕定位。 In response to the user pointing device such as a mouse movement signals, cursor shift (i.e., freely moves) on the screen to the desired screen positioning. 在光标运动期间或之后,使用者可给出用户事件信号(例如鼠标键钮“定位”和“制动”)以选择和操纵对象,如本领域所公知的。 During or after cursor movement, the user may be given a user event signals (e.g. mouse key button "positioning" and "brake") for selecting and manipulating objects, as is well known in the art. 例如,窗口可以关闭,再定大小,或通过“定位”(选择)上卷屏幕组件。 For example, the window can be closed, and then sized, or (selecting) screen components scroll through "Location." 键笔画等效,包括键盘加速或“热键”被提供用来完成这些和其它通过键盘的用户操作。 Equivalent key strokes, including keyboard accelerator or "hot key" is provided to accomplish these and other user through operation of the keyboard. 因此,窗口接口提供了与控制计算机交流的更直接的途经。 Therefore, the window interface provides a more direct communication and control via computer.

窗口接口下面是一条信息或事件驱动体系结构。 Here is an informational window interfaces or event-driven architecture. 这个模型可以通过将其操作与传统使用的模态或顺序体系结构的操作对比而给出最好的描述,如图25中的命令接口所示例的。 This model can operate by comparison of its operation mode with the use of a conventional architecture or order to give the best description of the interface 25 a command example of FIG. 在这一方式中,读数器可以有利于增加的事件驱动系统的灵活性和复杂性。 In this way, the reader can help to increase the flexibility and complexity of the event-driven system.

模态程序包括一系列在开始,中间进程,和结束时定义好的离散操作模块或模。 Mode program comprises a series of at the beginning, the middle of processing, and end-defined modules or discrete operating mode. 因此程序按照完全固定的操作顺序,在进行下一步之前每一步骤必须完成。 Thus according to a program completely fixed operation sequence, each step before proceeding to the next must be completed.

模态程序相对容易设计和执行的同时,通常并不容易使用。 Modal relatively easy to design and implement the program at the same time, not always easy to use. 设计当然保证所有所需情报进入,只是在使用户以程序方式操作的花费上进入。 Of course, designed to ensure that all the required information to enter, but the program allows the user to enter the mode of operation cost. 特别是,由于程序建立了一套预安排模,用户不首先完成预需模就不能由一个模进入另一个。 In particular, since a pre-established program schedule mode, the user is not required to first complete the pre-molding can not be molded by one into another. 不允许用户对此顺序有任何偏差。 This order does not allow users of any deviation. 这种模态程序的不灵活性不利于处理现实任务。 This inflexibility modal program is not conducive to real tasks.

另一方面,事件驱动体系结构避免预选择顺序,优化(opting)取代“事件循环”。 On the other hand, event-driven architecture to avoid pre-selection order, optimization (opting) substituted "event loop." 事件循环是处理关于用户和系统事件信息的中心化机理制。 Event loop is the processing center of the mechanism of the system user and system information about the event. 它包括事件排队和进程,用来检索并发送信息至各窗口组。 It includes an event queue and processes to retrieve and send messages to each window group.

信息是操作系统怎样设法使多种应用与硬件同步,如鼠标定位和敲击键盘,MS-窗口的信息被转化为窗口事件处理程序。 Operating system how information is managed to a variety of applications and hardware synchronization, such as positioning the mouse and the keyboard, MS- information window is a window into the event handler. 从程序来看,一条信息只是包含特别事件情报的数字结构。 From the program point of view, a message only contains information of special events digital architecture. 信息结构可以含有用作特别事件恒定标记的信息识别。 Configuration information may contain information identifying the particular event as constant markers. 例如,来自窗口对象的信息可以包括关于窗口产生,关闭,移动,和变大小的信息。 For example, the information from the window object may include information window generator, close, move, size and variable information. 辅助事件数据可以作为信息参数而得到;给定参数的确切解释根据每一所代表的事件类型而变化。 Secondary event data obtained can be used as information parameter; a precise explanation given parameter varies according to the type of each event represented.

提供进程以检索来自系统排队的信息并将安们发送到合适的应用,接着应用可以开始处理任何到达的信息。 Providing processes to retrieve information from the system and security are queued sent to the appropriate application, then the application can begin processing any information arrives. 每个窗口属于一特别窗口类型,其定义了某些这种类型窗口共有的特征。 Each window belongs to a particular type of window, which window defines certain common features of this type. 与每个类型相联系的是具有送到这类窗口的所有信息的窗口功能。 Associated with each type having all of such information to the window function window. 提供应用排队,其中Windows可以放置属于特定应用的信息。 Providing queuing, which Windows can place the information belonging to a specific application. 当该应用已收到输入时,它只读在等待的信息。 When the application has been received input, it waits in the read-only information. 如果没有找到或如果以较高优先性存在,其它应用信息,Windows通过其它应用控制。 If none is found or if there is a higher priority, other information applications, Windows applications by other control.

在事件系统中检索并发送信息的一般进程,如Mi-crosoftRWindowsTM,是本领域公知的,参见例如Petzold,C.,Programming Windows,第二版,Microsoft Press,1990和Custer,H.,Inside Windows NT,Microsoft Press,1993.辅助情报可以在由Microsoft Corp.,Redmond,WA购得的Mi-crosoft Window Software Development中发现。 In the event retrieval system and send a message of general processes, such as Mi-crosoftRWindowsTM, are known in the art, see 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 by Microsoft Corp., Redmond, WA purchased Microsoft Window Software Development in. 上文所引的每个文献的公开内容为所有的目的引作参考。 Disclosure of each document cited above as incorporated by reference for all purposes.

图33说明在控制计算机上实现的GUI。 33 illustrates a GUI implemented in a control computer. 该GUI包括有工作空间1303的矩形窗口1301。 The GUI workspace 1303 including the rectangular window 1301. 在窗口顶部是选择单条线1305,其有用户命令选择1306和1313。 In the top of the window 1305 to select a single line, it has to select a user command 1306 and 1313. 每个这些命令选择包括含有控制合成仪操作命令的亚菜单。 Each of these contains a control command options include a command operation of synthesizers sub menu. 这些命令选择通过用鼠标调用合适的命令给用户提供了自动或手工完成合成的灵活性。 These commands selected by calling the appropriate mouse commands manually or automatically provides synthetic flexibility to the user.

GUI设计注意到给用户一个好的环境,因此减小了程序控制合成仪的努力。 GUI design notice to the user a good environment, thus reducing the effort of program-controlled synthesizer. 例如一个对话框对象或一套对话框对象与每个命令相联系。 For example linked to a dialog object or set of objects and dialog boxes for each command. 当调用一个命令时,合适的对话对象显示在工作空间,人机联作地提醒使用者输入所需情报。 When you call a command, the appropriate dialog object displayed in the work space, man-machine joint operation to alert the user to enter the required information. 一个Help命令1313提供情报有助于用户通过该程序。 Help command provides a 1313 intelligence helps the user through the program. 使用对话对象,用户不用使用成文命令即可操作合成仪。 Use dialogue object, the user can operate without the use of written commands synthesizer.

图34说明了与宏指令(Macros)选择相联系的亚菜单1320。 Figure 34 illustrates a linked macro (Macros) selected sub-menu 1320. 用户可以通过分别在亚菜单上题目1321或1322的鼠标定位而调用Learn或Run命令。 Users can call the Learn or Run command by positioning the mouse were the subject of the 1321 or 1322 in the sub-menu. 在Windows环境中,宏指令是包含一套离散命令的对象。 In a Windows environment, macro that contains a set of discrete command object. 如与图16-24控制合成仪相关的讨论过的命令。 As discussed commands associated with the control synthesizer 16-24 in FIG. 使用Learn命令1321,用户可定义一宏指令来完成特定功能。 1321 using the Learn command, the user can define a macro to perform specific functions. 亚菜单也可以识别调用可得命令设计的键笔画等效。 Sub menu can also be called the equivalent key stroke command designed to identify available. 例如“Alt+L”用来执行Learn命令。 For example "Alt + L" Learn to execute the command.

宏指令一旦“Learned”,Run命令1322可以被选择执行程序设计学习过的宏指令和其它已预先定义的宏指令。 Once the macro "Learned", Run command 1322 may be selected to perform programming learned macros and other macros are pre-defined. 图35例示了当调用Run命令时显示的对话框对象。 Figure 35 illustrates a dialog object when calling the Run command is displayed. 对话框对象包括1325(结合框),其列出了可得的宏指令。 It includes dialog object 1325 (in conjunction with block), which lists the available macros. 为了选择一宏指令,用户在Selet Macro空间1324输入宏指令的名字。 To select a macro, the user name of the macro enter 1324 in Selet Macro space. 或者用户通过鼠标定位和制动上卷,直到选择空间1325中所需宏指令文件。 Or the user positioning the mouse on the volume and the brake, until the selection of the desired macro file 1325. 然后用户在空间1326输入宏指令将被重复的次数。 The user then inputs a space 1326 to be repeated the number of times the macro. 最后运行宏指令,用户在RunMacro按钮1327或RunSMacro按钮1328上定位鼠标。 Finally run the macro, the user positioning the mouse over the button RunMacro 1327 or 1328 RunSMacro button. RunSMacro命令使系统顺序完成宏指令功能,即一次一个反应器。 RunSMacro complete sequence macro commands the system to function, i.e., a primary reactor. 当选择Cancel选择1329时,退出对话框对象1326。 When you select Cancel 1329 when selected, exit the dialog object 1326. Help选择1330根据对话框对象提供情报。 Help choose 1330 to provide information in accordance with the dialog object.

图36说明当选择单线条上Groups选择1310被选择时显示的亚菜单1335。 36 illustrates Groups selected when the selection menu 1335 is displayed when the alkylene 1310 is selected on a single line. 亚菜单1330包括Define命令1331,它被用来定义一套与一组特定反应器相连接的阀。 Define command 1330 includes sub-menu 1331, which is used to define a specific set of a valve of the reactor is connected. 一旦定义后,阀组就被作为一组对象储存在储存器中。 Once defined, the valve block was stored in the reservoir as a set of objects. 计算机储存器可以含有很多组对象,每一对象定义一特别的阀组。 Computer storage group may contain many objects, each object defines a particular valve.

亚菜单1330也包含一Open/Pulse命令1332以在预定时间周期装填和排泄选择反应器。 1330 also includes a sub-menu Open / Pulse command 1332 and loaded to a predetermined time period selected excretion reactor. 当调用时,显示与Open/Pules命令相关的对话对象框。 When invoked, the object to display the dialog box associated with Open / Pules command. 这个对话对象框有些类似于图35中所说明的,含有一结合框,其列出从中选择的可得组对象。 This dialog box is somewhat similar to the object 35 illustrated in FIG., In conjunction with a block containing that group object lists available from which to choose. 用户选择所需组并输入脉冲由对话对象提供的设计空间中选择阀所需时间周期。 And enter the user selects a desired set of pulses provided by the design space session object selected time period required for the valve. 为了装填反应器,用户在Open按钮上定位表明阀要开启。 To a reactor charged, the user is positioned on the Open button indicate the valve to open. 接着用户通过定位Pulse或Spules(顺序装填反应器)按钮开始装填过程。 Then the user or by positioning Pulse Spules (sequential packed reactor) button to start the filling process. 为排空反应器,用户定位Close按钮和Pules或SPules按钮。 The reactor is evacuated, and Close button user location or SPules Pules button.

使用Fill/Drain命令1333,使用者能用传感器自动装填或排空容器。 Use Fill / Drain command 1333, a user can automatically filling or emptying the container sensor. 当选择Fill/Drain命令时,显示了与Open/Pules命令相似的对话框对象。 When selecting Fill / Drain command, and displays the Open / Pules command similar to the dialog object. 用户选择一组阀,并定位AutoFill或SAutoFill按钮完成装入功能或定位AutoDrain或SAutoDrain按钮排空反应器。 The user selects a set of valves, and the positioning or SAutoFill AutoFill button or location AutoDrain completion of the loading or emptying button SAutoDrain reactor. 在一些实例中,可提供Time Delay选择,在触发传感器后推迟阀闭合或开启。 In some examples, selection may be provided Time Delay, delayed trigger sensor after the valve open or closed. 这一功能在传感器被触动时反应器还没有限好地达到所需含量的情况下特别有用。 This feature is particularly useful in the case where the sensor is not limited touched reactor well desired content. 通过建立有特定延迟周期的延迟选择,反应器可适当地被装填。 Selecting specific setup delay by the delay period, the reactor may be charged appropriately. 可以看出,与Group菜单相关的命令在选择组合合成中应用反应器仅给用户提供了灵活性。 As can be seen, the command associated with the menu selection Group combinatorial synthesis reactor only application provides the user with flexibility.

图37说明了含有适于变化菜单选择的选择的亚菜单1340。 FIG 37 illustrates a sub-menu containing the menu selection is adapted to change the selection 1340. 产生的命令1341定义了一个可在宏指令中执行的变量。 The command generator 1341 define a variable that may be performed in the macro. 变量可典型地,例如在值,如时间,可以从一个合成至另一个变化的情况下应用。 Variables may typically be, for example, in value, such as time, can be applied from a case to another variation of the synthesis. 代替产生每个时间值的宏指令,一个变量可简单地定义成相应时间。 Instead of generating macro for each time value, a variable may simply define the corresponding time. 使用建立的命令1342在每一合成循环之前,可将变量建立成所需值。 1342 using the established command prior to each synthesis cycle, the variables can be established to a desired value.

图38说明与Diagnostics选择相关的亚菜单1345。 38 illustrates Diagnostics and select the relevant sub-menu 1345. 亚菜单1345包括Valves 1346,Sensor 1347,和Mix 1348命令,这些命令传递给用户提示控制合成仪并存联关于诊断目的合成仪的情报。 1345 Asian menu includes Valves 1346, Sensor 1347, and Mix 1348 command, pass these commands to prompt the user to control synthesizer coexist with information about diagnostic purposes synthesizer. 当选择Mix命令1348时,开动涡旋马达混合反应器,进行用户规定的时间周期。 When the selected time period Mix command 1348, the start motor hybrid vortex reactor, a predetermined user.

参照图39,当选择Valves命令时,显示Valve Diagnostic对话框对象1390。 Referring to FIG 39, when the selected command Valves, Valve Diagnostic display dialog object 1390. 用户通过对话框对象1390通过选择所需阀对应的入口,可以控制合成仪中任何阀的操作。 User dialog object 1390 by selecting the entry corresponding to the desired valve, can control the operation of the synthesizer of any valve. 例如库1中的阀100可以通过在框1391上定位光标而开启。 1, for example, the library may be opened by valve 100 to position a cursor on a block 1391. 为了方便,所有阀可以通过选择Close All命令1397而闭合。 For convenience, all valves may be closed by selecting the command 1397 Close All. 一Can-cel命令1398关闭对话框对象1390。 1398 Can-cel a command to close the dialog object 1390.

图40说明相应于传感器命令的Sensor Diagnostics对话框对象1391。 Figure 40 illustrates Sensor Diagnostics dialog object 1391 corresponding to the command sensor. 如所述,对话框对象1391含有一与每个传感器相关的框1396。 As described, the dialog object 1391 comprises a sensor associated with each block 1396. 框1396通知用户相应传感器状态。 Block 1396 notify the user of the state of the respective sensor. 例如,如果框中是“X”,长时相应传感器接通。 For example, if the box is "X", the length of the respective sensor is turned on. 相反,空框表明传感器关闭。 Conversely, an empty box indicates that the sensor is closed.

图41说明当选择File选择时显示亚菜单1350。 FIG. 41 illustrates a display 1350 when the sub-menu selection Select File. 新命令1351产生一新合成建立文件控制进行合成,Modify命令1352使用户选择编辑用预存在合成文件。 1351 new command file for generating a new synthesis to establish control synthesis, Modify command 1352 with a prestored user to select to edit the file in the synthesis. 一旦合成建立文件完成,用户调用Save 1354或Save As命令1355将文件存在储存器中。 Upon completion of the synthesis create documents, the user calls the Save As Save 1354 or 1355 there will be a command storage file. Print命令1357打印选择合成文件。 1357 Print command to print selected synthesis file. Print Setup命令1358配置打印机至所需规格,因此打印文件以美观规格出来。 1358 Print Setup command to configure the printer to the desired specifications, so beautiful print file specification to come out. 调用Exit命令1359退出File选择。 1359 File Exit command to exit the call options.

在某些实例中,如每一合成之前,或当选择一组新阀时,用户可能希望通过调用Load CFG文件命令1356而配置该系统。 In certain instances, such as before each synthesis, or when selecting a new valve, the user may wish to configure the system by calling the command 1356 Load CFG file. 系统装载合适的文件告知哪个是要使用的合适的阀。 The system loads the appropriate file suitable valve which is used to inform. 事实上,CFG文件变换或将阀与每个选择反应器“联系”上。 Indeed, CFG files or transformed with each selector valve reactor "link."

图42-44说明用在产生和修饰一合成建立文件中的对话对象。 42-44 illustrate dialogue object is used in the generation and synthesis of a modified build file. 参照图42,Set Assozciatedi对话框1360让用户通过检查含在空间1361中的合适的框选择所需反应器。 Referring to FIG. 42, Set Assozciatedi dialog 1360 lets the user select the desired reactor by checking the appropriate boxes contained in the space 1361. 为了方便,提供Check All按钮1362和Uncheck All按钮1362容易地选择或不选择所有反应器。 For convenience, there is provided Check All Uncheck All button 1362 and a button 1362 to easily select or deselect all of the reactors. Check Bank按钮1364a-1364d让用户选择所有属于特定库的反应器。 Check Bank 1364a-1364d button allows the user to select all of the reactors belonging to a particular library. 当选择Cancel按钮时,退出合成建立程序。 When you select the Cancel button to exit the synthesis of established procedures. 如先前解释的,Help按钮1369提供帮助用户通过该程序的情报。 As previously explained, Help buttons 1369 to help users through the program information. 为继续合成建立过程,用户定位关闭Set Associate对话框对象的OK按钮1365并显示Syn-thesis Setup对话框对象。 In order to continue the process of establishing a synthetic, user location close Set Associate dialog object 1365 and the OK button to display the Syn-thesis Setup dialog object.

图43说明Synthesis Setup对话框对象1370,用它来定义合成的Start Macro 1371,Loop Macro 1372和End Macro1372。 FIG 43 illustrates Synthesis Setup dialog object 1370, uses it to define a synthetic Start Macro 1371, Loop Macro 1372 and End Macro1372. Start Macro和End Macro,例如,包括每一合成步骤之前或之后洗涤反应器的命令。 Start Macro and End Macro, for example, each command comprises synthetic steps before or after the washing of the reactor. Loop Macro含有进行合成的命令。 Loop Macro commands containing synthesis. 这些命令包括用氨基酸结构单元和寡核苷酸注入反应器,在母体容器中合并反应器内含物并混合它们生成小球悬浮液。 These commands include into the reactor using amino acid building blocks and oligonucleotides, the reactor contents were combined and mixed that they produce beads in suspension in the mother vessel. 将小球悬浮液分配给所选择反应器。 The pellet suspension was assigned to the selected reactor. 注入,合并和分配步骤构成一个合成循环。 Injection, merging and distributing steps constituting a synthesis cycle. 根据在Number Synthesis Steps参数1374中输入的数重复合成循环。 Synthesis cycle is repeated according to the number of input parameters Number Synthesis Steps in 1374. 一旦定义了宏指令,用户通过定位OK按钮1375存起Synthesis Setup对话框对象内容并继续建立过程。 Once the macro is defined, the user save 1375 from Synthesis Setup dialog object content and continue building process by positioning the OK button. 用户还是可以定位Cancel按钮1376终止该过程。 Users can also locate the Cancel button 1376 to terminate the process.

图44说明了Amino & Oligo Setup对话框对象1380。 FIG 44 illustrates Amino & amp; Oligo Setup dialog object 1380. 可见,框含有与每个反应器相关的入口1381。 Visible, comprising an inlet block 1381 associated with each reactor. 用户在入口输入所需氨基酸符号和寡核苷酸码。 User input at the inlet amino acid code symbols and the desired oligonucleotide. 例如,反应器1(RVo1)入口含有“V ATGCCGA”,将使合成仪向反应器1中注入氨基酸V和寡核苷酸ATGCCGA。 For example, the reactor 1 (RVo1) comprising an inlet "V ATGCCGA", will cause the synthesizer and the oligonucleotide injection ATGCCGA amino acid V into the reactor 1. 输入合适码后,用户定位OK按钮1382完成建立的过程。 After entering the proper code, the user positioning the OK button 1382 to complete the build process. 提供Cancel钮1383退出该程序。 1383 provide Cancel button to exit the program. 可以看到,用合成建立程序容易建立合成库。 It can be seen easily establish procedures established synthetic synthesis library.

一旦完成合成建立,用户可以开始合成过程。 Upon completion of synthesis up, you can begin the process of synthesis. 参照图45,用户首先选择Synthesis选择1308,然后是Go命令1391开始该方法。 Referring to FIG. 45, the user first selects choice Synthesis 1308, 1391 and the Go command is the start of the method. 或者,用户可建立一个单宏指令完成相似于合成文件中储存的功能。 Alternatively, the user can create a single macro similar to the synthesis completed file storage function.

参照图46,GUI显示在合成或宏指令执行期间状态屏幕1400。 Referring to FIG. 46, GUI shows the synthesis or during the macro execution status screen 1400. 如所示,状态屏幕分成两个分离的区1401和1402。 As shown, the status screen is divided into two separate regions 1401 and 1402. 第一区1401显示与合成相关的情报,例如列出Start Macro,Loop Macro,End Macro的名字,另外Loop Number程序行1408通知用户合成中保持的循环的次数。 The first display area 1401 associated with the synthesis of intelligence, for example, lists the Start Macro, Loop Macro, End Macro name, in addition to the number of 1408 to inform the user of the synthesis loop to maintain the Loop Number program line.

第二区1402显示现在正在执行的Macro文件名。 The second display area 1402 Macro file name is now being executed. 如上所述,一个宏指令可以与另一宏指令嵌套以完成高水平功能。 As described above, a macro can be nested with other macro functions to perform a high level. 设计状态屏幕列出被称为10嵌套水平的1409a-1409j宏指令。 Design condition screen lists 1409a-1409j macro 10 is called nested levels. 可以提供Command程序行1410,Timing程序行1411,Message程序行1412,和RV′S程序行1413,来显示辅助状态或配置情报。 Command lines may be provided program 1410, Timing program line 1411, Message program line 1412, line 1413 and RV'S program, to display the auxiliary status or configuration information. 例如,Command程序行可以显示哪个宏指令命令正在执行,当使用Message程序行时,显示用户预程序的成文信息。 For example, Command-line program which can display the macro command is executed when the program is run using the Message, to display user information pre-written program. Timing程序行显示阀开启或闭合保持的时间。 Timing displayed program line valve opening or closing the holding time. RV′S程序行告知用户使用哪个反应器。 RV'S inform the user which program line reactor use. Time to Complete线条1414告知用户合成完成前剩余时间的百分比。 Time to Complete lines 1414 to inform the user prior to completion of the synthesis percentage of the remaining time.

在合成或宏命令执行期间的任何时候,用户可以按预设计的功能键,即F12,接通“User Abort”状态。 Synthesis or at any time during execution of the macro command, the user can press the function key pre-designed, i.e. F12, ON "User Abort" state. User Abort状态使用户暂停或执行不作为合成一部分定义的宏指令,而不用退出合成程序。 User Abort status allows the user to pause or not to perform as part of the definition of synthetic macro, without leaving the synthesis procedure. 当调用时,系统暂时地暂停执行,并显示UserAbort对话框对象1420,如图47说明的。 When called, the system is temporarily suspended, and displays UserAbort dialog object 1420, 47 illustrated in FIG. 在此刻,用户可插入执行任何宏指令。 At this point, the user can perform any macro inserted. 可通过在Select Macro框1421中敲入所需宏指令名称和在Run Macro按钮1422上定位鼠标而进行。 By typing the desired macro name and location in the mouse button 1422 Run Macro Select Macro block 1421 is performed.

User Abort对话框对象也提供了一个Ignore按钮1424,它使用户继续使用合成程序,就好象该系统从未进入过UserAbort模态。 User Abort also provides a dialog object 1424 Ignore button, which allows the user to continue using the synthetic procedure, as if the system is never entered UserAbort mode. 一个跳跃选择1423指令系统在继续合成之前第一次跳跃宏指令中的当前命令。 1423 hop select a first jump instruction in the current macro command before continuing the synthesis. Retry按钮1425使系统从头执行当前宏指令。 Retry button 1425 to make the system re-execution of the current macro. Abort按钮1426让用户退出合成。 Abort button 1426 allows the user to exit the synthesis.

图48说明与Edit命令相关的亚菜单1430。 Figure 48 illustrates the Edit command associated with the sub-menu 1430. Edit命令使用户很容易通过选择列在亚菜单上的合适标题存取和改变各种对象。 Edit command allows the user to easily by selecting an appropriate sub-menu on the header row access and change various objects. 当选择Associate命令1431时,显示在储存器中储存的列为Set Associate对象的对话框对象。 1431 Associate command when selected, displays a dialog box as the object of the Set Associate objects stored in the reservoir. 通过选择所需对象,显示Set Associate对话框对象。 By selecting the desired object, display Set Associate dialog object. 此时用户可用鼠标编辑对话框对象的内容。 At this point the user can edit the contents of the mouse dialog object. 完成后,用户定位OK按钮存起修改文件。 When finished, locate the OK button to save the user from modifying the file. 类似地,用户可以通过选择所需对象编辑Group,Macro,Vari-able,和Code对象。 Similarly, the user may select objects by editing Group, Macro, Vari-able, and Code desired object. 然后将选择对象放在编辑器中,让用户修改文件。 Then select objects in the editor, allowing users to modify the file.

如上所提到的,传感器可以由于液滴的存在而偶然触动,这是一个问题,特别是当合成循环中使用的溶液含有高浓度气泡。 As mentioned above, the sensor may be due to the presence of the droplet is accidentally touched, which is a problem, particularly when using solution synthesis loop containing a high concentration of bubbles. 为避免或减缓传感器的偶然触发,其灵敏性可设计程序。 To avoid or mitigate accidental triggering sensor, sensitivity can be designed program.

传感器用Machine Config命令1456设计程序。 1456 sensor design program with command Machine Config. 这一命令显示有几个入口,Fill%,Drain%,Time Out%,和PtsAve的Config对话对象框。 This command displays the objects have Config dialog box several entrances, Fill%, Drain%, Time Out%, and the PtsAve. Fill%规定传感器保持接通测定何时反应器注满的百分数。 Fill% remains on a predetermined sensor to determine when the percentage of the reactor filled. 例如,如果进入80%,传感器一定会接通其读数的80%。 For example, if access to 80%, the sensor will be turned on 80% of its reading. Drain%规定传感器保持关闭测定什么时候反应器排空的百分数。 Drain% remains closed when a predetermined sensor reactor emptied percent measured. 如一个实施例,如果进入20%,传感器一定是关闭其读数的80%。 As one example, if access to 20%, the sensor must be closed 80% of its reading. Time out规定在传感器接通或关闭之前延长的时间周期。 Time out before a predetermined extended period of time the sensor on or off. PtsAve规定用来测定传感器接通或关闭的时间百分数的点数或读数。 PtsAve predetermined time used to determine the percentage of the sensor on or off or reading points. 用户也可规定要读数的传感器或传感器组。 The user may also provide for the reading of a sensor or set of sensors. 之后,用户定位OK按钮,其根据参数自动配置系统。 Thereafter, the user positioning the OK button, the system automatically configured based on the parameters.

图49是说明如图33-48所描述的控制软件的事件驱动体系程序框图。 FIG 49 is a system block diagram in FIG 33-48 as an event-driven control software described herein. 如图所示,作为合成仪的GUI心脏的I/OObject程序块有利于各对象程序块之间的接通。 As shown, the GUI of the heart synthesizer I / OObject blocks facilitate turned between target blocks. 当启动这个控制软件时,一个程序块1460将配置文件装载到I/O Object程序块上。 When the control software starts, a block 1460 the configuration file is loaded onto the I / O Object blocks. 这些文件被用来配置Variable Object程序块1456,Lookup Table程序块1457,Macro Objects程序块1458,Group Objects程序块1459,Valve Object Array程序块1461,和Sensor Object Array程序块1462。 These files are used to configure Variable Object block 1456, Lookup Table block 1457, Macro Objects block 1458, Group Objects block 1459, Valve Object Array block 1461, and block 1462 Sensor Object Array.

Lookup Table Objects程序块包括一用来指令阀和传感器对应其相应反应器的文件,这样以减少人工记录每一阀或传感器相应于特定反应器的地址。 Lookup Table Objects comprises a block valve and a sensor for instructions corresponding to their respective files reactor, so as to reduce artificial valve or sensor record for each address corresponding to a specific reactor. Variable ObjectS程序块存储定义的变量,而定义的组对象存在Group Objects程序块中。 Variable ObjectS group of objects stored in the block defined variables, the presence of defined blocks in Group Objects.

Valve Object Array程序块存有识别系统中每一个阀的数组。 Valve Object Array block identification system there array of each valve. 为关闭或开启一个阀,I/O Oject程序块扫描Valve Ob-ject Array程序块输出一个控制特定阀的信号。 A valve is closed or opened, I / O Oject block scan Valve Ob-ject Array control block outputs a signal of a particular valve. 为控制一组阀,I/O程序块与Group Objects程序块一起扫描Valve ObjectArray,让它发出合适的控制阀的信号。 A set of valves to control, I / O blocks together with the Group Objects Valve ObjectArray scan block, it emits a suitable signal control valve.

Sensor Object Array程序块存有识别系统中每一个传感器的数组。 Sensor Object Array block there recognition system each sensor array. I/O程序块可以通过扫描Sensor Object Array程序块读一个传感器,直到发现一个使其读一个特定传感器的匹配。 I / O blocks can be read by a scanning sensor Sensor Object Array block, until it finds a particular sensor reading a match it. 为了读一组传感器,I/O和Group Objects程序块扫描Sensor Object Array程序块来测定要读的合适的传感器。 In order to read a set of sensors, I / O block and the Group Objects to determine suitable sensor to read the scan Sensor Object Array block.

GUI也包括Dialog Box Objects程序块1453和SynthesisObject程序块1454。 Dialog Box Objects GUI also includes blocks 1453 and 1454 SynthesisObject block. Dialog Box Objects程序块含有当调用某些命令时显示的对话框对象。 Dialog Box Objects block containing dialog object invoked when certain commands displayed. Synthesis Object程序块存有当前合成建立文件。 Synthesis Object block the synthesis of the establishment of the current file there. 为完成不同合成,GUI将所需合成建立文件由存储器读入Synthesis Object程序块。 To complete the synthesis of different, GUI will create a file by the synthesis of the desired memory block is read Synthesis Object. 相反,Synthesis Ob-ject程序块的内容被写入储存器存入合成建立文件。 Instead, the content Synthesis Ob-ject block is written into the reservoir synthesis create a document.

Main Window Object(MWO)程序块1452与在屏幕上显示GUI′主窗口(Fig,33)的View Object程序块1455接通。 Main Window Object (MWO) blocks 1452 and displayed on the screen GUI 'main window (Fig, 33) of the View Object block 1455 is turned on. 响应用户命令的一条Message被发送到MWO。 In response to a user command is sent to the Message MWO. 分析这一信息给出合适的要执行的命令。 Analysis of this information is given appropriate command to be executed. 例如,如果接到Macro命令,MWO指令I/O对象从Dialog Box Objects程序块检索宏指令对话框对象,并从Macro Objects程序块检索宏指令目录,View Object程序块接到来自I/O程序块的情报并将其显示在GUI上。 For example, if the received command Macro, MWO command I / O objects retrieved from the macro dialog object Dialog Box Objects block, and retrieves the directory from the macro block Macro Objects, View Object block received from the I / O program block the information and displays it on the GUI.

在一实施方案中,如果计算机发生障碍,I/O程序块含有一Watchdog途经来关闭合成仪。 In one embodiment, the disorder occurs if the computer, I / O blocks containing a Watchdog to close via synthesizer. 例如,I/O对象程序块可以设计程序每2秒钟给合成仪一个脉冲。 For example, I / O target program can be designed to block synthesizer pulse every 2 seconds. 如果因为某些原因合成仪未收到这些脉冲,则可假设控制计算机发生故障而将关闭。 If for some reason do not receive the pulse synthesizer can assume control of the computer fails and will be closed.

实施例下面实施例更详细说明本发明但不限制本发明。 EXAMPLES The following Examples further illustrate the present invention but do not limit the present invention. 实施例1:库的制备和筛选本实施例说明树脂球上可组合肽合成产物怎样通过命名寡核苷配识别器标记接触与合成中每一氨基酸偶联步骤共同存在的小球而明确地给定的。 Example 1: Preparation and screening of libraries This example illustrates the combination of the resin beads may be designated by how the product peptide synthesis oligonucleotide with identification tags in contact with each amino acid coupling step in the synthesis of the co-presence of a pellet and clearly given. 每个标记转运在合成的特定步骤中被偶联的氨基酸单体,可以通过阅读小球上的标记来推断小球上肽的总序列。 Each specific tag in a transport step of the synthesis is the coupling of amino acid monomers, the total sequence of the peptide may be inferred from the beads by reading indicia on pellet. 收集的小球可以筛选,用荧光激活细胞分选仪(FACS)与荧光标记的抗肽抗体结合。 The collected pellets can be screened by fluorescence-activated cell sorter (FACS) binding to fluorescently labeled anti-peptide antibodies. 那些抗体与其紧密结合的小球可以用FACS分离,与各被分选的小球接触的寡核苷酸识别符可通过PCR扩增。 Those antibodies bind to its compact pellets can be separated by FACS, in contact with the beads be sorted identifier oligonucleotides can be amplified by PCR. 扩增DNA序列被测定具有与具有高亲和性抗体结合的肽序列的同一性。 Amplified DNA sequence is determined to have identity with a peptide sequence having a high binding affinity antibodies. 通过结合高容,寡核苷酸编码为基础的信息储存,扩增方法学,和以荧光为基础的分选,本发明提供了一种确定由天然或非天然化学高分子链节合成的庞大分子库中每个分子本性的方法,及有效分离具有生物受体高亲和性配体的各小球的方法。 By combining the high capacity, the oligonucleotide encoding the information stored based amplification methodologies, and fluorescence-based sorting, the present invention provides a method of determining chemically synthesized from natural or unnatural polymer chain link huge method molecules per molecule library nature and method of efficient separation of each pellet having high affinity for a biological receptor ligands.

在该实施例中,单链寡核苷酸被用来编码使用L和D-氨基酸两者结构单元和10μm直径聚苯乙烯小球的可组合肽合成。 In this embodiment, a single-stranded oligonucleotides were used to encode both L and D- amino acids and structural units 10μm diameter polystyrene beads may be combined with peptide synthesis. 寡核苷酸标记物具有高情报内含,适合于非常高灵敏性检测和译码,并在本发明方法中,对用于肽合成的试剂是稳定的。 The labeled oligonucleotide has a high intelligence contained, suitable for very high sensitivity detection and decoding, and in the process of the present invention, the reagents for peptide synthesis are stable. 肽和寡核苷酸平行装配交替合成,这样每个小球具有许多单肽序列和独特寡核苷酸识别标记的复制物。 Peptide and oligonucleotide synthesis are alternately fitted parallel, so that each bead has a single peptide sequence, and a number of unique identification mark oligonucleotide replicates. 寡核苷酸共享通常的5′-和3′-PCR引发位点,因此小球可作为PCR的模板。 Oligonucleotides generally share the 5'- and 3'-PCR priming sites, so pellets can be used as a template for PCR. 编码合成库含有大约8.2×105个七肽并筛选,以结合到抗强啡肽B单克隆抗体D32.39(参见Barrett &Goldstein,1985,Neuropeptides 6:113-120,这里引作参考),用荧光激活细胞分选仪(FACS)选择牢固结合抗体的各小球。 Encoding the synthesis library containing approximately 8.2 × 105 heptad and screened, to bind to the anti-dynorphin B monoclonal antibody D32.39 (see Barrett & amp; Goldstein, 1985, Neuropeptides 6: 113-120, incorporated herein by reference), sorter (FACS) to select each of tightly bound antibody by fluorescence-activated cell pellet. 分选球上寡核苷酸PCR扩增作用后,DNA被测定序列以测定肽配体的特性。 After PCR amplification of the oligonucleotides ball sorting, DNA sequence to be measured to determine the characteristics of peptide ligands.

A.试剂和一般方法用在该项工作中的单分散10μm直径小球原料是由Pharmacia购得的,商业上合成的用1,12-二氨基十二烷连接剂功能化的大孔苯乙烯-二乙烯苯共聚物。 A. Reagents and general procedure used in this work 10μm diameter monodisperse beads which are made commercially available Pharmacia, with a synthetic macroporous styrene-1,12-diamino-dodecane linker functionalized commercially - divinylbenzene copolymer. 该小球是没有用Pharmacia′s Gene Assembler Support连接剂衍生化的Phar-macia MonobeadsTM。 The pellet is not Gene Assembler Support linker derivatized Pharmacia MonobeadsTM with Pharmacia's. 参见Ugelstad和Mork,1980,Adv.ColloidInterface Sci.,13:101-140,这里引作参考。 See Ugelstad and Mork, 1980, Adv.ColloidInterface Sci, 13:. 101-140, incorporated herein by reference.

所有保护氨基酸由Bachem Bioscience Inc.获得。 Inc. All it protected amino acids obtained from Bachem Bioscience. PCR和测序引物用Applid Biosystems model 394寡苷酸合成仪合成。 PCR and sequencing primers nucleotide model 394 oligonucleotide synthesizer using Applid Biosystems. 一些肽的可靠样品是用Applied Blosystems model 431A肽合成仪合成的,使用Fmoc-保护氨基酸,HBTU/HOBt就地化学活化,并用40∶1∶1TFA/水/乙二硫醇去保护。 Some peptides are authentic sample synthesizer of Applied Blosystems model 431A peptide was used Fmoc- protected amino acid, HBTU / HOBt activation in situ chemical, and washed with 40:1:1TFA / water / ethanedithiol deprotection. 这些肽通过在Rainin C18反相柱上的HPLC(>95%纯度)进行纯化,用水/乙腈/0.1%TFA为洗脱剂,用质谱证实结构。 These peptides by Rainin C18 reverse phase column of HPLC (> 95% purity) purified water / acetonitrile as eluent /0.1%TFA, structure confirmed by mass spectrometry.

B.69碱基寡核苷酸和阿片样肽强啡肽B的平行合成阿片样肽强啡呔B的C末端七氨基酸片段H-Arg-Gin-Phe-Lys-Val-Val-Thr-NH2(RQFKVVT)(SEQ IDNO:2)与69链节寡核苷酸(ST08)在10μm直径小球上平行合成,ST08的序列是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)该序列划线部分相应于PCR引物位点,而括号中的区与用于测序模板的引物同源。 C-terminal parallel synthetic opioid peptide dynorphin B B.69 base oligonucleotide and the opioid peptide dynorphin B heptad fragment H-Arg-Gin-Phe-Lys-Val-Val-Thr-NH2 (RQFKVVT) (SEQ IDNO: 2) and 69 mer oligonucleotide (ST08) in a parallel synthesis on 10μm diameter pellets, ST08 sequence is 5'-ATC CAA TCT CTC CAC (ATC TCTATA CTA TCA) TCA CC [ TT TIAC TA TC CT aT] CTC ACTCAC TTC CAT TCC AC-3 '(SEQ ID NO: 20) the underlined sequence corresponds to the PCR primer sites, and brackets region homologous to the primer for sequencing templates. 括号中14碱基序列代表模板编码区。 14 representative of a template nucleotide sequence of the coding region in parentheses.

首先用琥珀酰亚胺4-O-DMT-氧化丁酸酯(分子探针)和N-Fmoc-2,4-二甲氧-4′-(羧甲基氧)-二苯甲基胺(即酸裂解Knorr羧酰胺连接剂)或N-Fmoc-Thr(tBu)-OH(用于非裂解实验)的1-氧化苯并三唑酯的混合物处理小球。 First, with succinimidyl 4-O-DMT- oxide butyrate (Molecular Probes) and N-Fmoc-2,4- dimethoxy-4 '- (carboxymethyl oxo) - benzhydrylamine ( i.e., a mixture of acid cleavable linker Knorr carboxamide), or N-Fmoc-Thr (tBu) -OH (for non-cleavage experiments) of 1-benzotriazole ester oxidation process pellets. 分光光度测得小球上Fmoc-保护氨基与DMT-保护羟基残基的比例大约为20∶1。 Spectrophotometrically measured on pellets and Fmoc- protected amino protecting the hydroxyl residues DMT- ratio is about 20:1. 使用下面核苷的3′-甲基-N,N-二异丙基氨基磷酸酯在自动合成仪上,使小球进行寡核苷酸合成的20次循环:N6-Bz-5′-O-DMT-(7-去氮杂)-2′-脱氧腺苷(Berry和Associates,Ann Arbor,Michi-gan),N4-Bz-5′-O-DMT-2′-脱氧胞苷和胞5′-O-DMT-胸腺嘧啶脱氧核)苷(Glen Research)。 Use the following nucleosides 3'-methyl -N, N- diisopropyl phosphoramidate in an automatic synthesizer of the small balls 20 oligonucleotide synthesis cycles: N6-Bz-5'-O -DMT- (7- deaza) -2'-deoxyadenosine (Berry and Associates, Ann Arbor, Michi-gan), N4-Bz-5'-O-DMT-2'- deoxycytidine and 5 cells '-O-DMT- nuclear thymidine) glycosides (Glen Research).

然后将小球从合成仪中取出并用10%哌啶的DMF溶液处理5分钟示除Fmoc保护基。 The pellet was then removed from the synthesizer, and the other for 5 minutes illustrates Fmoc protecting group with 10% piperidine in DMF. 偶联上第一个氨基酸残基后(N-Fmoc-Thr(tBu)-OH),用乙酐和1-甲基咪唑的DMF溶液处理小球以覆盖未反应的胺。 A first coupling of the amino acid residues (N-Fmoc-Thr (tBu) -OH), with acetic anhydride and 1-methylimidazole in DMF was treated beads to cover the unreacted amine. 所有的肽偶联反应进行20分钟,并含有0.11M Fmoc-氨基酸,0.1M HBTU,0.1M HOBt和0.3MDIEA的DMFF溶液。 All peptide coupling reaction for 20 minutes, and containing 0.11M Fmoc- amino acid, DMFF solution of 0.1M HBTU, 0.1M HOBt and the 0.3MDIEA. 然后让小球进行两次合成仪上核苷酸加成的循环(用TCA进行脱三苯甲基作用,四唑催化的亚磷酸基化作用,用乙酐覆盖,用乙腈/水中的碘氧化)。 Then let the pellets for two cycles of addition of nucleotides synthesizer (for detritylation with the TCA, phosphorous acid group of tetrazole catalytic action, covered with acetic anhydride, using acetonitrile / water oxidation iodine ). 重复氨基酸偶联和二核苷酸加成的顺序步骤,直到完成肽序列RQFKVVT(SEQ ID NO:2)和寡核苷酸编码区构建的合成。 And dinucleotide repeat amino acid coupling step sequence of addition, until the peptide sequence RQFKVVT (SEQ ID NO: 2) Construction of the coding region and oligonucleotide synthesis. 进行另外的35次寡核苷酸合成循环后,依次用哌啶/DMF(1∶9,8分钟),硫代苯酚/三乙胺/二噁烷(1∶2∶2,4小时),亚乙基二胺/乙醇(1∶1,55℃下处理5小时),和TFA/水(20∶1,1小时)处理以对肽和寡核苷酸两条链完全去保护。 After 35 additional oligonucleotide synthesis cycle, sequentially with piperidine / DMF (1:9,8 minutes), thiophenol / triethylamine / dioxane (1:2:2,4 hours), ethylenediamine / ethanol (treatment at 1:1,55 ℃ 5 hours), and TFA / water (20:1,1 hours) to treatment peptide and oligonucleotide deprotection of both strands completely. 在使用酸裂解连接剂的实验中,真空下浓缩TFA去保护反应上清液,然后分离的粗肽产品用HPLC分析。 In an experiment using an acid cleavable linker in, TFA deprotection reaction was concentrated in vacuo supernatant was isolated crude peptide product was analyzed by HPLC.

C.编码库的构建上面划线的平行合成化学用在了该库的构建中。 C. Construction of encoded library above scribing parallel synthetic chemistry used in the construction of the library. 通过将N-Fmoc-Thr(tBu)-OBt和琥珀酰亚胺4-O-DMT氧代丁酸酯的混合物偶联到所有小球上,如上所述,使本实验中肽合成位点不同于DNA合成位点。 By adding a mixture N-Fmoc-Thr (tBu) -OBt and succinimidyl 4-O-DMT-oxobutanoate all coupled to beads, as described above, the synthetic peptide of the present experiment different sites sites in DNA synthesis. 与ST08不同的库中寡核苷酸标记物序列只在编码区内。 ST08 different libraries with labeled oligonucleotide sequences in the coding region only. 寡核苷酸ST08的3′-保守区首先在35mg(~1.75×108个小球)总球量上合成。 3'-conserved region of the oligonucleotide ST08 is first synthesized in 35mg (~ 1.75 × 108 balls) the total amount of the ball. 去除Fmoc保护基,将小球质量分为七等份。 Remove Fmoc protecting groups, the quality of the ball into seven equal parts. 每一等份偶联-个七个不同2-N-Fmoc-保护氨基酸(侧链保护基在括号中表示):Ary(NG-Pmc),Gln(Trt),Phe,Lys(tBoc),Val,D-Val和Thr(tBoc).然后让每一部分进行两次自动寡核苷酸合成。 Each aliquot coupling - of seven different 2-N-Fmoc- protected amino acids (side-chain protecting groups shown in brackets): Ary (NG-Pmc), Gln (Trt), Phe, Lys (tBoc), Val , D-Val and Thr (tBoc). each portion is then let automatic two synthetic oligo nucleotide. 唯一确定每个不同氨基酸残基的增补二核苷酸的各序列是TA,TC,CT,AT,TI,CA和AC。 Uniquely identify each of the different amino acid residues supplement each sequence is dinucleotide TA, TC, CT, AT, TI, CA and AC. 然后将小球合并,充分混合,将全部小球进行Fmoc去保护。 The pellets were then combined, mixed thoroughly, all small balls go Fmoc protection.

小球分配,肽偶联,寡核苷酸二聚体合成,小球再化合,Fmoc去除这一循环总共重复七次。 Pellet distribution, peptide coupling, synthetic oligonucleotide duplex, pellets recombination, Fmoc removal This cycle was repeated seven times in total. 最后的Fmoc保护基未去除。 The final Fmoc protecting group is not removed. 或者,让合并的大量小球进行35次寡核苷酸合成循环。 Or, let's merge large number of small balls 35 times oligonucleotide synthesis cycle. 然后如上所述,库被完全去保护。 Then as mentioned above, go to the library is fully protected.

D.库染色和FACS分析将库的一部分(典型地0.5-2mg小球)悬浮在封阻缓冲剂(PBS,1%BSA,0.05%Tween-20)中并在室温下保温1小时。 D. Library staining and FACS analysis part of the library (typically 0.5-2mg pellet) was suspended and incubated at room temperature in blocking buffer (PBS, 1% BSA, 0.05% Tween-20) for 1 hour. 通过离心小球沉淀成片状并再悬浮在mAbD32,39溶液(10mg/mL封阻缓冲剂)中。 The precipitate pellet into a sheet by centrifugation and resuspended in mAbD32,39 solution (10mg / mL blocking buffer) was. 悬浮液在冰浴上保温30分钟,离心成片状沉淀,并用封阻缓冲剂洗涤。 The suspension was incubated on ice for 30 minutes, centrifuged to a pellet, and washed with blocking buffer. 然后在冰浴上将小球悬浮在藻红蛋白缀合的羊抗鼠抗体(分子探针)溶液中二十分钟。 Then the ice bath for pellet was suspended anti-mouse antibody (Molecular Probes) was twenty minutes in sheep conjugated to phycoerythrin. 小球在封阻缓冲剂中洗涤并在PBS中稀释,送至荧光激活细胞分洗(FACS)(Becton Dickinson FACStar Plus)。 Pellet was washed in PBS and diluted in blocking buffer, to the fluorescence activated cell wash (FACS) (Becton Dickinson FACStar Plus). 通过它们需要的荧光鉴别已与mABD32,39结合的球。 They require identification by fluorescence bound with mABD32,39 ball. 来自最明显染色的库的0.17%和具有最低荧光量区(大约98%)两者的各小球被分类进入PCR miscrofuge小瓶。 0.17% and having a minimum amount of fluorescence region (about 98%) from the most significant staining in each library both pellets were classified into the PCR miscrofuge vial. D32.39对小球的特性结合通过mAb与可溶肽Ac-RQFKVVT-OH(SEQ IDNO:2)D XTU IPE YA O 10M的预保温而被封阻。 D32.39 properties of the pellets by binding of mAb to soluble peptides Ac-RQFKVVT-OH (SEQ IDNO: 2) is blocked preincubated D XTU IPE YA O 10M of.

E.球结合模板PCR在生产者提供的具有0.2mM dATP,dCTP和dGTP,0.8mM dVTP,2mM每种引物,3单位Tag聚合酶(Promega),和1单位尿嘧啶-DNA糖苷酶(Gibco BRL)的缓冲系统(50mM KCl,10mM Tris-HCl,pH9.o,0.1%Triton X-100,2mM MgCl2)(总体积为70L)中进行PCR扩增。 E. Ball PCR template provided in conjunction with producers 0.2mM dATP, dCTP and dGTP, 0.8mM dVTP, 2mM each primer, 3 units of Tag polymerase (Promega), and 1 unit of Uracil -DNA glycosylase (Gibco BRL ) buffer system (50mM KCl, PCR amplification was 10mM Tris-HCl, pH9.o, 0.1% Triton X-100,2mM MgCl2) (total volume of 70L) in. 引物序列5′-ATC CAA TCT CTC CAC-3′(SP13)(SEQ ID NO:21)和5′-(生物素)-GTG-GAA-TGG-AAG-TGA-3′(SP14)(SEQ ID NO:22)分别与ST08模板同源或互补。 Primer sequence 5'-ATC CAA TCT CTC CAC-3 '(SP13) (SEQ ID NO: 21) and 5' - (biotin) -GTG-GAA-TGG-AAG-TGA-3 '(SP14) (SEQ ID NO: 22) are homologous or complementary to the template ST08. PCR反应由45个在95℃变性30秒,在50℃引物退火1分钟,在72℃延伸反应1分钟循环构成。 PCR reaction was 45 cycles composed of 1 min 95 deg.] C denaturation for 30 sec, primer annealing at 50 deg.] C 1 minute, extension at 72 ℃. 用20%丙烯酰胺或2%低熔点琼脂糖凝胶中的电泳分析反应。 With 20% acrylamide or a 2% low melting point agarose gel electrophoresis analysis of the reaction.

F.测定PCR产物序列用链霉抗生素蛋白包裹的磁珠(Dynal,Inc.)分离来自各反应的生物素化的PCR产物。 F. Determination of protein sequences of PCR products wrapped with streptavidin magnetic beads (Dynal, Inc.) Biotinylated PCR products from each reaction. 非生物素化链碱性洗脱后洗涤,每一小球样品用测序混合物处理。 Washed pellet of each sample was sequenced mixture was treated with non-biotinylated strand alkaline elution. 根据生产者的说明,使用引物5′-ATC TCT ATA CTA TCA-3′(SP15)(SEQ IDNO:23)和Bst聚合酶(Bio-Rad)进行双脱氧序列测定,不同的是使用了1∶100比例的脱氧与双脱氧核苷酸三磷酸酯(Pharmacia)。 According to manufacturer's instructions using primers 5'-ATC TCT ATA CTA TCA-3 '(SP15) (SEQ IDNO: 23) and Bst polymerase (Bio-Rad) for dideoxy sequencing, except that the 1: 100 deoxidation ratio and dideoxynucleotide triphosphates (Pharmacia).

G.肽结合亲和性的测定各种肽对单克隆抗体D32.39的结合亲和性在一竞争结合实验中被测定。 G. Determination of peptide binding affinity of various peptides on the binding affinity of D32.39 monoclonal antibody was determined in a competition binding experiments. 含有与CAMP依赖蛋白激酶共有区底物序列融合的D32.39的已知表位的痕量肽(LRRASL GGGRRGFKVVT(SEQ ID NO:24;50pM)被用[g-33P]ATP放射标记至高特异活性(参见Li etal.,1989,Proc.Natl,Acad,Sci.USA 86:558-562,这里引作参考),并与各种浓度待测肽混合(10μM-1pM)。将肽混合物加入到用D32.39(0.1Gmg/mL)涂层的聚苯乙烯井中,样品在4℃保温2小时,用PBS洗涤井,记数与每个井有关的放射活性并用来制成竞争结合曲线。在测定条件下,IC50应接近肽的相关常数(Kd)。实施例2:噻唑烷并酮(thiazolidinones)的合成和稳定性研究下面实施例涉及用本发明方法合成噻唑烷并酮。这一合成在US专利申请号No.08/256090,申请申1994年6月23日的专利中更详细地说明,本文为所有目的引作参考。 Trace known epitope peptides CAMP-dependent protein kinase comprising the consensus sequence of the fusion region of the substrate D32.39 (LRRASL GGGRRGFKVVT (SEQ ID NO: 24; 50pM) was used [g-33P] ATP radiolabeled to high specific activity (see Li etal, 1989, Proc.Natl, Acad, Sci.USA 86:. 558-562, incorporated herein by reference)., and mixed with various concentrations of peptide tested (10μM-1pM) was added to the mixture with peptide D32.39 (0.1Gmg / mL) coated polystyrene wells, and samples were incubated for 2 hours at 4 ℃, wells were washed with PBS, count associated with each well and radioactivity was used to make competitive binding curve assay under the conditions, IC50 should be related constant (Kd) close peptide Example 2: Example relates to a method of the present invention thiazolidin-and-one synthesis and stability of thiazolidine and one (thiazolidinones) following the implementation of this synthesized in US. Patent application No. No.08 / 256090, Shen patent application June 23, 1994 in more detail, the paper incorporated by reference for all purposes.

A.制备双标记噻唑烷并酮H2N-S-TentaGel(500mg),一种商购聚苯乙烯树脂(Rapp Pdymere,Tubingen,Germany,1g,0.30mmol/g装载),用Fmoc-Gly-OH在α-碳上进行标记(2-13C,99%,由Cambridge Isotope Laboratories Inc.,And Over,MA购得)。 A. Preparation of doubly labeled thiazolidine-one and H2N-S-TentaGel (500mg), one kind of commercially available polystyrene resin (Rapp Pdymere, Tubingen, Germany, 1g, 0.30mmol / g loading), with Fmoc-Gly-OH marking the α- carbon (2-13C, 99%, of Cambridge Isotope Laboratories Inc., And over, MA commercially available). 树脂用Ac2O覆盖,用哌啶去保护,以其OBt-活化的酯偶联Fmol-光连接剂。 Ac2O covered with the resin, deprotected with piperidine, activated ester thereof OBt- Fmol- optical coupling linking agent. 树脂再被覆盖,去保护,并与未标记的Fmoc-Gly-OH的酸酐反应。 Resin was covered, deprotected, and reacted with unlabeled Fmoc-Gly-OH anhydride. 再进行覆盖和去保护生成游离胺树脂。 And then covering the free deprotected amine resin generated. 与0.75M在碳基上标记的PhCHO(羰基-13C,99%;购自Cambridge Isotope Laboratorios,Inc.,Andover,MA)和2.0巯基乙酸的含3分子的ACN溶液在70℃反应2小时,生成双标记噻唑烷并酮树脂。 0.75M marked on the carbon-based PhCHO (carbonyl -13C, 99%; available from Cambridge Isotope Laboratorios, Inc., Andover, MA) ACN solution containing 2.0 3 thioglycolic acid molecules reacted at 70 ℃ 2 hours. generating a dual-labeled thiazolidine and ketone resins. 将树脂充分洗涤(3×5mlCH2Cl2,3×5ml DMF,3×5ml CH2Cl2,3×5ml MeOH,3×5mlCH2Cl2,3×5ml Et2O)并在真空下干燥。 The resin was washed thoroughly (3 × 5mlCH2Cl2,3 × 5ml DMF, 3 × 5ml CH2Cl2,3 × 5ml MeOH, 3 × 5mlCH2Cl2,3 × 5ml Et2O) and dried under vacuum.

B.TFA稳定性研究一部分树脂(20mg)用95%TFA 15%H2O处理1小时,然后用CH2Cl2,MeOH和Et2O洗涤。 Stability B.TFA part of the resin (20mg) for 1 hour with 95% TFA 15% H2O, then washed with CH2Cl2, MeOH and with Et2O. Gel-13CNMR分析树脂表明没有噻唑烷并酮的损失,如通过两个标记碳相对整合作用所证实的。 Gel-13CNMR analysis of the resin indicated no loss of thiazolidine and ketones, such as by two opposing marks carbon demonstrated integration effect. 见条1,图30。 See Article 1, 30 in FIG. 希望光连接剂或噻唑烷并酮的任何破坏会导致苄位碳上的整合作用的减少。 Desirable optical connection or any damage thiazolidine-one and leads to a reduction of the effect on the integration of the benzylic carbon. 这一实验表明噻唑烷酮的光连接剂对TFA处理是稳定的。 This experiment shows that the optical connector thiazolidinone TFA treatment agent is stable.

C.DNA合成稳定性研究将一部分树脂(20mg)装载到标准DNA合成柱上,并以其氨基磷酸酯的形成(Phosphoramidites)应用A,C,和T核苷进行DNA合成的40次循环。 Synthesis Stability C.DNA the part of the resin (20mg) is loaded into a standard DNA synthesis column, and its form phosphoramidate (of the phosphoramidites) application A, C, and T nucleoside 40 cycles of DNA synthesis. 每一循环后用碘氧化。 With iodine oxidation after each cycle. 在每一循环开始用2%TFA/CH2Cl2处理树脂去除“模型”二对甲氧三苯甲基(DMT)。 In each cycle begins processing with 2% TFA / CH2Cl2 resin removal "model" two pairs of dimethoxytrityl (DMT). 由柱中取出树脂,用DMF洗涤,用硅胶-13CNMR谱分析。 Withdrawn from the column resin was washed with DMF, spectral analysis on silica gel -13CNMR. 见图30,条A。 Figure 30, Article A. 所得图谱表明几乎设有或设有光连接剂或噻唑烷并酮分子的破坏。 The resulting spectra showed little or is provided with an optical damage or thiazolidine-linking agent and one molecule.

一部分树脂(2mg)也在pH7.4 PBS缓冲液中进行了三小时光解,用HPLC分析释放出的噻唑烷二酮。 Part of the resin (2mg) were also three pH7.4 PBS buffer solution hr, by HPLC analysis thiazolidinedione released. 见图31和图32,条A。 See FIG. 31 and FIG. 32, Article A. 数据表明噻唑烷并酮在高纯度下释放,并且用标准DNA合成试剂处理,光连接剂和噻唑烷二酮两者都不明显变化。 Data show that the release of thiazolidine-one and at a high purity, and both treated with standard DNA synthesis reagents, linker and light thiazolidinedione not change significantly. 实施例3:可组合合成用合成装置可进行YGGFL的可组合合成。 Synthesis of combinatorial synthesis may be combined with a device may YGGFL synthesized: Example 3. 合成在反应器1-6中进行,7-9是不连接的。 Synthesis is carried out in a reactor 1-6, 7-9 is not connected. 向小球中加入的六种氨基酸是L,E,G,Y,A和F。 Was added to the pellet six amino acid is L, E, G, Y, A and F. YGGFL和其它肽一起规定。 And other peptides with predetermined YGGFL. 将小球加到母体容器中(29.5mg)并悬浮在DMF中。 The pellets were added to the mother vessel (29.5 mg) and suspended in DMF. 每次合成循环包括再分布,肽偶联,覆盖,胺去保护,去保护后FMOC的收集,用DMF淋洗,及在母体容器中再组合这几步。 Each synthesis cycle comprises a redistribution, peptide coupling, covering, amine deprotection, deprotection of the FMOC collected, rinsed with DMF, and then a combination of the steps in the mother vessel.

合成后,将标记Hettz抗体引入混合的小球。 After synthesis, the labeled antibody into the mixing Hettz pellets. Hertz抗体大部分与YGGFL结合。 Most of Hertz antibody binds to YGGFL. FACS分析证明YGGFL的存在,证明这种规定结合是在合成仪中合成的。 FACS analysis demonstrated the presence of YGGFL, demonstrate such a provision is incorporated in the synthetic synthesizers. 本实验证明肽的异收集,包括YGGFL链,可通过合成仪规定并合成。 This experiment demonstrates heterologous peptide were collected, including YGGFL chain, and can be synthesized by a predetermined synthesizer.

尽管本发明为了清楚和理解的目的以说明书和实施例的方式详细进行了说明,但很明显在增补的权利要求书范围内可进行一些变化和修饰。 Although the present invention is for purposes of clarity and understanding of the specification and in the embodiment has been described in detail, but it is clearly within the scope of claims of claim supplement certain changes and modifications may be made. 实施例4:使用本发明仪器测定小球分布为测定母体容器中的混合小球是否被均匀地分布到反应器中,将一些小球生物素化。 Example 4: Determination of pellet distribution measurement parent container whether mixed pellets uniformly distributed to the reactor, some of the pellets using biotinylated apparatus of the present invention. 人工将生物化球放在一个反应器中。 The artificial ball in a biochemical reactor. 非生物素化球人工放置在另8个反应器中。 Non-biotinylated artificial ball 8 placed in the other reactors. 合成仪将9个反应器中的球送至母体容器。 The synthesizer 9 to the reactor ball parent container. 从母体容器中取一样品,让带荧充的链霉抗生物素蛋白与生物素化小球上的生物素结合。 Sample was taken from the mother vessel, so that the charging with fluorescent streptavidin biotin-binding avidin with the biotinylated bio beads. 荧光激活细胞分选仪(FACS)分析证明母体容器中大约9.1%的小球被生物素化。 Fluorescence activated cell sorter (FACS) analysis demonstrated that approximately 9.1% of the parent container of the pellets were biotinylated. 然后将小球再分配于9个反应器,每个容器中生物素化小球对总的小球的百分比用FACS分析仪测定。 The pellet is then redistributed to the reactor 9, each container biotinylated beads percentage of the total of the pellets were measured by FACS analysis. 表5表明每个反应器中混合小球具有与母体容器大约相同的生物素化小球与总的小球的比例。 Table 5 shows that each reactor having a mixing ratio of the pellet of approximately the same parent container biotinylated beads and the total pellet.

表5 table 5

应该明白上述描述只为了说明而不作为限制。 It should be understood that the foregoing description is only for purposes of illustration and not limitation. 通过上述说明,很多具体实例对于本领域熟练技术人员来说明显而易见的,因此本发明范围应该不受上述说明的局限,而应该参照权利要求书以及等价于权利要求书权利的全部范围。 By the above description, numerous specific examples of ordinary skill in the art will be described with apparent that the scope of the present invention should not limited to the above description, but should refer to the claims and equivalents of the claims to the full scope of the claims.

序列表(1)综合信息:(I)申请人:AFFYMAX TECHNOLOGIES NV SEQUENCE LISTING (1) General Information: (I) Applicant: AFFYMAX TECHNOLOGIES NV

(II)发明名称:Synthesizing and Screening Molecular Di-versity(III)序列数目:24(IV)联系地址:(A)地址:Townsend and Townsend Khourie and Crew(B)街道:One Market Plaza,Steuart Tower,Suite2000(C)城市:San Francisco(D)州:California(E)国家:USA(F)邮编:94105(V)计算机可读形式(A)媒体类型:Floppy盘(B)计算机:IBM PC兼容机(C)操作系统:PC-DOS/MS-DOS(D)软件:PatentIn Release # 1.0,Version # 1.25(VI)流程应用数据:(A)申请号: (II) Title: Number (III) sequence Synthesizing and Screening Molecular Di-versity: 24 (IV) Address: (A) Address: Townsend and Townsend Khourie and Crew (B) Street: One Market Plaza, Steuart Tower, Suite2000 (C) city: San Francisco (D) states: California (E) State: USA (F) Zip Code: 94105 (V) computer readable form (A) media type: Floppy disk (B) computer: IBM PC compatible ( C) operating system: PC-DOS / MS-DOS (D) software: PatentIn Release # 1.0, Version # 1.25 (VI) process application data: (A) application No.:

(B)申请日:(C)分类:(VII)优先权数据(A)申请号:US 08/146,886(B)申请日:1993.11.02(VII)优先权数据(A)申请号:US 08/149,675(B)申请日:1993.11.02(VIII)代理人/事务所情报(A)名称:Norviel,Vernon A. (B) Application date: (C) Classification: (VII) Priority data (A) Application No.: US 08 / 146,886 (B) Application date: 1993.11.02 (VII) Priority data (A) Application No.: US 08 / 149,675 (B) filing date: 1993.11.02 (VIII) Agent / Office Intelligence (A) name: Norviel, Vernon A.

(B)登记号:32,483(C)参考/摘要号:16528J-000740PC(IX)电讯信息:(A)电话:415-326-2400(B)电传:415-326-2422(2)SEQ ID NO:1的情报:(I)序列特征(A)长度:13氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:1:Tye Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Val Thr (B) Registration Number: 32,483 (C) Reference / Docket No.: 16528J-000740PC (IX) Telecommunication Information: (A) Telephone: 415-326-2400 (B) Fax: 415-326-2422 (2) SEQ ID NO: Information 1: (the I) sEQUENCE cHARACTERISTICS (a) length: 13 amino acids (B) type: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI ) sEQUENCE dESCRIPTION: SEQ ID NO: 1: Tye Gly Gly Phe Leu Arg Arg Gln Phe Lys Val Val Thr

1 5 10(2)SEQ ID NO:2的情报:(I)序列特征(A)长度:7氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:2:Arg Gln Phe Lys Val Val The1 5(2)SEQ ID NO:3的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:3:Thr Phe Arg Gln Phe Lys Val Thr1 5(2)SEQ ID NO:4的情报:(I)序列特征(A)长度:8氨基酸 1 5 10 (2) SEQ ID NO: Information 2: (the I) SEQUENCE CHARACTERISTICS (A) Length: 7 amino acids (B) TYPE: amino acid (C) Number of shares: single-stranded (D) TOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 2: Arg Gln Phe Lys Val Val The1 5 (2) SEQ ID NO: Information 3: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) type: amino acid (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 3: Thr Phe Arg Gln Phe Lys Val Thr1 5 (2) SEQ ID NO: 4 INFORMATION: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids

(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:4:Thr Thr Arg Arg Phe Arg Val Thr1 5(2)SEQ ID NO:5的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:5:Thr Val Arg Gln Phe Lys Thr Thr1 5(2)SEQ ID NO:6的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽 (B) TYPE: amino acid (C) Number of shares: single-stranded (D) TOPOLOGY: linear (II) MOLECULE TYPE: peptide (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Thr Thr Arg Arg Phe Arg Val Thr1 5 ( 2) SEQ ID NO: Intelligence 5: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) type: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 5: Thr Val Arg Gln Phe Lys Thr Thr1 5 (2) SEQ ID NO: Intelligence 6: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) tYPE: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide

(XI)序列描述:SEQ ID NO:6:Gln Val Arg Gln Phe Lys Thr Thr1 5(2)SEQ ID NO:7的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:7:Arg Gln Phe Arg Thr Val Gln Thr1 5(2)SEQ ID NO:8的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:8:Lys Gn Phe Lys Val Thr Lys Thr1 5(2)SEQ ID NO:9的情报: (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Gln Val Arg Gln Phe Lys Thr Thr1 5 (2) SEQ ID NO: INFORMATION 7: (the I) SEQUENCE CHARACTERISTICS (A) Length: 8 amino acids (B) TYPE: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 7: Arg Gln Phe Arg Thr Val Gln Thr1 5 (2) SEQ ID NO: Intelligence 8: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) tYPE: amino acid (C) number of shares: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 8: Lys Gn Phe Lys Val Thr Lys Thr1 5 (2) SEQ ID NO: 9 INFORMATION:

(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:9:Gln Gln Phe Lys Val Val Gln Thr1 5(2)SEQ ID NO:10的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:10:Lys Gln Phe Lys Val Thr Gln Thr1 5(2)SEQ ID NO:11的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链 (I) SEQUENCE CHARACTERISTICS (A) Length: 8 amino acids (B) Type: amino acid number (C) Unit: single-stranded (D) TOPOLOGY: linear (II) MOLECULE TYPE: peptide (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Gln Gln Phe Lys Val Val Gln Thr1 5 (2) SEQ ID NO: INFORMATION 10: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) tYPE: amino acid (C) number of shares: single-stranded (D ) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 10: Lys Gln Phe Lys Val Thr Gln Thr1 5 (2) SEQ ID NO: INFORMATION 11: (the I) sEQUENCE cHARACTERISTICS ( A) lENGTH: 8 amino acids (B) tYPE: amino acid (C) number of shares: single-stranded

(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:11:Thr Gln Phe Lys Val Thr Lys Thr1 5(2)SEQ ID NO:12的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:12:Thr Phe Arg Val Phe Arg Val Thr1 5(2)SEQ ID NO:13的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:13:Phe Arg Arg Gln Phe Arg Val Thr (D) TOPOLOGY: linear (II) MOLECULE TYPE: peptide (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Thr Gln Phe Lys Val Thr Lys Thr1 5 (2) SEQ ID NO: INFORMATION 12: (the I) the sequence wherein (A) length: 8 amino acids (B) type: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 12: Thr Phe Arg Val Phe Arg Val Thr1 5 (2) SEQ ID NO: INFORMATION 13: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) tYPE: amino acid (C) number of shares: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 13: Phe Arg Arg Gln Phe Arg Val Thr

1 5(2)SEQ ID NO:14的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:14:Arg Gln PHe Lys Gln Val Gln Thr1 5(2)SEQ ID NO:15的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:15:Gln Thr Val Thr Val Lys Lys Thr1 5(2)SEQ ID NO:16的情报:(I)序列特征(A)长度:8氨基酸 1 5 (2) SEQ ID NO: INFORMATION 14: (the I) SEQUENCE CHARACTERISTICS (A) Length: 8 amino acids (B) TYPE: amino acid (C) Number of shares: single-stranded (D) TOPOLOGY: linear (II) molecule tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 14: Arg Gln PHe Lys Gln Val Gln Thr1 5 (2) SEQ ID NO: INFORMATION 15: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) type: amino acid (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 15: Gln Thr Val Thr Val Lys Lys Thr1 5 (2) SEQ ID NO: 16 INFORMATION: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids

(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:16:Gln Gln Val Gln Arg Gln Thr Thr1 5(2)SEQ ID NO:17的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:17:Lys Thr gln Val Val Gln Phe Thr1 5(2)SEQ ID NO:18的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽 (B) type: amino acid (C) Unit: single-stranded (D) TOPOLOGY: linear (II) MOLECULE TYPE: peptide (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Gln Gln Val Gln Arg Gln Thr Thr1 5 ( 2) SEQ ID NO: INFORMATION 17: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) type: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 17: Lys Thr gln Val Val Gln Phe Thr1 5 (2) SEQ ID NO: INFORMATION 18: (the I) sEQUENCE cHARACTERISTICS (a) length: 8 amino acids (B) tYPE: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide

(XI)序列描述:SEQ ID NO:18:Gln Val Thr Gln Val Arg Val Thr1 5(2)SEQ ID NO:19的情报:(I)序列特征(A)长度:8氨基酸(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:19:Phe Val Val Thr Val Arg Val Thr1 5(2)SEQ ID NO:20的情报:(I)序列特征(A)长度:69碱基对(B)类型:核酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:DNA(寡核苷酸)(XI)序列描述:SEQ ID NO:20:ATCCAATCTC TCCACATCTC TATACTATCA TCACC-TATCC TATTITTACC TCACTCACIT CCATTCCAC(2)SEQ ID NO:21的情报: (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Gln Val Thr Gln Val Arg Val Thr1 5 (2) SEQ ID NO: INFORMATION 19: (the I) SEQUENCE CHARACTERISTICS (A) Length: 8 amino acids (B) TYPE: amino acid number (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 19: Phe Val Val Thr Val Arg Val Thr1 5 (2) SEQ ID NO: Intelligence 20: (the I) sEQUENCE cHARACTERISTICS (a) length: 69 base pairs (B) type: number of nucleic acid (C) Unit: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: the DNA (oligonucleotide acid) (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 20: ATCCAATCTC TCCACATCTC TATACTATCA TCACC-TATCC TATTITTACC TCACTCACIT CCATTCCAC (2) SEQ ID NO: 21 INFORMATION:

(I)序列特征(A)长度:15碱基对(B)类型:核酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:DNA(寡核苷酸)(XI)序列描述:SEQ ID NO:21:ATCCAATCTC TCCAC(2)SEQ ID NO:22的情报:(I)序列特征(A)长度:15碱基对(B)类型:核酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:DNA(寡核苷酸)(XI)序列描述:SEQ ID NO:22:GTGGAATGGA AGTGA(2)SEQ ID NO:23的情报:(I)序列特征(A)长度:15碱基对(B)类型:核酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:DNA(引物) (I) SEQUENCE CHARACTERISTICS (A) Length: 15 base pairs (B) Type: Number of nucleic acid (C) Unit: single-stranded (D) TOPOLOGY: linear (II) MOLECULE TYPE: the DNA (oligonucleotide) (XI ) sEQUENCE dESCRIPTION: SEQ ID NO: 21: ATCCAATCTC TCCAC (2) SEQ ID NO: 22 INFORMATION: (the I) sEQUENCE cHARACTERISTICS (a) length: 15 base pairs (B) type: number of nucleic acid (C) Unit: single chain (D) tOPOLOGY: linear (II) mOLECULE tYPE: the DNA (oligonucleotide) (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 22: GTGGAATGGA AGTGA (2) SEQ ID NO: 23 INFORMATION: (the I) the sequence wherein (A) length: 15 base pairs (B) tYPE: nucleic acid (C) number of shares: single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: the DNA (primer)

(XI)序列描述:SEQ ID NO:23:ATCTCTATAC TATCA(2)SEQ ID NO:24的情报:(I)序列特征(A)长度:17碱基对(B)类型:氨基酸(C)股数:单链(D)拓扑结构:线性(II)分子类型:肽(XI)序列描述:SEQ ID NO:24:Leu Arg Arg Ala Ser Leu Gly Gly Gly1 5Arg Arg Gln Phe Lys Val Val Thr10 15 (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 23: ATCTCTATAC TATCA (2) SEQ ID NO: 24 INFORMATION: (the I) SEQUENCE CHARACTERISTICS (A) Length: 17 base pairs (B) Type: amino acid number (C) Unit : single-stranded (D) tOPOLOGY: linear (II) mOLECULE tYPE: peptide (XI) sEQUENCE dESCRIPTION: SEQ ID NO: 24: Leu Arg Arg Ala Ser Leu Gly Gly Gly1 5Arg Arg Gln Phe Lys Val Val Thr10 15

Claims (39)

1.一种合成大量底物上多种分子的方法,由以下步骤组成:将所述底物分布在多个反应器中;用不同试剂在每个所述反应器中将所述各种分子的第一部分与所述反应器中的所述底物偶联;将所述底物通过流送管送到混合器再中并混合所述底物;将所述底物通过所述流送管从所述混合器分布到所述反应器中;将所述各种分子的第二部分与所述分子的所述第一部分偶联生成所述底物上的各种分子。 CLAIMS 1. A method of synthesizing a large amount of various molecules on a substrate, comprising the steps of: the substrate is distributed in a plurality of reactors; the various molecules with different reagents in each of the reactor in the the substrate is coupled to a first portion of the reactor; the substrate by a flow line to the mixer and mixed and then the substrate; the substrate through said flow line distribution from the mixer to the reactor; the second portion of the various molecules of the molecule generating a first portion of the coupling of various molecules on the substrate.
2.权利要求1的方法,其中所述再分布步骤。 The method of claim 1, wherein said redistribution step. 包括通过共用管道再分布至少一部分所述底物。 Through a common conduit comprises at least a portion of the redistribution substrate.
3.一种固相载体上平行偶联反应的仪器,包括:母体容器;至少一个与母体容器相连的反应器库,所述反应库含有大量平行进行偶联反应的反应器;所述母体容器和反应器间有大量流送管,所述流送管形成所述反应器和所述母体容器间的流路;将试剂送至所述反应器库的输送系统;和在所述母体容器和所述至少一个反应器库之间协调转移所述固相载体的可编程计算机。 Parallel to the coupling reaction on solid support An apparatus, comprising: a parent container; at least one reactor vessel is connected to the parent library, the library contains a large number of parallel reaction for the coupling reaction reactor; the parent container and a large number of reactors flowline, the flowline forming a flow path between said reactor vessel and said precursor; fed to the reagent delivery system of the reactor bank; and the mother vessel and the coordinated transfer between the at least one reactor programmable computer library of solid phase support.
4.权利要求3的仪器,进一步包括一共用管道,所述共用管道与至少一个所述大量流送管连接。 4. The instrument of claim 3, further comprising a common conduit, said common conduit and at least one of said plurality of flowline connector.
5.权利要求3的仪器,其中所述输送系统进一步包括将试剂由仪器的第一部分输送到仪器第二部分的装置,所述第一部分具有比第二部分高的压力。 5. The instrument of claim 3, wherein the delivery system further comprises a second portion of the instrument means transferring the reagents to the first part of the instrument, the first portion having a higher pressure than the second portion.
6.权利要求3的仪器,其中所述反应器库进一步包括平行搅拌所述大量反应器内含物的装置。 6. The instrument of claim 3, wherein said reactor further comprises means for parallel library agitating the reactor contents is large.
7.权利要求3的仪器,其中所述反应器库进一步包括响应来自所述可编程计算机命令信号的阀装置,用以以平行方式以第一模式将所述试剂输送到选择所述大量反应器中,并用以以第二模式顺序将试剂输送至选择所述大量顺序反应器中。 7. The instrument of claim 3, wherein said reactor further comprises a database responsive valve means from said programmable computer command signals, in a parallel manner to the first mode selecting said plurality of agent delivered to the reactor in a second mode and for sequentially selecting said plurality of reagent delivery into the reactor in order.
8.权利要求3的仪器,其中所述母体容器,所述的应器库,和所述输送系统在所述平行偶联合成反应中密闭隔离大气。 8. The instrument of claim 3, wherein said parent container, the library should be, and the delivery system of the parallel coupling in the synthesis reaction atmosphere hermetic isolation.
9.权利要求4的仪器,进一步包括在所述母体容器和所述大量反应器之间移运所述固相载体的阀,所述阀是在所述母体容器和所述大量反应器间移运期间所述载体通过的唯一阀。 9. The apparatus as claimed in claim 4, further comprising phase shifting said solid support valve operation between the parent and the large reactor vessel, the valve is a shift between the precursor and the large reactor vessel the only support by the valve during operation.
10.权利要求9的仪器,其中所述反应器库进一步包括响应来自所述可编程计算机命令信号的阀装置,用以以平行方式以第一模式将所述试剂输送到选择所述大量反应器中,并用以以第二模式顺序将试剂输送至选择所述大量顺序反应器中。 9 of significant reactor apparatus as claimed in claim 10, wherein said reactor further comprises a database responsive valve means from said programmable computer command signals, in a parallel manner to the first mode selection agent delivered to the in a second mode and for sequentially selecting said plurality of reagent delivery into the reactor in order.
11.一种在小球上平行进行偶联反应的方法,所述方法包括:将所述小球悬浮液由母体容器转移到大量反应器中;在所述大量反应器中在所述小球上进行所述偶联反应;将所述小球由大量反应器转移到所述母体容器;并且混合所述小球。 11. A method of coupling reactions in parallel on a small ball, the method comprising: the bead suspension was transferred from the parent vessel into the large reactor; the reactor at the large number of pellets the coupling reaction is performed on; the pellets were transferred to the reactor by a large parent container; and mixing said pellets.
12.权利要求11的方法,其中所述步骤重复预测定次数。 12. The method of claim 11, wherein said predetermined number of prediction steps are repeated.
13.权利要求11的方法,其中所述转移步骤进一步包括通过共用管道转移至少一部分所述小球步骤。 13. The method of claim 11, wherein said transferring step further comprises the step of at least a portion of the pellets through a common transfer pipe.
14.一种在检测基本上半透明管中液体存在的用于光学检测器的光顺序序列模块,所述光顺序序列模块包括:控制来自所述光检测器发送机的光束通过所述管中心到所述光检测器接收器部分的设置;和抑制由所述光检测器发送机发射的除所述充束外的到达所述检测器接收器部分。 14. A detector tube substantially translucent liquid present in the sequence order of the optical module for an optical detector, the sequence order of the optical module comprising: a control light beam from the light detector transmitter through the tube center to the light detector receiving portion is provided; and inhibition emitted by the light detector reaches the detector transmitter receiver portion of the other outer beam of charged.
15.权利要求14的光顺序序列模块,其中所述控制设置包括一通过光顺序序列模块的针孔开口。 15. The sequence order of the optical module of claim 14, wherein the control arrangement comprises a sequence order of the optical module through the pinhole opening.
16.权利要求15的光顺序序列模块,其中所述光学检测器有两个插头,发送机连在第一插头上,接收器连在第二插头上,所述光学顺序序列模块进一步包括:摩擦接合所述两个插头之间的光顺序序列模块的设置;和放置所述管使所述针孔开口纵向轴和所述管径向轴以90度角交切的设置。 16. The sequence order of the optical module of claim 15, wherein said optical detector has two plugs, a transmitter attached to the first plug, the receiver attached to the second plug, the sequence order of the optical module further comprises: a friction the order of the sequence joining the optical module is provided between two plugs; and the placement of the tube and the longitudinal axis of the pinhole aperture diameter 90 degree angle to the axis of the cross-cut setting.
17.权利要求14的光顺序序列模块,其中所述控制设置进一步包括在发送机和所述光学检测器的接收器之间定位所述管的装置。 17. The sequence order of the optical module of claim 14, wherein said control further comprises means disposed in the tube between the receiver and the transmitter of the optical detector is positioned.
18.输送试剂的输送系统,包括:一个具有第一端口和第二端口的2-端口阀;一个具有一个第一通道和一个第三端口的3-端口阀;响应可编程计算机的设置,用来选择地允所述第三端口与所述第一通道联通;响应所述可编程计算机的设置,用以选择地允许所述第一端口与所述第二端口联通;和将所述第一端口与所述第一通道密封连接而形成管道的设置。 18. The delivery agent delivery system, comprising: a first port and 2-port valve having a second port; a first passage having a valve port and a third port 3-; in response to a programmable computer provided with to selectively allow said third port and the first passage Unicom; provided responsive to said programmable computer, for selectively permitting said first port and said second port Unicom; and the first port and the first sealing passage is formed connecting duct provided.
19.权利要求14的系统,其中所述第三端口与载有试剂的第一管连接,而另一所述第一端口和所述第二端口与载有氩气的第二管连接,其中所述第一管载着所述试剂到所述管道或流出所述管道。 19. The system of claim 14, wherein the third port is connected with the first tube containing a reagent, and the other of the first port and the second port and the second pipe is connected with a carrier gas of argon, wherein the tube carrying the first agent to the outflow duct or the duct.
20.一种在小球上进行偶联反应的可组合合成设备,包括:至少一个反应器库,所述反应器库包括大量反应器和大量单体储器,每个所述储器与一个所述反应器相关联;和一个共用试剂储器,所述共用试剂储器与所述至少一个反应器库连接通过共用管道将共用试剂输送到所述大量反应器中。 20. A coupling reaction in the pellets may be combined synthesizing apparatus, comprising: at least one reactor library, said library comprising a large number of reactors and reactor monomers large reservoirs, each with a reservoir the reactor is associated; and a common reagent reservoir, the common reagent reservoir and the at least one reactor connected to the common library agent delivered to the reactor through a large number of common conduit.
21.一种合成标记分子库的方法,其中库中每一不同分子与一固相载体共价连接,并用一个或多个不同标记物标记,其中每个所述一个或多个不同标记物含有一变化烃区和一分子钩,每个标记物也是共价与所述固相载体连接,所述方法包括:(a)以随机方式在大量反应器间分配载体;(b)将每个反应器中的载体暴露于第一化学结构单元;(c)合并载体;(d)在大量反应器间随机分配载体;(e)将每个反应器中的载体暴露于一个化学结构单元;和(f)重复步骤(a)至(e)至少一次至二十次。 21. A method of synthesizing labeled molecule library, a library in which each distinct molecule with a solid support covalently attached, and one or more labeled with different labels, wherein each of the one or more different markers comprising a change in the hydrocarbon zone and the hook part of each label is covalently connected with said solid support, said method comprising: (a) a random manner between a large number of assigned carriers in a reactor; (b) of each reaction the carrier is exposed to the chemical structure of the first unit; (c) combined vector; (d) randomly assigned among a large number of carriers reactor; (e) each carrier in the reactor is exposed to a chemical structural unit; and ( f) repeating steps (a) to (e) at least one to twenty times.
22.权利要求21的方法,其中所述固相载体是一接头。 22. The method of claim 21, wherein said solid support is a linker.
23.权利要求21的方法,其中所述固相载体是MonobeadTM。 23. The method of claim 21, wherein said solid support is MonobeadTM.
24.权利要求21的方法,其中所述分子通过一接头与所述固相载体连接。 24. The method of claim 21, wherein said molecule to the solid support via a linker.
25.权利要求24的方法,其中接头是可裂解的。 25. The method of claim 24, wherein the linker is cleavable.
26.权利要求21的方法,其中每个所述一个或多个不同标记物含有:一个将每个所述一个或多个不同标记物与所述固相载体连接的可裂解接头;一个分子钩;和一个连接所述接头和所述分子钩的可变长度烃链。 26. The method of claim 21, wherein each of the one or more different labels comprising: each one of said one or more different labels and connected to the solid support cleavable linker; a molecule hook ; and attached to the linker and a molecular hook of the variable-length hydrocarbon chain.
27.权利要求21的方法,其中所述每个一个或多个不同标记物具有下式:其中n是1至10的整数,X是可裂解接头,R是一分子钩。 27. The method of claim 21, wherein each of said one or more different labels having the formula: wherein n is an integer from 1 to 10, X is a cleavable linker, R is a part of the hook.
28.权利要求27的方法,其中X是光裂解接头。 28. The method of claim 27, wherein X is a photocleavable linker.
29.权利要求21的方法,其中所述分子钩选自生物素,可活化基团和高缔合肽组成的组。 29. The method of claim 21, wherein the biotin molecule from the VGH, activatable group and the high group of associated peptides.
30.一种筛选权利要求25标记分子库的方法,其中所述分子从所述固相载体上裂解,并与受体在有利于将配体结合到所述受体上的条件下温育。 Method 25 30. A labeled molecule library screening claim, wherein the molecule is cleaved from the solid support and the receptor facilitates the binding ligand to the receptor is incubated on the condition.
31.权利要求30的方法,其中所述分子是肽,所述固相载体是直径为大约50至大约500μm的小球,所述可裂解接头是一可裂解接头的混合物,而且只有所述小球上的一部分所述分子在所述温育步骤前裂解。 31. The method of claim 30, wherein said molecule is a peptide, the solid support having a diameter of about 50 to about 500μm pellets, the cleavable linker is a cleavable linker mixture, and only a small the ball portion of the molecule on the cleaved prior to said incubation step.
32.一种合成含有大量不同成员合成肽库的方法,每一成员包括由与小球连接的氨基酸单体序列合成的肽,所述小球也连接一个或多个寡核苷酸识别标记物,识别所述肽的单体顺序,其中所述氨基酸单体用Fmoc保护并用哌啶去除Fmoc保护基,改进措施包括用5-15%哌啶处理5-60分钟或用15-30%哌啶处理1至30分钟有效去除Fmoc。 32. A method of synthesizing synthetic peptides containing a large number of different members of the library, each member comprising a synthetic peptide with the amino acid sequence of a monomer of the ball connection, the ball is also connected to one or more oligonucleotide identification marker identifying the peptide monomer sequence, wherein the amino acid monomers protected with Fmoc and removing Fmoc protecting group with piperidine, the improvement comprising 5-15% piperidine was treated with 5 to 60 minutes or 15 to 30% piperidine 1-30 minutes treatment effectively remove Fmoc.
33.权利要求32的改进措施,其中所述小球具有大约10μm直径,并且由十二烷基胺接头衍生的大孔苯乙烯-二乙烯苯共聚物组成。 33. Improvement as claimed in claim 32, wherein said beads having a diameter of about 10μm, and by the joint dodecylamine derivatized macroporous styrene - divinylbenzene copolymer.
34.权利要求32的改进措施,其中所述小球是MonobeaduTM。 34. Improvement as claimed in claim 32, wherein the pellets are MonobeaduTM.
35.权利要求32的改进措施,其中所述氨基酸单体具有侧链(tBu)侧链保护基,用TFA去除所述(tBu)侧链保护基,且所述寡核苷酸标记物包括7-去氮杂-2′-脱氧腺苷。 35. Improvement as claimed in claim 32, wherein said monomer having the amino acid side chains (tBu) side chain protecting group, removing the (tBu) side chain protecting groups with TFA, and the oligonucleotide comprises a label 7 - deaza-2'-deoxyadenosine.
36.一种检测权利要求26方法中存在一种或几种不同标记物的方法,该方法包括:从所述固相载体上裂解一个或多个不同标记物;固定一个或多个不同标记物;在所述一个或多个不同标记物上偶联一可扩增的,可检测的基团到所述分子钩上;扩增该可扩增,可检测基团;并且检测所述可扩增可检测基团的存在,其中存在可扩增可检测基团说明存在一个或多个不同标记物。 The method of one or more of several different markers in the presence of 26 36. A method as claimed in claim detected, the method comprising: said solid support from the cleaved one or more different labels; one or more fixed different markers ; on the one or more different labels can be conjugated to an amplification of the detectable group on the hook molecule; amplifying the amplifiable, detectable group; and detecting the expansive by the presence of the detectable group, wherein the presence of amplification detectable group described the presence of one or more different labels.
37.权利要求35的方法,其中所述可扩增,可检测基团含有能与所述分子钩结合的寡核苷酸序列。 37. The method of claim 35, wherein said amplifiable, detectable group comprises an oligonucleotide sequence capable of binding the molecule hook.
38.一种测定连接到权利要求21标记分子库中固相载体的分子的合成序列的方法,该方法包括各个测定所述一个或多个不同的与所述固相载体连接的标记物的存在,所述一个或多个不同标记物的存在或不存在表明是否发生了所述分子合成顺序中特定的合成步骤。 38. A method of measuring the phase vector of claim connected to synthetic sequences of molecular markers molecule libraries of the solid in claim 21, the method comprising determining the one or more respective different labels coupled to the solid support in the presence of the presence of one or more different labels or absence indicate whether the sequence of the synthetic molecule specific synthetic steps occur.
39.权利要求38的方法,其中所述一个或多个不同标记物的所述各个检测包括:根据其结构,物理分离所述一个或多个不同标记物,所用分离形式来自所述一个或多个不同标记物;固定所述一个或多个不同标记物,以保持所述分离形式;用寡核苷酸序列处理所述标记物,所用寡核苷酸序列选择地结合所述一个或多个不同标记物;扩增所述寡核苷酸序列;检测所述寡核苷酸序列的存在,其中所述寡核苷酸序列的存在与不存在表示出所述一个或多个不同标记物的存在与不存在;通过在所述分离形式中所述一个或多个不同标记物的相对位置识别所述一个和多个不同标记物的存在;和将所述一个或多个不同标记物的存在与不存在与所述分子合成顺序中特定合成步骤的发生联系起来。 39. The method of claim 38, wherein the one or more different labels for each detecting comprises: according to their structures, the one or more physical separation of the different markers used in the form isolated from the one or more different labels; fixing said one or more different labels, to hold the isolated form; a nucleotide sequence oligonucleotide treated with the marker, the oligonucleotide sequences bind selectively with the one or more different labels; amplifying the oligonucleotide sequence; detecting the presence of an oligonucleotide sequence, wherein the presence of said oligonucleotide sequence is shown with the one or more different labels absence and the presence of one or more different markers; the presence and absence; by the presence in the isolated form or in a plurality of relative positions of the identification of the different markers and a plurality of different markers linked to the occurrence of a particular synthetic sequence molecule synthetic steps and does not exist.
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