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CN117384089A - A probe that can be used for reversible chemical modification of protein cysteine residues and its preparation method - Google Patents

A probe that can be used for reversible chemical modification of protein cysteine residues and its preparation method Download PDF

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CN117384089A
CN117384089A CN202311301203.XA CN202311301203A CN117384089A CN 117384089 A CN117384089 A CN 117384089A CN 202311301203 A CN202311301203 A CN 202311301203A CN 117384089 A CN117384089 A CN 117384089A
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protein
reversible
chemical modification
probe
modification
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万川
胡俊青
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Shenzhen Technology University
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Abstract

The invention provides a probe for reversible chemical modification of protein cysteine residues, which has a structural general formula shown in a formula III:the invention also provides a reversible modification method of the protein, the protein to be modified is added, and the chemical modification site is protein cysteine residue; the charge of probe III for chemical modification of cysteine residues of proteins is 0.1 to 200 equivalents of protein; the modification can be removed using a thiol-containing reducing agent. According to the chemical selective modification technology and the application requirement of reversible modification of protein cysteine residue, the invention provides a high-efficiency selective reversible chemical modification method of protein cysteine residue with halopyridine salt as an active functional group.

Description

一种可用于蛋白质半胱氨酸残基可逆化学修饰的探针及其制 备方法A probe that can be used for reversible chemical modification of protein cysteine residues and its preparation method

技术领域Technical Field

本发明属于生物化学领域，涉及一种蛋白质修饰的方法，具体来说是一种可用于蛋白质半胱氨酸残基可逆化学修饰的探针及其制备方法，The present invention belongs to the field of biochemistry and relates to a method for protein modification, in particular to a probe that can be used for reversible chemical modification of protein cysteine residues and a preparation method thereof.

背景技术Background Art

对蛋白质的可逆化学修饰在化学生物学和药物科学研究中起着重要的作用。例如，抗体-药物偶联物发挥作用的过程就包括抗体蛋白和药物之间链接的断裂以释放细胞毒性药物，达到杀伤靶标癌细胞的作用；对蛋白质化学修饰标记物的时空控制的可逆性释放，是现代化学生物学研究中重要的手段和工具。因此，对蛋白质的可逆化学修饰方法的研究开发对于此类生物学和药学应用至关重要，是研究蛋白质立体结构与生理功能的重要手段，也是定向改造蛋白质性质的一种方法，在蛋白质工程和组学研究中有着广泛的应用前景。Reversible chemical modification of proteins plays an important role in chemical biology and pharmaceutical science research. For example, the process of antibody-drug conjugates includes the breaking of the link between the antibody protein and the drug to release the cytotoxic drug, thereby killing the target cancer cells; the spatiotemporal controlled reversible release of protein chemical modification markers is an important means and tool in modern chemical biology research. Therefore, the research and development of reversible chemical modification methods for proteins is crucial for such biological and pharmaceutical applications. It is an important means to study the three-dimensional structure and physiological function of proteins, and a method for the directional modification of protein properties. It has broad application prospects in protein engineering and omics research.

芳香亲核取代反应在近年来常被用于蛋白质的亲核氨基酸残基的化学修饰。因为此类反应通常具有高反应性和高化学选择性等优点。吡啶盐类化合物因为自身的强缺电子性，使其具有优越的亲核反应性。因此，卤代吡啶盐类化合物是潜在的优秀芳香亲核取代反应底物。又因为烷硫基吡啶盐也具有一定的亲核反应性，所以卤代吡啶盐与半胱氨酸的巯基侧链的反应产物是可以被游离巯基进一步取代的半活性化合物，即可以发生硫醇交换的可逆反应。通过合理调节吡啶盐底物的反应活性，可以很好的控制吡啶盐底物对蛋白质半胱氨酸的化学修饰以及可逆反应活性。另外，吡啶盐类化合物通常具有良好的生物相容性、较低的生物毒性和更高的内吸性等优点。因此卤代吡啶盐类化合物在蛋白质半胱氨酸残基可逆化学修饰的应用上具有广阔的前景。Aromatic nucleophilic substitution reactions have been frequently used in recent years for the chemical modification of nucleophilic amino acid residues of proteins. This is because such reactions usually have the advantages of high reactivity and high chemical selectivity. Pyridinium salt compounds have excellent nucleophilic reactivity due to their strong electron deficiency. Therefore, halogenated pyridinium salt compounds are potential excellent aromatic nucleophilic substitution reaction substrates. Because alkylthiopyridinium salts also have certain nucleophilic reactivity, the reaction product of halogenated pyridinium salts with the thiol side chain of cysteine is a semi-active compound that can be further replaced by free thiol groups, that is, a reversible reaction of thiol exchange can occur. By rationally regulating the reactivity of pyridinium salt substrates, the chemical modification of protein cysteine and the reversible reaction activity of pyridinium salt substrates can be well controlled. In addition, pyridinium salt compounds usually have the advantages of good biocompatibility, low biotoxicity and higher systemicity. Therefore, halogenated pyridinium salt compounds have broad prospects for the application of reversible chemical modification of protein cysteine residues.

发明内容Summary of the invention

针对现有技术中的上述技术问题，本发明提供了一种可用于蛋白质半胱氨酸残基可逆化学修饰的探针及其制备方法，所述的这种可用于蛋白质半胱氨酸残基可逆化学修饰的探针及其制备方法要解决现有技术中蛋白质可逆修饰反应的可逆性不强且难以调节的缺点。In view of the above technical problems in the prior art, the present invention provides a probe that can be used for the reversible chemical modification of cysteine residues in proteins and a method for preparing the same. The probe that can be used for the reversible chemical modification of cysteine residues in proteins and a method for preparing the same are intended to overcome the shortcomings of the prior art that the reversible modification reaction of proteins is not highly reversible and is difficult to regulate.

本发明提供了一种可用于蛋白质半胱氨酸残基可逆化学修饰的探针，具有如式III所示的结构通式：The present invention provides a probe that can be used for reversible chemical modification of cysteine residues in proteins, having a general structural formula as shown in Formula III:

其中：R1、R2分别选自：氢、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、C1-C6烷胺基、C1-C6酰胺基、C1-C6卤代烷氧基、C2-C6烯基、C2-C6卤代烯基、C2-C6炔基、C2-C6卤代炔基、羟基、氨基、C3-C6环烷基、苯基、卤素、氰基、羧基或者醛基；Wherein: R 1 and R 2 are independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, amino, C3-C6 cycloalkyl, phenyl, halogen, cyano, carboxyl or aldehyde;

所述的卤素是氟、氯、溴或碘；The halogen is fluorine, chlorine, bromine or iodine;

所述烷基、烯基或炔基为直链的或支链的烷基；烷基本身或作为其它取代基的部分选自甲基、乙基、丙基、丁基、戊基、己基或者其异构体，其异构体选自异丙基、异丁基、仲丁基、叔丁基、异戊基或叔戊基；The alkyl, alkenyl or alkynyl group is a straight-chain or branched alkyl group; the alkyl group itself or as a part of other substituents is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl or isomers thereof, and the isomers thereof are selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or tert-pentyl;

所述卤代烷基基团选自含一个或多个相同或不同的卤素原子的基团，所述卤代烷基选自CH2Cl、CHCl2、CCl3、CH2F、CHF2、CF3、CF3CH2、CH3CF2、CF3CF2或CCl3CCl2；The haloalkyl group is selected from a group containing one or more halogen atoms which are the same or different, and the haloalkyl group is selected from CH 2 Cl, CHCl 2 , CCl 3 , CH 2 F, CHF 2 , CF 3 , CF 3 CH 2 , CH 3 CF 2 , CF 3 CF 2 or CCl 3 CCl 2 ;

所述环烷基本身或作为其它取代基的部分选自环丙基、环丁基、环戊基或环己基；The cycloalkyl group itself or as part of other substituents is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;

所述烯基本身或作为其它取代基的部分选自乙烯基、烯丙基、1-丙烯基、丁烯-2-基、丁烯-3-基、戊烯-1-基、戊烯-3-基、己烯-1-基或4-甲基-3-戊烯基；The alkenyl group itself or as part of other substituents is selected from vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten-3-yl, hexen-1-yl or 4-methyl-3-pentenyl;

所述炔基本身或作为其它取代基的部分选自乙炔基、丙炔-1-基、丙炔-2-基、丁炔-1-基、丁炔-2-基、1-甲基-2-丁炔基、己炔-1-基或1-乙基-2-丁炔基。The alkynyl group itself or as part of another substituent is selected from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl-2-butynyl, hexyn-1-yl or 1-ethyl-2-butynyl.

X选自：烷基酸根、取代烷基酸根、卤素或者其含氧酸根、磷酸根、硫酸根、磺酸根或者硼酸根；X is selected from: alkyl acid radical, substituted alkyl acid radical, halogen or its oxygen-containing acid radical, phosphate, sulfate, sulfonate or borate;

所述烷基酸根、取代烷基酸根选自C1-C6烷基酸根或者C1-C6卤代烷基酸根；The alkyl acid radical and substituted alkyl acid radical are selected from C1-C6 alkyl acid radical or C1-C6 halogenated alkyl acid radical;

所述卤素或者其含氧酸根选自氟、氯、溴或碘、次氯酸根、亚氯酸根、氯酸根、高氯酸根、次溴酸根、亚溴酸根、溴酸根、高溴酸根、次碘酸根、亚碘酸根、碘酸根或者高碘酸根；The halogen or its oxygen-containing acid radical is selected from fluorine, chlorine, bromine or iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromite, bromate, perbromate, hypoiodite, iodite, iodate or periodate;

所述磷酸根选自磷酸一氢根、磷酸二氢根、焦磷酸根、偏磷酸根、次磷酸根、亚磷酸根、多聚磷酸根、磷酸根或者六氟磷酸根；The phosphate is selected from monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, phosphate or hexafluorophosphate;

所述硫酸根选自硫负离子、硫酸氢根、硫酸根、亚硫酸氢根、亚硫酸根、焦硫酸根、连二硫酸根、硫代硫酸根、连二亚硫酸根或者过硫酸根；The sulfate group is selected from sulfide anion, bisulfate, sulfate, bisulfite, sulfite, pyrosulfate, dithionate, thiosulfate, dithionite or persulfate;

所述磺酸根选自三氟甲磺酸根、甲磺酸根、苯磺酸根或者对-甲基苯磺酸根；The sulfonate group is selected from trifluoromethanesulfonate, methanesulfonate, benzenesulfonate or p-toluenesulfonate;

所述硼酸根选自硼酸根或者四氟硼酸根；The borate is selected from borate or tetrafluoroborate;

Y选自：卤素。Y is selected from: halogen.

所述的卤素是氟、氯、溴或碘。The halogen is fluorine, chlorine, bromine or iodine.

本发明的可用于蛋白质半胱氨酸残基可逆化学修饰的探针III的合成方法如下：The synthesis method of the probe III that can be used for reversible chemical modification of protein cysteine residues of the present invention is as follows:

其中，取代基如前所述R1优选自：苯基、取代苯基、甲基；R2优选自：甲基；X优选自：碘、三氟甲磺酸根；Y优选自：氧、硫。Wherein, the substituents are as described above, R1 is preferably selected from: phenyl, substituted phenyl, methyl; R2 is preferably selected from: methyl; X is preferably selected from: iodine, trifluoromethanesulfonate; Y is preferably selected from: oxygen, sulfur.

本发明还公开了上述的可用于蛋白质半胱氨酸残基化学修饰的探针的制备方法，包括以下步骤：The present invention also discloses a method for preparing the above-mentioned probe that can be used for chemical modification of protein cysteine residues, comprising the following steps:

在一个反应容器中加入化合物Add the compound to a reaction vessel

和烷基化试剂II R2X，所述的烷基化试剂II选自：碘甲烷、三氟甲磺酸甲酯、三氟甲磺酸乙酯、苯磺酸甲酯或者三甲基氧鎓四氟硼酸盐，反应溶剂选自：二氯甲烷、甲苯、乙腈、乙醚或者四氢呋喃，得到可用于蛋白质半胱氨酸残基可逆化学修饰的探针and an alkylating agent II R 2 X, wherein the alkylating agent II is selected from: iodomethane, methyl trifluoromethanesulfonate, ethyl trifluoromethanesulfonate, methyl benzenesulfonate or trimethyloxonium tetrafluoroborate, and the reaction solvent is selected from: dichloromethane, toluene, acetonitrile, ether or tetrahydrofuran, to obtain a probe that can be used for reversible chemical modification of protein cysteine residues

，产物以沉淀的形式直接析出，或添加反应溶剂等体积的乙醚促进产物的析出。The product is directly precipitated in the form of a precipitate, or an equal volume of ether is added to the reaction solvent to promote the precipitation of the product.

本发明还提供了上述的可用于蛋白质半胱氨酸残基化学修饰的探针的含卤素的吡啶盐类化合物或者其衍生物。其反应通式如下：The present invention also provides the above-mentioned halogen-containing pyridinium salt compound or its derivative which can be used as a probe for chemical modification of protein cysteine residues. The general reaction formula is as follows:

本发明还提供了上述的可用于蛋白质半胱氨酸残基化学修饰的探针或者其中间体在蛋白质半胱氨酸可逆修饰上的用途。The present invention also provides the use of the above-mentioned probe for chemical modification of protein cysteine residues or its intermediate in the reversible modification of protein cysteine.

本发明还提供了一种蛋白质的可逆修饰方法，加入需要修饰的蛋白质，所述的化学修饰位点为蛋白质半胱氨酸残基；可用于蛋白质半胱氨酸残基可逆化学修饰的探针III投料为蛋白质的0.1当量至200当量；使用含巯基的还原剂可以移除该修饰。其反应通式如下：The present invention also provides a method for reversible modification of proteins, wherein a protein to be modified is added, wherein the chemical modification site is a cysteine residue of the protein; the probe III that can be used for reversible chemical modification of the cysteine residue of the protein is added in an amount of 0.1 equivalent to 200 equivalents of the protein; and the modification can be removed by using a thiol-containing reducing agent. The general reaction formula is as follows:

进一步的，反应溶剂为水或极性有机溶剂：所述的溶剂选自水、乙腈、甲醇、乙醇、异丙醇、叔丁醇、乙二醇、甘油、三氟乙醇、六氟异丙醇、二甲基亚砜、N,N-二甲基甲酰胺中的任意一种或者它们任意两种的混合溶剂。Furthermore, the reaction solvent is water or a polar organic solvent: the solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide, N,N-dimethylformamide, or a mixed solvent of any two of them.

进一步的，所述的反应时间为0.1小时至100小时。Furthermore, the reaction time is 0.1 hour to 100 hours.

进一步的，所述的反应温度为-20至50摄氏度。Furthermore, the reaction temperature is between -20 and 50 degrees Celsius.

进一步的，所述的含巯基的还原剂选自丙硫醇、巯基乙醇、2-巯基吡啶、半胱氨酸、谷胱甘肽中的任意一种。Furthermore, the thiol-containing reducing agent is selected from any one of propylmercaptan, mercaptoethanol, 2-mercaptopyridine, cysteine, and glutathione.

本发明对可用于蛋白质半胱氨酸残基可逆化学修饰的探针III进行了合成研究，并对可用于蛋白质半胱氨酸残基可逆化学修饰的探针III进行了化学生物学活性的探索。具体的，本发明提改了一种含有卤素的吡啶盐类衍生物，可用于可逆修饰蛋白质半胱氨酸(Cys，C)残基。The present invention has conducted a synthetic study on a probe III that can be used for reversible chemical modification of a protein cysteine residue, and has explored the chemical biological activity of the probe III that can be used for reversible chemical modification of a protein cysteine residue. Specifically, the present invention has provided a pyridinium salt derivative containing a halogen that can be used for reversible modification of a protein cysteine (Cys, C) residue.

本发明公开了上述化合物的结构通式、合成方法与用作蛋白质半胱氨酸残基可逆化学修饰的探针的用途，其与化学生物学上可接受的探针以及与商品化赖氨酸探针、酪氨酸探针和半胱氨酸探针等组合使用在多肽、蛋白质、细胞水平上的修饰蛋白质组学和生物医药上的用途和制备方法。The present invention discloses the general structural formula, synthesis method and use of the above-mentioned compound as a probe for reversible chemical modification of protein cysteine residues, and the use and preparation method of the compound in combination with chemically biologically acceptable probes and commercially available lysine probes, tyrosine probes and cysteine probes in modified proteomics and biomedicine at the polypeptide, protein and cell levels.

本发明和已有技术相比，其技术进步是显著的。根据蛋白质半胱氨酸残基的化学选择性和可逆修饰技术和应用的需求，本发明提供了一种以含卤素的吡啶盐为活性官能团的蛋白质半胱氨酸残基的高效选择性和可逆的化学修饰方法。Compared with the prior art, the present invention has significant technical progress. According to the requirements of chemical selectivity and reversible modification technology and application of protein cysteine residues, the present invention provides a highly efficient, selective and reversible chemical modification method of protein cysteine residues using halogen-containing pyridinium salt as an active functional group.

附图说明：Description of the drawings:

图1是可用于蛋白质半胱氨酸残基化学修饰的探针III与多肽的反应。FIG1 shows the reaction between probe III, which can be used for chemical modification of cysteine residues in proteins, and a polypeptide.

图2是可用于蛋白质半胱氨酸残基化学修饰的探针III与多肽的可逆反应。FIG. 2 is a diagram showing the reversible reaction of probe III, which can be used for chemical modification of cysteine residues in proteins, with a polypeptide.

图3是可用于蛋白质半胱氨酸残基化学修饰的探针III与蛋白质的可逆反应的凝胶内荧光分析。FIG. 3 is an in-gel fluorescence analysis of the reversible reaction between probe III, which can be used for chemical modification of cysteine residues of proteins, and proteins.

图4是可用于蛋白质半胱氨酸残基化学修饰的探针III与蛋白质的可逆反应的一级质谱。FIG. 4 is a primary mass spectrum of the reversible reaction of probe III, which can be used for chemical modification of cysteine residues of proteins, with proteins.

图5是可用于单克隆抗体半胱氨酸残基化学修饰的探针III与蛋白质的可逆反应的一级质谱。FIG. 5 is a primary mass spectrum of the reversible reaction of probe III, which can be used for chemical modification of cysteine residues of monoclonal antibodies, with proteins.

具体实施方式：Specific implementation method:

本发明通过特定制备和化学生物学实施例更加具体说明可用于蛋白质半胱氨酸残基可逆化学修饰的探针III的合成与生物应用，所述实施例仅用于具体说明本发明而非限制本发明，尤其是生物应用仅是举例说明，而非限制本专利，具体实施方式如下：The present invention further specifically illustrates the synthesis and biological application of probe III that can be used for reversible chemical modification of protein cysteine residues through specific preparation and chemical biology examples. The examples are only used to specifically illustrate the present invention but not to limit the present invention. In particular, the biological application is only an example and not to limit the present patent. The specific implementation methods are as follows:

实施例1：化合物Ⅲ的制备：Example 1: Preparation of Compound III:

在250毫升双口圆底烧瓶中加入1.13克2氯-吡啶I和100毫升反应溶剂二氯甲烷。冰浴下，滴加2.8克碘甲烷。有沉淀析出，减压抽滤，用少量二氯甲烷洗涤沉淀，收集沉淀即为产物。产物为黄色粉末1.6克，收率65％。该化合物的核磁数据如下：1H NMR(400MHz,D2O)δ8.92(dd,J＝6.1,1.7Hz,1H),8.51(dd,J＝8.1,1.6Hz,1H),7.96(td,J＝7.9,1.7Hz,1H),7.87(ddd,J＝7.8,6.1,1.5Hz,1H),4.38(s,3H).Add 1.13 g of 2-chloro-pyridine I and 100 ml of dichloromethane as the reaction solvent to a 250 ml double-necked round-bottom flask. Add 2.8 g of iodomethane dropwise under ice bath. A precipitate is precipitated, and it is filtered under reduced pressure, washed with a small amount of dichloromethane, and the precipitate is collected as the product. The product is 1.6 g of yellow powder, with a yield of 65%. The NMR data of the compound are as follows: 1 H NMR (400 MHz, D 2 O) δ 8.92 (dd, J = 6.1, 1.7 Hz, 1H), 8.51 (dd, J = 8.1, 1.6 Hz, 1H), 7.96 (td, J = 7.9, 1.7 Hz, 1H), 7.87 (ddd, J = 7.8, 6.1, 1.5 Hz, 1H), 4.38 (s, 3H).

同类的化合物合成及表征数据：Synthesis and characterization data of similar compounds:

黄色粉末，收率85％.1H NMR(400MHz,D2O)δ8.91(dd,J＝6.1,1.7Hz,1H),8.51(dd,J＝8.1,1.6Hz,1H),7.96(td,J＝7.9,1.7Hz,1H),7.87(ddd,J＝7.8,6.1,1.5Hz,1H),4.37(s,3H). Yellow powder, yield 85%. 1 H NMR (400 MHz, D 2 O) δ8.91 (dd, J=6.1, 1.7 Hz, 1H), 8.51 (dd, J=8.1, 1.6 Hz, 1H), 7.96 (td, J=7.9, 1.7 Hz, 1H), 7.87 (ddd, J=7.8, 6.1, 1.5 Hz, 1H), 4.37 (s, 3H).

棕色粉末，收率73％.1H NMR(400MHz,D2O)δ8.91(dd,J＝6.2,1.7Hz,1H),8.51(dd,J＝8.1,1.6Hz,1H),7.96(td,J＝7.9,1.7Hz,1H),7.87(ddd,J＝7.8,6.1,1.5Hz,1H),4.37(s,3H). Brown powder, yield 73%. 1 H NMR (400 MHz, D 2 O) δ8.91 (dd, J=6.2, 1.7 Hz, 1H), 8.51 (dd, J=8.1, 1.6 Hz, 1H), 7.96 (td, J=7.9, 1.7 Hz, 1H), 7.87 (ddd, J=7.8, 6.1, 1.5 Hz, 1H), 4.37 (s, 3H).

白色粉末，收率82％.1H NMR(400MHz,D2O)δ9.04(d,J＝1.8Hz,1H),8.73(d,J＝6.1Hz,1H),8.64(dd,J＝8.3,1.9Hz,1H),7.93-7.85(m,1H),4.31(s,3H). White powder, yield 82%. 1 H NMR (400 MHz, D 2 O) δ 9.04 (d, J = 1.8 Hz, 1H), 8.73 (d, J = 6.1 Hz, 1H), 8.64 (dd, J = 8.3, 1.9 Hz, 1H), 7.93-7.85 (m, 1H), 4.31 (s, 3H).

棕色粉末，收率87％.1H NMR(400MHz,D2O)δ8.59-8.53(m,2H),8.23-8.17(m,2H),4.24(s,3H). Brown powder, yield 87%. 1 H NMR (400 MHz, D 2 O) δ 8.59-8.53 (m, 2H), 8.23-8.17 (m, 2H), 4.24 (s, 3H).

白色粉末，收率89％.1H NMR(400MHz,D2O)δ8.57-8.51(m,2H),8.21-8.15(m,2H),4.23(s,3H). White powder, yield 89%. 1 H NMR (400 MHz, D 2 O) δ 8.57-8.51 (m, 2H), 8.21-8.15 (m, 2H), 4.23 (s, 3H).

棕色粉末，收率84％.1H NMR(400MHz,D2O)δ8.42-8.36(m,2H),8.31(d,J＝6.7Hz,2H),4.18(s,3H). Brown powder, yield 84%. 1 H NMR (400 MHz, D 2 O) δ 8.42-8.36 (m, 2H), 8.31 (d, J = 6.7 Hz, 2H), 4.18 (s, 3H).

白色粉末，收率90％.1H NMR(400MHz,D2O)δ8.39(d,J＝6.8Hz,2H),8.34-8.28(m,2H),4.18(s,3H). White powder, yield 90%. 1 H NMR (400 MHz, D 2 O) δ 8.39 (d, J = 6.8 Hz, 2H), 8.34-8.28 (m, 2H), 4.18 (s, 3H).

白色粉末，收率87％.1H NMR(400MHz,D2O)δ7.63(d,J＝7.1Hz,1H),7.24(d,J＝2.1Hz,1H),6.97(dd,J＝7.2,2.1Hz,1H),3.66(s,3H). White powder, yield 87%. 1 H NMR (400 MHz, D 2 O) δ7.63 (d, J=7.1 Hz, 1H), 7.24 (d, J=2.1 Hz, 1H), 6.97 (dd, J=7.2, 2.1 Hz, 1H), 3.66 (s, 3H).

原料由2-氨基-4-氯吡啶与4-炔基戊酸缩合反应制备得来。白色粉末，82％.1H NMR(400MHz,D2O)δ8.50(d,J＝6.9Hz,1H),8.28(d,J＝2.3Hz,1H),7.71(dd,J＝6.9,2.4Hz,1H),4.09(s,3H),2.81(t,J＝6.8Hz,2H),2.53(td,J＝6.8,2.7Hz,2H),2.32(t,J＝2.6Hz,1H). The raw material was prepared by condensation reaction of 2-amino-4-chloropyridine and 4-alkynylpentanoic acid. White powder, 82%. 1 H NMR (400MHz, D 2 O) δ8.50 (d, J = 6.9 Hz, 1H), 8.28 (d, J = 2.3 Hz, 1H), 7.71 (dd, J = 6.9, 2.4 Hz, 1H), 4.09 (s, 3H), 2.81 (t, J = 6.8 Hz, 2H), 2.53 (td, J = 6.8, 2.7 Hz, 2H), 2.32 (t, J = 2.6 Hz, 1H).

原料由2-氨基-4-溴吡啶与4-炔基戊酸缩合反应制备得来。白色粉末，收率80％.1H NMR(400MHz,DMSO-d6)δ8.70(d,J＝6.9Hz,1H),8.52(d,J＝2.2Hz,1H),8.07(d,J＝6.8Hz,1H),4.09(s,3H),2.90(t,J＝2.6Hz,1H),2.83(t,J＝7.2Hz,2H),2.54(d,J＝2.6Hz,1H),2.50(s,1H). The raw material was prepared by condensation reaction of 2-amino-4-bromopyridine and 4-alkynylpentanoic acid. White powder, yield 80%. 1 H NMR (400MHz, DMSO-d 6 )δ8.70(d, J＝6.9Hz,1H),8.52(d, J＝2.2Hz,1H),8.07(d, J＝6.8Hz,1H),4.09(s,3H),2.90(t, J＝2.6Hz,1H),2.83(t, J＝7.2Hz,2H),2.54(d, J＝2.6Hz,1H),2.50(s,1H).

白色粉末，79％.1H NMR(400MHz,D2O)δ8.92(d,J＝6.6Hz,1H),8.87(d,J＝2.3Hz,1H),8.55(dd,J＝6.6,2.3Hz,1H),4.46(s,3H). White powder, 79%. 1 H NMR (400 MHz, D 2 O) δ8.92 (d, J=6.6 Hz, 1H), 8.87 (d, J=2.3 Hz, 1H), 8.55 (dd, J=6.6, 2.3 Hz, 1H), 4.46 (s, 3H).

白色粉末，收率75％.1H NMR(400MHz,D2O)δ8.75(d,J＝2.4Hz,1H),8.70(d,J＝6.6Hz,1H),8.36(dd,J＝6.7,2.4Hz,1H),4.39(s,3H),3.99(s,3H). White powder, yield 75%. 1 H NMR (400 MHz, D 2 O) δ 8.75 (d, J = 2.4 Hz, 1H), 8.70 (d, J = 6.6 Hz, 1H), 8.36 (dd, J = 6.7, 2.4 Hz, 1H), 4.39 (s, 3H), 3.99 (s, 3H).

白色粉末，收率80％.1H NMR(400MHz,D2O)δ9.13(d,J＝6.4Hz,1H),8.73(d,J＝1.9Hz,1H),8.29(dd,J＝6.5,1.9Hz,1H),4.41(s,3H). White powder, yield 80%. 1 H NMR (400 MHz, D 2 O) δ 9.13 (d, J = 6.4 Hz, 1H), 8.73 (d, J = 1.9 Hz, 1H), 8.29 (dd, J = 6.5, 1.9 Hz, 1H), 4.41 (s, 3H).

黄色油状物，收率77％.1H NMR(400MHz,D2O)δ8.74(d,J＝6.5Hz,1H),8.14(d,J＝1.9Hz,1H),7.87(dd,J＝6.6,1.9Hz,1H),4.38(s,1H),4.24(s,3H). Yellow oil, yield 77%. 1 H NMR (400 MHz, D 2 O) δ 8.74 (d, J = 6.5 Hz, 1H), 8.14 (d, J = 1.9 Hz, 1H), 7.87 (dd, J = 6.6, 1.9 Hz, 1H), 4.38 (s, 1H), 4.24 (s, 3H).

黄色油状物，收率79％.1H NMR(400MHz,D2O)δ8.86(d,J＝6.5Hz,1H),8.36(d,J＝1.9Hz,1H),7.97(dd,J＝6.7,1.9Hz,1H),6.06(s,1H),4.33(s,3H). Yellow oil, yield 79%. 1 H NMR (400 MHz, D 2 O) δ 8.86 (d, J = 6.5 Hz, 1H), 8.36 (d, J = 1.9 Hz, 1H), 7.97 (dd, J = 6.7, 1.9 Hz, 1H), 6.06 (s, 1H), 4.33 (s, 3H).

白色粉末，87％.1H NMR(400MHz,D2O)δ9.25(d,J＝2.1Hz,1H),8.70(dd,J＝8.6,2.2Hz,1H),8.22(d,J＝8.6Hz,1H),4.38(s,3H),2.89(s,3H).原料由2-氨基-4-氯吡啶与乙酸缩合反应制备得来。白色粉末，85％.1HNMR(400MHz,D2O)δ8.47(d,J＝6.9Hz,1H),8.30(d,J＝2.4Hz,1H),7.69(dd,J＝6.9,2.4Hz,1H),4.08(s,3H),2.28(s,3H). White powder, 87%. 1 H NMR (400 MHz, D 2 O) δ9.25 (d, J=2.1 Hz, 1H), 8.70 (dd, J=8.6, 2.2 Hz, 1H), 8.22 (d, J=8.6 Hz, 1H), 4.38 (s, 3H), 2.89 (s, 3H). The raw material is prepared by condensation reaction of 2-amino-4-chloropyridine and acetic acid. White powder, 85%. 1 HNMR (400MHz, D 2 O) δ8.47 (d, J＝6.9Hz, 1H), 8.30 (d, J＝2.4Hz, 1H), 7.69 (dd, J＝6.9, 2.4Hz, 1H), 4.08 (s, 3H), 2.28 (s, 3H).

白色粉末，89％.1H NMR(400MHz,D2O)δ8.56(d,J＝2.9Hz,1H),8.03(dd,J＝9.3,2.9Hz,1H),7.97(d,J＝9.3Hz,1H),4.28(s,3H),3.94(s,3H).白色粉末，80％.1H NMR(400MHz,D2O)δ8.05(d,J＝2.8Hz,1H),7.66(d,J＝9.1Hz,1H),7.59(dd,J＝9.1,2.8Hz,1H),4.14(s,3H). White powder, 89%. 1 H NMR (400 MHz, D 2 O) δ8.56 (d, J=2.9 Hz, 1H), 8.03 (dd, J=9.3, 2.9 Hz, 1H), 7.97 (d, J=9.3 Hz, 1H), 4.28 (s, 3H), 3.94 (s, 3H). White powder, 80%. 1 H NMR (400 MHz, D 2 O) δ8.05 (d, J=2.8 Hz, 1H), 7.66 (d, J=9.1 Hz, 1H), 7.59 (dd, J=9.1, 2.8 Hz, 1H), 4.14 (s, 3H).

白色粉末，79％.1H NMR(400MHz,DMSO-d6)δ10.25(s,1H),8.91(s,1H),8.49(dd,J＝8.3,1.1Hz,1H),8.36-8.25(m,2H),8.10(ddd,J＝8.3,6.2,1.9Hz,1H),4.47(s,3H). White powder, 79%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.25 (s, 1H), 8.91 (s, 1H), 8.49 (dd, J = 8.3, 1.1 Hz, 1H), 8.36-8.25 (m, 2H), 8.10 (ddd, J = 8.3, 6.2, 1.9 Hz, 1H), 4.47 (s, 3H).

白色粉末，收率73％.1H NMR(400MHz,DMSO-d6)δ10.29(s,1H),9.04(s,1H),8.47(dd,J＝8.4,1.1Hz,1H),8.35-8.23(m,2H),8.09(ddd,J＝8.2,6.6,1.5Hz,1H),4.50(s,3H). White powder, yield 73%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.29 (s, 1H), 9.04 (s, 1H), 8.47 (dd, J = 8.4, 1.1 Hz, 1H), 8.35-8.23 (m, 2H), 8.09 (ddd, J = 8.2, 6.6, 1.5 Hz, 1H), 4.50 (s, 3H).

该产物由新鲜制备的2-(2-(2-(丙-2-炔-1-基氧基)乙氧基)乙氧基)三氟甲磺酸乙酯与2-溴吡啶制备。无色油状物，61％.1H NMR(400MHz,D2O)δ8.87(dt,J＝6.3,1.2Hz,1H),8.31-8.28(m,2H),7.96-7.92(m,1H),4.98-4.92(m,2H),4.12(d,J＝2.4Hz,2H),4.02-3.95(m,2H),3.59(ddd,J＝6.4,4.8,2.8Hz,4H),3.56-3.47(m,4H),2.80(t,J＝2.4Hz,1H). The product was prepared from freshly prepared ethyl 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)trifluoromethanesulfonate and 2-bromopyridine. Colorless oil, 61%. 1 H NMR (400 MHz, D 2 O) δ 8.87 (dt, J = 6.3, 1.2 Hz, 1H), 8.31-8.28 (m, 2H), 7.96-7.92 (m, 1H), 4.98-4.92 (m, 2H), 4.12 (d, J = 2.4 Hz, 2H), 4.02-3.95 (m, 2H), 3.59 (ddd, J = 6.4, 4.8, 2.8 Hz, 4H), 3.56-3.47 (m, 4H), 2.80 (t, J = 2.4 Hz, 1H).

该产物由新鲜制备的2-(2-(2-(丙-2-炔-1-基氧基)乙氧基)乙氧基)三氟甲磺酸乙酯与4-溴吡啶制备。无色油状物，55％.1H NMR(400MHz,D2O)δ8.67-8.60(m,2H),8.24(dd,J＝7.3,2.6Hz,2H),4.66(d,J＝4.9Hz,2H),4.14(d,J＝2.4Hz,2H),3.93(d,J＝4.9Hz,2H),3.61(td,J＝3.9,1.5Hz,4H),3.55-3.53(m,4H),2.81(t,J＝2.4Hz,1H). The product was prepared from freshly prepared ethyl 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)trifluoromethanesulfonate and 4-bromopyridine. Colorless oil, 55%. 1 H NMR (400 MHz, D 2 O) δ 8.67-8.60 (m, 2H), 8.24 (dd, J = 7.3, 2.6 Hz, 2H), 4.66 (d, J = 4.9 Hz, 2H), 4.14 (d, J = 2.4 Hz, 2H), 3.93 (d, J = 4.9 Hz, 2H), 3.61 (td, J = 3.9, 1.5 Hz, 4H), 3.55-3.53 (m, 4H), 2.81 (t, J = 2.4 Hz, 1H).

白色粉末，72％.1H NMR(400MHz,D2O)δ8.26(d,J＝8.2Hz,2H),8.06(t,J＝8.2Hz,1H),4.54(s,3H). White powder, 72%. 1 H NMR (400 MHz, D 2 O) δ 8.26 (d, J = 8.2 Hz, 2H), 8.06 (t, J = 8.2 Hz, 1H), 4.54 (s, 3H).

白色粉末，77％.1H NMR(400MHz,D2O)δ8.70(d,J＝6.7Hz,1H),8.59(d,J＝2.2Hz,1H),8.14(dd,J＝6.7,2.2Hz,1H),4.27(s,3H). White powder, 77%. 1 H NMR (400 MHz, D 2 O) δ 8.70 (d, J = 6.7 Hz, 1H), 8.59 (d, J = 2.2 Hz, 1H), 8.14 (dd, J = 6.7, 2.2 Hz, 1H), 4.27 (s, 3H).

灰色粉末，53％.1H NMR(400MHz,D2O)δ8.58(s,2H),4.44(s,3H).13CNMR(101MHz,D2O)δ142.83,140.17,136.09,50.83. Gray powder, 53%. 1 H NMR (400MHz, D 2 O) δ 8.58 (s, 2H), 4.44 (s, 3H). 13 CNMR (101MHz, D 2 O) δ 142.83, 140.17, 136.09, 50.83.

白色粉末，71％.1H NMR(400MHz,D2O)δ6.94(s,2H),3.97(s,3H).13CNMR(101MHz,D2O)δ158.68,145.93,109.93,40.44. White powder, 71%. 1 H NMR (400MHz, D 2 O) δ 6.94 (s, 2H), 3.97 (s, 3H). 13 CNMR (101MHz, D 2 O) δ 158.68, 145.93, 109.93, 40.44.

原料由2,6-二氯-4-羧基吡啶与苄胺缩合反应制备得来。白色粉末，60％.1H NMR(400MHz,D2O)δ8.37(s,2H),7.39-7.33(m,1H),7.33-7.23(m,4H),4.53(s,2H),4.43(s,3H). The raw material is prepared by condensation reaction of 2,6-dichloro-4-carboxypyridine and benzylamine. White powder, 60%. 1 H NMR (400MHz, D 2 O) δ8.37 (s, 2H), 7.39-7.33 (m, 1H), 7.33-7.23 (m, 4H), 4.53 (s, 2H), 4.43 (s, 3H).

原料由2,6-二氯-4-羧基吡啶与2-氨甲基萘缩合反应制备得来。白色粉末，63％.1H NMR(400MHz,DMSO-d6)δ9.41(t,J＝5.9Hz,1H),9.03(s,1H),7.94-7.83(m,3H),7.80(d,J＝1.8Hz,1H),7.56-7.45(m,3H),6.94(d,J＝1.8Hz,1H),6.86(d,J＝1.8Hz,1H),4.61(d,J＝5.9Hz,2H),3.59(s,3H). The raw material was prepared by condensation reaction of 2,6-dichloro-4-carboxypyridine and 2-aminomethylnaphthalene. White powder, 63%. 1 H NMR (400MHz, DMSO-d 6 )δ9.41(t, J＝5.9Hz, 1H), 9.03(s, 1H), 7.94-7.83(m, 3H), 7.80(d, J＝1.8Hz, 1H), 7.56-7.45(m, 3H), 6.94(d, J＝1.8Hz, 1H), 6.86(d, J＝1.8Hz, 1H), 4.61(d, J＝5.9Hz, 2H), 3.59(s, 3H).

白色粉末，68％.1H NMR(400MHz,DMSO-d6)δ8.19-8.12(m,1H),7.70(ddd,J＝8.3,7.0,1.4Hz,1H),7.63-7.56(m,1H),7.49(ddd,J＝8.2,7.0,1.3Hz,1H),6.91(s,1H),3.61(s,3H). White powder, 68%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.19-8.12 (m, 1H), 7.70 (ddd, J = 8.3, 7.0, 1.4 Hz, 1H), 7.63-7.56 (m, 1H), 7.49 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 6.91 (s, 1H), 3.61 (s, 3H).

实施例2：化合物Ⅲ与半胱氨酸IV偶联反应生成V的鉴定：Example 2: Identification of V generated by coupling reaction of compound III with cysteine IV:

在5毫升离心管中加入0.52克2氯-吡啶III、0.16克半胱氨酸IV、0.16克磷酸钠和1毫升反应溶剂去离子水。将离心管用恒温振荡器于室温下振荡反应30分钟。过滤反应液，直接用制备液相色谱分离产物。含有产物的馏分用冻干机冻干。产物为白色粉末0.38克，收率94％。该化合物的核磁和高分辨质谱数据如下：1H NMR(400MHz,D2O)δ8.63-8.57(m,1H),8.25(ddd,J＝8.8,7.5,1.6Hz,1H),7.98-7.91(m,1H),7.62(ddd,J＝7.6,6.3,1.3Hz,1H),4.57(dd,J＝8.2,4.5Hz,1H),4.12(s,3H),3.86(dd,J＝13.9,4.5Hz,1H),3.62(dd,J＝13.9,8.2Hz,1H),1.92(s,3H).13C NMR(101MHz,D2O)δ174.43,174.03,158.99,146.71,143.53,125.61,122.86,52.62,46.48,34.84,22.09.HRMS m/z(ESI-TOF):[M]+calcd forC11H15N2O3S+255.0798,found 255.0796.Add 0.52 g of 2-chloro-pyridine III, 0.16 g of cysteine IV, 0.16 g of sodium phosphate and 1 ml of deionized water as the reaction solvent to a 5 ml centrifuge tube. Shake the centrifuge tube at room temperature for 30 minutes using a constant temperature oscillator. Filter the reaction solution and directly separate the product using preparative liquid chromatography. The fraction containing the product is freeze-dried using a freeze dryer. The product is 0.38 g of white powder with a yield of 94%. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1 H NMR (400 MHz, D 2 O) δ8.63-8.57 (m, 1H), 8.25 (ddd, J = 8.8, 7.5, 1.6 Hz, 1H), 7.98-7.91 (m, 1H), 7.62 (ddd, J = 7.6, 6.3, 1.3 Hz, 1H), 4.57 (dd, J = 8.2, 4.5 Hz, 1H), 4.12 (s, 3H), 3.86 (dd, J = 13.9, 4.5 Hz, 1H), 3.62 (dd, J = 13.9, 8.2 Hz, 1H), 1.92 (s, 3H). 13 C NMR (101 MHz, D 2 O)δ174.43,174.03,158.99,146.71,143.53,125.61,122.86,52.62,46.48,34.84,22.09.HRMS m/z(ESI-TOF):[M] + calcd forC 11 H 15 N 2 O 3 S + 255.0798, found 255.0796.

同类的偶联产物表征数据：Characterization data of similar coupling products:

无色油状物，93％.1H NMR(400MHz,D2O)δ9.02(d,J＝2.1Hz,1H),8.49(dt,J＝8.8,1.6Hz,1H),8.02(d,J＝8.9Hz,1H),4.78(dd,J＝8.5,4.6Hz,1H),4.16(s,3H),3.97(dd,J＝14.1,4.7Hz,1H),3.76-3.65(m,1H),2.85(s,3H),1.91(s,3H).13C NMR(101MHz,D2O)δ174.29,172.13,164.26,162.07,146.46,140.61,128.74,124.91,50.87,46.56,33.88,26.53,21.58.HRMS m/z(ESI-TOF):[M]+calcd forC13H18N3O4S+312.1013,found 312.1011. Colorless oil, 93%. 1 H NMR (400 MHz, D 2 O) δ9.02 (d, J = 2.1 Hz, 1H), 8.49 (dt, J = 8.8, 1.6 Hz, 1H), 8.02 (d, J = 8.9 Hz, 1H), 4.78 (dd, J = 8.5, 4.6 Hz, 1H), 4.16 (s, 3H), 3.97 (dd, J = 14.1, 4.7 Hz, 1H), 3.76-3.65 (m, 1H), 2.85 (s, 3H), 1.91 (s, 3H). 13 C NMR (101 MHz, D 2 O)δ174.29,172.13,164.26,162.07,146.46,140.61,128.74,124.91,50.87,46.56,33.88,26.53,21.58.HRMS m/z(ESI-TOF):[M] + calcd forC 13 H 18 N 3 O 4 S + 312.1013, found 312.1011.

无色油状物，89％.1H NMR(400MHz,D2O)δ8.41(t,J＝1.7Hz,1H),7.92(d,J＝1.6Hz,2H),4.63(dd,J＝8.2,4.5Hz,1H),4.15(s,3H),3.89(s,3H),3.81(dd,J＝14.4,4.5Hz,1H),3.55(dd,J＝14.4,8.2Hz,1H),1.89(s,3H).13C NMR(101MHz,D2O)δ174.13,172.72,155.84,148.45,133.80,130.92,128.29,57.09,51.82,46.89,34.96,21.58.HRMS m/z(ESI-TOF):[M]+calcd for C12H17N2O4S+285.0904,found 285.0903. Colorless oil, 89%. 1 H NMR (400 MHz, D 2 O) δ8.41 (t, J = 1.7 Hz, 1H), 7.92 (d, J = 1.6 Hz, 2H), 4.63 (dd, J = 8.2, 4.5 Hz, 1H), 4.15 (s, 3H), 3.89 (s, 3H), 3.81 (dd, J = 14.4, 4.5 Hz, 1H), 3.55 (dd, J = 14.4, 8.2 Hz, 1H), 1.89 (s, 3H). 13 C NMR (101 MHz, D 2 O)δ174.13,172.72,155.84,148.45,133.80,130.92,128.29,57.09,51.82,46.89,34.96,21.58.HRMS m/z(ESI-TOF):[M] + calcd for C 12 H 17 N 2 O 4 S + 285 .0904, found 285.0903.

无色油状物，61％.1H NMR(400MHz,D2O)δ8.03(d,J＝2.7Hz,1H),7.73(d,J＝9.1Hz,1H),7.53(dd,J＝9.1,2.7Hz,1H),4.57(dd,J＝8.0,4.4Hz,1H),4.10(s,3H),3.65(dd,J＝14.6,4.5Hz,1H),3.42(dd,J＝14.6,8.1Hz,1H),1.88(s,3H).13C NMR(101MHz,D2O)δ174.07,172.67,145.97,140.54,132.51,130.79,129.15,52.20,46.85,35.62,21.57.HRMS m/z(ESI-TOF):[M]+calcd for C11H16N3O3S+270.0907,found 270.0906. Colorless oil, 61%. 1 H NMR (400 MHz, D 2 O) δ8.03 (d, J = 2.7 Hz, 1H), 7.73 (d, J = 9.1 Hz, 1H), 7.53 (dd, J = 9.1, 2.7 Hz, 1H), 4.57 (dd, J = 8.0, 4.4 Hz, 1H), 4.10 (s, 3H), 3.65 (dd, J = 14.6, 4.5 Hz, 1H), 3.42 (dd, J = 14.6, 8.1 Hz, 1H), 1.88 (s, 3H). 13 C NMR (101 MHz, D 2 O)δ174.07,172.67,145.97,140.54,132.51,130.79,129.15,52.20,46.85,35.62,21.57.HRMS m/z(ESI-TOF):[M] + calcd for C 11 H 16 N 3 O 3 S + 270.0907, found 270.0906.

白色粉末，88％.1H NMR(400MHz,DMSO-d6)δ9.21(d,J＝6.6Hz,1H),8.80(d,J＝1.8Hz,1H),8.64(d,J＝8.2Hz,1H),8.28(dd,J＝6.5,1.7Hz,1H),4.68(td,J＝8.0,5.2Hz,1H),4.20(s,3H),3.97(dd,J＝13.8,5.3Hz,1H),3.77(dd,J＝13.8,8.1Hz,1H),1.85(s,3H).13C NMR(101MHz,DMSO-d6)δ171.36,170.39,161.61,148.75,129.77,125.39,124.51,115.24,51.01,47.54,34.88,22.85,22.71.HRMS m/z(ESI-TOF):[M]+calcd for C12H14N3O3S+280.0750,found280.0748. White powder, 88%. 1 H NMR (400MHz, DMSO-d 6 ) δ9.21 (d, J = 6.6 Hz, 1H), 8.80 (d, J = 1.8 Hz, 1H), 8.64 (d, J = 8.2 Hz,1H),8.28(dd,J＝6.5,1.7Hz,1H),4.68(td,J＝8.0,5.2Hz,1H),4.20(s,3H),3.97(dd,J＝13.8,5.3Hz ,1H),3.77(dd,J＝13.8,8.1Hz,1H),1.85(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ171.36,170.39,161.61,148.75,129.77,125.39,124.51,115.24,51.01,47.54,34.88,22.85,22.71.HRMS m/z(ESI-TOF):[M] + calcd for C 12 H 14 N 3 O 3S + 280.0750,found280.0748.

无色油状物，90％.1H NMR(400MHz,D2O)δ8.31(d,J＝6.6Hz,2H),7.69(d,J＝6.5Hz,2H),4.68(q,J＝5.6Hz,1H),4.09(s,3H),3.77-3.67(m,1H),3.47(dd,J＝14.6,8.3Hz,1H),1.87(s,3H).13C NMR(101MHz,D2O)δ174.18,172.71,161.70,142.92,122.89,51.26,46.68,31.92,21.55.HRMS m/z(ESI-TOF):[M]+calcd forC11H15N2O3S+255.0798,found 255.0797. Colorless oil, 90%. 1 H NMR (400 MHz, D 2 O) δ 8.31 (d, J = 6.6 Hz, 2H), 7.69 (d, J = 6.5 Hz, 2H), 4.68 (q, J = 5.6 Hz, 1H), 4.09 (s, 3H), 3.77-3.67 (m, 1H), 3.47 (dd, J = 14.6, 8.3 Hz, 1H), 1.87 (s, 3H). 13 C NMR (101 MHz, D 2 O) δ 174.18, 172.71, 161.70, 142.92, 122.89, 51.26, 46.68, 31.92, 21.55. HRMS m/z (ESI-TOF): [M] + calcd for C 11 H 15 N 2 O 3 S + 255.0798, found 255.0797.

无色油状物，89％.1H NMR(400MHz,D2O)δ8.73(d,J＝6.9Hz,1H),8.46(d,J＝2.4Hz,1H),8.08(dd,J＝6.8,2.4Hz,1H),4.85-4.83(m,1H),4.42(s,3H),3.90(dd,J＝14.2,5.1Hz,1H),3.69(dd,J＝14.2,8.2Hz,1H),2.04(s,3H).13C NMR(101MHz,D2O)δ174.16,172.27,164.48,145.95,129.32,125.43,125.08,110.27,50.84,46.74,32.51,21.62.HRMS m/z(ESI-TOF):[M]+calcd for C12H14N3O3S+280.0750,found 280.0749. Colorless oil, 89%. 1 H NMR (400 MHz, D 2 O) δ8.73 (d, J = 6.9 Hz, 1H), 8.46 (d, J = 2.4 Hz, 1H), 8.08 (dd, J = 6.8, 2.4 Hz, 1H), 4.85-4.83 (m, 1H), 4.42 (s, 3H), 3.90 (dd, J = 14.2, 5.1 Hz, 1H), 3.69 (dd, J = 14.2, 8.2 Hz, 1H), 2.04 (s, 3H). 13 C NMR (101 MHz, D 2 O)δ174.16,172.27,164.48,145.95,129.32,125.43,125.08,110.27,50.84,46.74,32.51,21.62.HRMS m/z(ESI-TOF):[M] + calcd for C 12 H 14 N 3 O 3 S + 28 0.0750, found 280.0749.

白色粉末，76％.1H NMR(400MHz,D2O)δ8.47(d,J＝6.8Hz,1H),8.24(d,J＝2.5Hz,1H),7.85(dd,J＝6.8,2.6Hz,1H),4.75(d,J＝4.7Hz,1H),4.27(s,3H),3.97(s,3H),3.79(dd,J＝14.4,4.8Hz,1H),3.56(dd,J＝14.4,7.9Hz,1H),1.90(s,3H).13C NMR(101MHz,D2O)δ174.23,172.43,163.92,160.67,146.39,140.43,125.76,124.19,54.58,51.16,47.13,32.18,21.59.HRMS m/z(ESI-TOF):[M]+calcd forC13H17N2O5S+313.0853,found 313.0854. White powder, 76%. 1 H NMR (400MHz, D 2 O) δ8.47 (d, J = 6.8 Hz, 1H), 8.24 (d, J = 2.5 Hz, 1H), 7.85 (dd, J = 6.8, 2.6Hz,1H),4.75(d,J＝4.7Hz,1H),4.27(s,3H),3.97(s,3H),3.79(dd,J＝14.4,4.8Hz,1H),3.56(dd, J＝14.4,7.9Hz,1H),1.90(s,3H). 13 C NMR (101MHz, D 2 O)δ174.23,172.43,163.92,160.67,146.39,140.43,125.76,124.19,54.58,51.16,47.13,32.18,21.59.HRMS m/z(ESI-TOF):[M] + calcd forC 13 H 17 N 2 O 5S + 313.0853,found 313.0854.

无色油状物，83％.1H NMR(400MHz,D2O)δ8.03-7.96(m,1H),7.20(d,J＝6.4Hz,2H),4.69(d,J＝4.8Hz,1H),4.13(s,3H),3.79(s,3H),3.74(dd,J＝14.7,4.8Hz,1H),3.48(dd,J＝14.6,8.0Hz,1H),1.91(s,3H).13C NMR(101MHz,D2O)δ174.23,172.71,163.37,159.23,141.27,114.99,104.67,58.68,51.38,40.22,31.92,21.57.HRMS m/z(ESI-TOF):[M]+calcd for C12H17N2O4S+285.0904,found 285.0902. Colorless oil, 83%. 1 H NMR (400 MHz, D 2 O) δ 8.03-7.96 (m, 1H), 7.20 (d, J = 6.4 Hz, 2H), 4.69 (d, J = 4.8 Hz, 1H), 4.13 (s, 3H), 3.79 (s, 3H), 3.74 (dd, J = 14.7, 4.8 Hz, 1H), 3.48 (dd, J = 14.6, 8.0 Hz, 1H), 1.91 (s, 3H). 13 C NMR (101 MHz, D 2 O)δ174.23,172.71,163.37,159.23,141.27,114.99,104.67,58.68,51.38,40.22,31.92,21.57.HRMS m/z(ESI-TOF):[M] + calcd for C 12 H 17 N 2 O 4 S + 285 .0904, found 285.0902.

无色油状物，45％.1H NMR(400MHz,D2O)δ7.53(d,J＝7.2Hz,1H),6.68(d,J＝2.1Hz,1H),6.63(dd,J＝7.2,2.2Hz,1H),4.60(dd,J＝8.1,4.7Hz,1H),3.60(s,3H),3.52(dd,J＝14.4,4.8Hz,1H),3.29(dd,J＝14.4,8.2Hz,1H),1.87(s,3H).13CNMR(101MHz,D2O)δ174.08,172.73,156.14,153.12,138.95,111.60,107.03,51.44,40.37,31.58,21.54.HRMS m/z(ESI-TOF):[M]+calcd for C11H16N3O3S+270.0907,found 270.0905. Colorless oil, 45%. 1 H NMR (400 MHz, D 2 O) δ7.53 (d, J = 7.2 Hz, 1H), 6.68 (d, J = 2.1 Hz, 1H), 6.63 (dd, J = 7.2, 2.2 Hz, 1H), 4.60 (dd, J = 8.1, 4.7 Hz, 1H), 3.60 (s, 3H), 3.52 (dd, J = 14.4, 4.8 Hz, 1H), 3.29 (dd, J = 14.4, 8.2 Hz, 1H), 1.87 (s, 3H). 13 CNMR (101 MHz, D 2 O)δ174.08,172.73,156.14,153.12,138.95,111.60,107.03,51.44,40.37,31.58,21.54.HRMS m/z(ESI-TOF):[M] + calcd for C 11 H 16 N 3 O 3 S + 270.0907, found 270.0905.

白色粉末，83％.1H NMR(400MHz,D2O)δ9.95(d,J＝6.1Hz,1H),8.71(d,J＝6.2Hz,1H),8.41(t,J＝7.4Hz,1H),8.24(d,J＝6.0Hz,2H),8.01(td,J＝8.5,7.6,3.2Hz,1H),4.87-4.79(m,1H),4.53(d,J＝6.4Hz,3H),3.96(dd,J＝13.2,6.7Hz,1H),3.72(dt,J＝14.2,7.1Hz,1H),1.99(d,J＝6.4Hz,3H).13C NMR(101MHz,D2O)δ172.20,170.98,152.53,145.32,138.02,137.30,130.73,129.72,126.39,126.29,125.94,51.23,46.26,35.88,21.06.HRMS m/z(ESI-TOF):[M]+calcd for C15H17N2O3S+305.0954,found305.0952. White powder, 83%. 1 H NMR (400MHz, D 2 O) δ9.95 (d, J = 6.1 Hz, 1H), 8.71 (d, J = 6.2 Hz, 1H), 8.41 (t, J = 7.4 Hz ,1H),8.24(d,J＝6.0Hz,2H),8.01(td,J＝8.5,7.6,3.2Hz,1H),4.87-4.79(m,1H),4.53(d,J＝6.4Hz, 3H), 3.96 (dd, J＝13.2, 6.7Hz, 1H), 3.72 (dt, J＝14.2, 7.1Hz, 1H), 1.99 (d, J＝6.4Hz, 3H). 13 C NMR (101MHz, D 2 O)δ172.20,170.98,152.53,145.32,138.02,137.30,130.73,129.72,126.39,126.29,125.94,51.23,46.26,35.88,21.06.HRMS m/z(ESI-TOF):[M] + calc d for C 15 H 17 N 2 O 3 S + 305.0954,found305.0952.

白色粉末，54％.1H NMR(400MHz,DMSO-d6)δ8.57(dd,J＝9.4,7.5Hz,2H),8.02(d,J＝2.2Hz,1H),7.70(dd,J＝7.0,2.3Hz,1H),4.59(td,J＝8.4,4.9Hz,1H),4.04(s,3H),3.68-3.64(m,2H),2.89(t,J＝2.6Hz,1H),2.80(t,J＝7.2Hz,2H),2.09(s,1H),1.87(s,3H).13C NMR(101MHz,DMSO-d6)δ171.71,171.52,170.23,161.40,145.92,143.76,118.60,116.86,83.59,72.39,50.96,43.53,35.51,32.79,22.82,14.05.HRMS m/z(ESI-TOF):[M]+calcd for C16H20N3O4S+350.1169,found 350.1169. White powder, 54%. 1 H NMR (400MHz, DMSO-d 6 ) δ8.57 (dd, J = 9.4, 7.5 Hz, 2H), 8.02 (d, J = 2.2 Hz, 1H), 7.70 (dd, J ＝7.0,2.3Hz,1H),4.59(td,J＝8.4,4.9Hz,1H),4.04(s,3H),3.68-3.64(m,2H),2.89(t,J＝2.6Hz,1H) ,2.80(t,J=7.2Hz,2H),2.09(s,1H),1.87(s,3H). 13 C NMR(101MHz,DMSO-d 6 )δ171.71,171.52,170.23,161.40,145.92,143.76,118.60,116.86,83.59,72.39,50.96,43.53,35.51,32.79,22.82,14.05.HRMS m/z(ESI-TOF):[M] + calcd for C 16 H 20 N 3 O 4 S + 350.1169,found 350.1169.

双取代反应产物的半胱氨酸原料为2倍当量。无色油状物，84％.1H NMR(400MHz,D2O)δ8.03(t,J＝8.3Hz,1H),7.68(d,J＝8.4Hz,2H),4.68(dd,J＝8.6,4.5Hz,2H),4.07(s,3H),3.84(dd,J＝14.3,4.5Hz,2H),3.56(dd,J＝14.2,8.6Hz,2H),1.89(s,6H).13C NMR(101MHz,D2O)δ174.22,172.57,159.88,141.08,121.93,51.14,41.79,34.93,21.58.HRMS m/z(ESI-TOF):[M]+calcd for C16H22N3O6S2 +416.0945,found416.0952. The cysteine starting material of the disubstitution reaction product is 2 equivalents. Colorless oil, 84%. 1 H NMR (400 MHz, D 2 O) δ8.03 (t, J=8.3 Hz, 1H), 7.68 (d, J=8.4 Hz, 2H), 4.68 (dd, J=8.6, 4.5 Hz, 2H), 4.07 (s, 3H), 3.84 (dd, J=14.3, 4.5 Hz, 2H), 3.56 (dd, J=14.2, 8.6 Hz, 2H), 1.89 (s, 6H). 13 C NMR (101 MHz, D 2 O) δ174.22, 172.57, 159.88, 141.08, 121.93, 51.14, 41.79, 34.93, 21.58. HRMS m/z (ESI-TOF): [M] + calcd for C 16 H 22 N 3 O 6 S 2 + 416.0945,found416.0952.

双取代反应产物的半胱氨酸原料为2倍当量。无色油状物，85％.1HNMR(400MHz,DMSO-d6)δ8.69(d,J＝7.0Hz,1H),8.62(dd,J＝8.0,5.7Hz,2H),7.90(d,J＝2.2Hz,1H),7.71(dd,J＝6.9,2.1Hz,1H),4.58(ddd,J＝15.8,7.8,2.3Hz,2H),4.03(s,3H),3.87(dd,J＝13.9,5.3Hz,1H),3.73-3.67(m,2H),3.55(dd,J＝13.8,7.8Hz,1H),1.88(s,3H),1.83(s,3H).13C NMR(101MHz,DMSO-d6)δ171.70,171.46,170.37,159.92,156.53,145.43,120.50,119.03,116.23,51.35,51.32,45.41,34.42,32.37,22.79,22.70.HRMS m/z(ESI-TOF):[M]+calcd for C16H22N3O6S2 +416.0945,found 416.0954. The cysteine starting material of the disubstitution reaction product is 2 equivalents. Colorless oil, 85%. 1 HNMR (400MHz, DMSO-d 6 ) δ8.69 (d, J = 7.0 Hz, 1H), 8.62 (dd, J = 8.0, 5.7 Hz, 2H), 7.90 (d, J = 2.2 Hz, 1H), 7.71 (dd, J = 6.9, 2.1 Hz, 1H), 4.58 (ddd, J = 15. 13 C NMR DMSO-d 6 )δ171.70,171.46,170.37,159.92,156.53,145.43,120.50,119.03,116.23,51.35,51.32,45.41,34.42,32.37,22.79,22.70.HRMS m/z(ESI-TOF):[M] + calcd for C 16 H 22 N 3 O 6 S 2 + 416.0945, found 416.0954.

三取代反应产物的半胱氨酸原料为3倍当量。无色油状物，81％.1H NMR(400MHz,DMSO-d6)δ8.64(d,J＝7.8Hz,1H),8.59(d,J＝8.0Hz,2H),7.69(s,2H),4.62-4.53(m,3H),3.99(s,3H),3.84(dd,J＝13.9,5.2Hz,2H),3.74-3.58(m,4H),1.88(s,3H),1.84(s,6H).13C NMR(101MHz,DMSO-d6)δ171.69,171.47,170.46,170.38,157.82,157.53,117.84,51.53,51.29,41.78,35.49,32.09,22.77,22.71.HRMS m/z(ESI-TOF):[M]+calcd for C21H29N4O9S3 +577.1091,found 577.1088. The cysteine starting material of the tri-substitution reaction product is 3 times equivalent. Colorless oil, 81%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.64 (d, J = 7.8 Hz, 1H), 8.59 (d, J = 8.0 Hz, 2H), 7.69 (s, 2H), 4.62-4.53 (m, 3H), 3.99 (s, 3H), 3.84 (dd, J = 13.9, 5.2 Hz, 2H), 3.74-3.58 (m, 4H), 1.88 (s, 3H), 1.84 (s, 6H). 13 C NMR (101 MHz, DMSO-d 6 )δ171.69,171.47,170.46,170.38,157.82,157.53,117.84,51.53,51.29,41.78,35.49,32.09,22.77,22.71.HRMS m/z(ESI-TOF):[M] + calcd for C 21 H 29 N 4 O 9 S 3 + 577.1091, found 577.1088.

实施例3：化合物V与谷胱甘肽VI硫醇交换可逆反应生成VII的鉴定：Example 3: Identification of the reversible thiol exchange reaction between compound V and glutathione VI to generate VII:

在5毫升离心管中加入0.25克吡啶盐-半胱氨酸偶联产物V、0.31克谷胱甘肽VI、0.16克磷酸钠和1毫升反应溶剂去离子水。将离心管用恒温振荡器于室温下振荡反应30分钟。过滤反应液，直接用制备液相色谱分离产物。含有产物的馏分用冻干机冻干。产物为白色粉末0.12克，收率30％。该化合物的核磁和高分辨质谱数据如下：1H NMR(400MHz,D2O)δ8.65-8.59(m,1H),8.32-8.23(m,1H),8.03-7.96(m,1H),7.64(ddd,J＝7.6,6.2,1.3Hz,1H),4.80(dd,J＝8.0,5.6Hz,1H),4.14(s,3H),3.92(d,J＝2.6Hz,2H),3.89-3.81(m,2H),3.66(dd,J＝14.1,8.0Hz,1H),2.48(td,J＝7.3,1.8Hz,2H),2.14-2.06(m,2H).13C NMR(101MHz,D2O)δ174.64,173.17,172.96,171.05,158.46,146.86,143.70,125.49,122.98,53.29,51.59,46.36,41.45,33.61,31.16,25.75.HRMS m/z(ESI-TOF):[M]+calcd forC16H23N4O6S+399.1333,found 399.1330.Add 0.25 g of pyridinium-cysteine coupling product V, 0.31 g of glutathione VI, 0.16 g of sodium phosphate and 1 ml of deionized water as the reaction solvent to a 5 ml centrifuge tube. Shake the centrifuge tube at room temperature for 30 minutes using a constant temperature oscillator. Filter the reaction solution and directly separate the product using preparative liquid chromatography. The fraction containing the product is freeze-dried using a freeze dryer. The product is 0.12 g of white powder with a yield of 30%. The NMR and high-resolution mass spectrometry data of the compound are as follows: 1 H NMR (400 MHz, D 2 O) δ8.65-8.59 (m, 1H), 8.32-8.23 (m, 1H), 8.03-7.96 (m, 1H), 7.64 (ddd, J = 7.6, 6.2, 1.3 Hz, 1H), 4.80 (dd, J = 8.0, 5.6 Hz, 1H), 4.14 (s, 3H), 3.92 (d, J = 2.6 Hz, 2H), 3.89-3.81 (m, 2H), 3.66 (dd, J = 14.1, 8.0 Hz, 1H), 2.48 (td, J = 7.3, 1.8 Hz, 2H), 2.14-2.06 (m, 2H). 13 C NMR (101 MHz, D 2 O)δ174.64,173.17,172.96,171.05,158.46,146.86,143.70,125.49,122.98,53.29,51.59,46.36,41.45,33.61,31.16,25.75.HRMS m/z(ESI-TOF):[M ] + calcd forC 16 H 23 N 4 O 6 S + 399.1333, found 399.1330.

同类的硫醇交换可逆反应产物表征数据：Characterization data of similar thiol exchange reversible reaction products:

白色粉末，22％.1H NMR(400MHz,D2O)δ8.39-8.32(m,2H),7.79-7.71(m,2H),4.78-4.74(m,1H),4.13(s,3H),3.89(s,2H),3.80-3.66(m,2H),3.49(dd,J＝14.3,8.1Hz,1H),2.50-2.37(m,2H),2.06(dtd,J＝9.1,6.6,2.3Hz,2H).13C NMR(101MHz,D2O)δ174.71,173.41,173.30,171.39,161.50,143.03,123.00,53.54,51.67,46.84,41.56,31.95,31.19,25.87.HRMS m/z(ESI-TOF):[M]+calcd for C16H23N4O6S+399.1333,found 399.1332. White powder, 22%. 1 H NMR (400MHz, D 2 O) δ8.39-8.32(m,2H),7.79-7.71(m,2H),4.78-4.74(m,1H),4.13(s,3H) ),3.89(s,2H),3.80-3.66(m,2H),3.49(dd,J＝14.3,8.1Hz,1H),2.50-2.37(m,2H),2.06(dtd,J＝9.1,6.6 ,2.3Hz,2H). 13 C NMR(101MHz,D 2 O)δ174.71,173.41,173.30,171.39,161.50,143.03,123.00,53.54,51.67,46.84,41.56,31.95,31.19,25.87.HRMS m/z(ESI-TOF):[M] + calcd for C 16 H 23 N 4 O 6 S + 399.1333, found 399.1332.

实施例4：本发明的可用于半胱氨酸残基化学修饰的探针III与多肽的反应结果：Example 4: Reaction results of the probe III for chemical modification of cysteine residues of the present invention with polypeptides:

为了验证可用于半胱氨酸残基化学修饰的探针与半胱氨酸(Cys)共价结合在多肽标记层面的反应性，采用蒸馏水将多肽溶解并配置成1mM的多肽溶液。准确称量可用于蛋白质半胱氨酸残基可逆化学修饰的探针III用PBS缓冲液或者蒸馏水配置成相应浓度的反应试液。将多肽溶液与10mM探针在PBS溶液中于37℃钟孵育10分钟。过滤反应液，使用液质联用色谱直接分析反应液。多肽可以为含半胱氨酸残基的3至50个氨基酸的不带保护基的多肽。In order to verify the reactivity of the probe that can be used for chemical modification of cysteine residues and covalent binding of cysteine (Cys) at the level of polypeptide labeling, the polypeptide was dissolved in distilled water and prepared into a 1mM polypeptide solution. Accurately weigh the probe III that can be used for reversible chemical modification of protein cysteine residues and prepare a reaction solution of corresponding concentration with PBS buffer or distilled water. Incubate the polypeptide solution with 10mM probe in PBS solution at 37°C for 10 minutes. Filter the reaction solution and directly analyze the reaction solution using LC-MS chromatography. The polypeptide can be a polypeptide with 3 to 50 amino acids containing cysteine residues without a protecting group.

图1显示了的多肽与探针III的直接修饰反应的色谱图，结果表明反应纯净单一。FIG1 shows the chromatogram of the direct modification reaction of the peptide with probe III, and the results show that the reaction is pure and single.

实施例5：本发明的可用于半胱氨酸残基化学修饰的探针III与多肽的硫醇交换可逆反应结果：Example 5: Results of the reversible thiol exchange reaction between the probe III of the present invention that can be used for chemical modification of cysteine residues and polypeptides:

为了验证可用于半胱氨酸残基化学修饰的探针与Cys共价结合在多肽标记层面的可逆反应性，采用蒸馏水将多肽溶解并配置成1mM的多肽溶液。准确称量可用于半胱氨酸残基可逆化学修饰的探针III用PBS缓冲液或者蒸馏水配置成相应浓度的反应试液。将多肽溶液与10mM探针在PBS溶液中于37℃中孵育10分钟。取一半反应液，用于可逆反应的研究。配制100mM谷胱甘肽(GSH)母液，直接在反应液中加入GSH母液，使GSH终浓度为20mM，继续于37℃中孵育2小时。分别过滤反应液，使用液质联用色谱直接分析反应液。多肽可以为含半胱氨酸残基的3至50个氨基酸的不带保护基的多肽。In order to verify the reversible reactivity of the probe that can be used for chemical modification of cysteine residues and covalent binding of Cys at the level of peptide labeling, the peptide was dissolved in distilled water and prepared into a 1mM peptide solution. Accurately weigh the probe III that can be used for reversible chemical modification of cysteine residues and prepare a reaction solution of corresponding concentration with PBS buffer or distilled water. Incubate the peptide solution and 10mM probe in PBS solution at 37°C for 10 minutes. Take half of the reaction solution for the study of reversible reaction. Prepare 100mM glutathione (GSH) mother solution, add GSH mother solution directly to the reaction solution to make the final concentration of GSH 20mM, and continue to incubate at 37°C for 2 hours. Filter the reaction solution separately and directly analyze the reaction solution using LC-MS chromatography. The polypeptide can be a polypeptide with 3 to 50 amino acids containing cysteine residues without a protecting group.

图2显示了的多肽与探针III的可逆修饰反应的色谱图，结果表明修饰反应纯净单一，可逆反应随着吡啶盐取代基的不同可以进行反应效率的调节。FIG2 shows the chromatogram of the reversible modification reaction of the peptide with probe III. The results show that the modification reaction is pure and single, and the reversible reaction efficiency can be adjusted with the different pyridinium salt substituents.

实施例6：本发明的可用于蛋白质半胱氨酸残基化学修饰的探针III与蛋白质的可逆反应凝胶内荧光分析：Example 6: In-gel fluorescence analysis of the reversible reaction between the probe III of the present invention that can be used for chemical modification of protein cysteine residues and proteins:

为了验证可用于蛋白质半胱氨酸残基化学修饰的探针与半胱氨酸(Cys)共价结合在蛋白标记层面的反应性，采用蒸馏水将蛋白溶解并配置成7.5μM的蛋白溶液。准确称量可用于蛋白质半胱氨酸残基可逆化学修饰的探针III用PBS缓冲液或者蒸馏水配置成相应浓度的反应试液。将蛋白溶液与50μM探针在PBS溶液中于37℃钟孵育10分钟。取一半反应液，用于可逆反应的研究。配制10mM GSH母液，直接在反应液中加入GSH母液，使GSH终浓度为100μM，继续于37℃钟孵育2小时。然后利用“click”反应分别给两份反应液中的蛋白标记荧光标签，具体做法是在反应体系中加入CuSO4(1mM)，TECP(1mM)，TBTA(100μM)，5-TAMRA-N3(100μM)，于25℃孵育2小时，反应结束。最后跑SDS-PAGE蛋白胶，观察胶内荧光。蛋白可以为牛血清蛋白(BSA)，马血清蛋白(HSA)，溶菌酶(Lysozyme)，胰岛素(Insulin)，碳酸酐酶(CA),肌红蛋白(Myoglobin),核糖核酸酶A(RNaseA)等。In order to verify the reactivity of the probe that can be used for chemical modification of protein cysteine residues and cysteine (Cys) covalently bound at the protein labeling level, the protein was dissolved in distilled water and prepared into a 7.5μM protein solution. The probe III that can be used for reversible chemical modification of protein cysteine residues was accurately weighed and prepared into a reaction solution of corresponding concentration using PBS buffer or distilled water. The protein solution and 50μM probe were incubated in PBS solution at 37℃ for 10 minutes. Half of the reaction solution was taken for the study of reversible reaction. A 10mM GSH mother solution was prepared and directly added to the reaction solution to make the final concentration of GSH 100μM, and continued to incubate at 37℃ for 2 hours. Then, the proteins in the two reaction solutions were labeled with fluorescent tags using the "click" reaction. Specifically, CuSO 4 (1mM), TECP (1mM), TBTA (100μM), and 5-TAMRA-N 3 (100μM) were added to the reaction system, and the reaction was terminated after incubation at 25℃ for 2 hours. Finally, run SDS-PAGE protein gel and observe the fluorescence in the gel. The protein can be bovine serum albumin (BSA), horse serum albumin (HSA), lysozyme, insulin, carbonic anhydrase (CA), myoglobin, ribonuclease A (RNaseA), etc.

图3显示了蛋白质与探针III的可逆反应的荧光胶图，结果表明修饰反应和可逆反应都纯净单一。FIG3 shows a fluorescent gel image of the reversible reaction of the protein with probe III, and the results show that both the modification reaction and the reversible reaction are pure and single.

实施例7：本发明的可用于蛋白质半胱氨酸残基化学修饰的探针III与蛋白质的可逆反应一级质谱研究：Example 7: Primary mass spectrometry study of the reversible reaction between the probe III of the present invention that can be used for chemical modification of protein cysteine residues and proteins:

为了验证可用于蛋白质半胱氨酸残基化学修饰的探针与半胱氨酸(Cys)共价结合在蛋白标记层面的反应性，采用蒸馏水将蛋白溶解并配置成7.5μM的蛋白溶液。准确称量可用于蛋白质半胱氨酸残基可逆化学修饰的探针III用PBS缓冲液或者蒸馏水配置成相应浓度的反应试液。将蛋白溶液与50μM探针在PBS溶液中于37℃钟孵育10分钟。取一半反应液，用于可逆反应的研究。配制10mM GSH母液，直接在反应液中加入GSH母液，使GSH终浓度为100μM，继续于37℃钟孵育2小时。然后直接使用超滤管，滤去反应液中的盐和小分子。所得纯净的蛋白质溶液进行ESI一级质谱测定。蛋白可以为牛血清蛋白(BSA)，马血清蛋白(HSA)，溶菌酶(Lysozyme)，胰岛素(Insulin)，碳酸酐酶(CA),肌红蛋白(Myoglobin),核糖核酸酶A(RNaseA)等。In order to verify the reactivity of the probe that can be used for chemical modification of protein cysteine residues and covalent binding of cysteine (Cys) at the protein labeling level, the protein was dissolved in distilled water and prepared into a 7.5μM protein solution. Accurately weigh the probe III that can be used for reversible chemical modification of protein cysteine residues and prepare the reaction solution of corresponding concentration with PBS buffer or distilled water. Incubate the protein solution with 50μM probe in PBS solution at 37℃ for 10 minutes. Take half of the reaction solution for the study of reversible reaction. Prepare 10mM GSH mother solution, add GSH mother solution directly to the reaction solution, make the final concentration of GSH 100μM, and continue to incubate at 37℃ for 2 hours. Then use ultrafiltration tube directly to filter out salts and small molecules in the reaction solution. The obtained pure protein solution is subjected to ESI primary mass spectrometry. The protein can be bovine serum albumin (BSA), horse serum albumin (HSA), lysozyme, insulin, carbonic anhydrase (CA), myoglobin, ribonuclease A (RNaseA), etc.

图4显示了蛋白质与探针III的可逆反应的一级质谱图，结果表明修饰反应和可逆反应都纯净单一。FIG4 shows the primary mass spectrum of the reversible reaction of the protein with probe III, and the results show that both the modification reaction and the reversible reaction are pure and single.

实施例8：本发明的可用于蛋白质半胱氨酸残基化学修饰的探针III与单克隆抗体的可逆反应一级质谱研究：Example 8: Primary mass spectrometry study of the reversible reaction of the probe III of the present invention that can be used for chemical modification of protein cysteine residues and monoclonal antibodies:

为了验证可用于单克隆抗体半胱氨酸残基化学修饰的探针与半胱氨酸(Cys)共价结合在蛋白标记层面的反应性，采用蒸馏水将单克隆抗体IgG1溶解并配置成7.5μM的抗体溶液。准确称量可用于单克隆抗体半胱氨酸残基可逆化学修饰的探针III用PBS缓冲液或者蒸馏水配置成相应浓度的反应试液。先在单克隆抗体溶液中加入50μM三(2-羧乙基)膦反应1小时，还原抗体中二硫键。然后加入50μM探针在PBS溶液中于37℃中孵育10分钟。取一半反应液，用于可逆反应的研究。配制10mM GSH母液，直接在反应液中加入GSH母液，使GSH终浓度为100μM，继续于37℃钟孵育2小时。使用N-糖苷酶F对单克隆抗体进行脱糖基处理。然后直接使用超滤管，滤去反应液中的盐和小分子。所得纯净的单克隆抗体溶液进行ESI一级质谱测定。In order to verify the reactivity of the probe that can be used for chemical modification of cysteine residues of monoclonal antibodies and covalent binding of cysteine (Cys) at the protein labeling level, the monoclonal antibody IgG1 was dissolved in distilled water and prepared into a 7.5μM antibody solution. The probe III that can be used for reversible chemical modification of cysteine residues of monoclonal antibodies was accurately weighed and prepared into a reaction solution of corresponding concentration using PBS buffer or distilled water. First, 50μM tris(2-carboxyethyl)phosphine was added to the monoclonal antibody solution for 1 hour to reduce the disulfide bonds in the antibody. Then 50μM probe was added to the PBS solution and incubated at 37℃ for 10 minutes. Half of the reaction solution was taken for the study of reversible reaction. A 10mM GSH mother solution was prepared and directly added to the reaction solution to make the final concentration of GSH 100μM, and then incubated at 37℃ for 2 hours. The monoclonal antibody was deglycosylated using N-glycosidase F. Then, the salt and small molecules in the reaction solution were filtered out directly using an ultrafiltration tube. The obtained pure monoclonal antibody solution was subjected to ESI primary mass spectrometry.

图5显示了单克隆抗体与探针III的可逆反应的一级质谱图，结果表明修饰反应和可逆反应都纯净单一。FIG5 shows the primary mass spectrum of the reversible reaction of the monoclonal antibody with probe III, and the results show that both the modification reaction and the reversible reaction are pure and single.

Claims (10)

1.一种可用于蛋白质半胱氨酸残基可逆化学修饰的探针，其特征在于，具有如式III所示的结构通式：1. A probe that can be used for reversible chemical modification of protein cysteine residues, characterized in that it has a general structural formula as shown in formula III: 其中：R1、R2分别选自：氢、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、C1-C6烷胺基、C1-C6酰胺基、C1-C6卤代烷氧基、C2-C6烯基、C2-C6卤代烯基、C2-C6炔基、C2-C6卤代炔基、羟基、氨基、C3-C6环烷基、苯基、卤素、氰基、羧基或者醛基；Among them: R 1 and R 2 are respectively selected from: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 amide, C1-C6 haloalkoxy Base, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, amino, C3-C6 cycloalkyl, phenyl, halogen, cyano, carboxyl or aldehyde group; 所述的卤素是氟、氯、溴或碘；The halogen is fluorine, chlorine, bromine or iodine; 所述烷基、烯基或炔基为直链的或支链的烷基；烷基本身或作为其它取代基的部分选自甲基、乙基、丙基、丁基、戊基、己基或者其异构体，其异构体选自异丙基、异丁基、仲丁基、叔丁基、异戊基或叔戊基；The alkyl, alkenyl or alkynyl group is a linear or branched alkyl group; the alkyl group itself or as part of other substituents is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl or Its isomer, its isomer is selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or tert-amyl; 所述卤代烷基基团选自含一个或多个相同或不同的卤素原子的基团，所述卤代烷基选自CH2Cl、CHCl2、CCl3、CH2F、CHF2、CF3、CF3CH2、CH3CF2、CF3CF2或CCl3CCl2；The haloalkyl group is selected from groups containing one or more identical or different halogen atoms, and the haloalkyl group is selected from CH 2 Cl, CHCl 2 , CCl 3 , CH 2 F, CHF 2 , CF 3 , CF 3 CH 2 , CH 3 CF 2 , CF 3 CF 2 or CCl 3 CCl 2 ; 所述环烷基本身或作为其它取代基的部分选自环丙基、环丁基、环戊基或环己基；The cycloalkyl itself or as part of other substituents is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; 所述烯基本身或作为其它取代基的部分选自乙烯基、烯丙基、1-丙烯基、丁烯-2-基、丁烯-3-基、戊烯-1-基、戊烯-3-基、己烯-1-基或4-甲基-3-戊烯基；The alkenyl group itself or as part of other substituents is selected from vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten- 3-yl, hexen-1-yl or 4-methyl-3-pentenyl; 所述炔基本身或作为其它取代基的部分选自乙炔基、丙炔-1-基、丙炔-2-基、丁炔-1-基、丁炔-2-基、1-甲基-2-丁炔基、己炔-1-基或1-乙基-2-丁炔基。The alkynyl group itself or as part of other substituents is selected from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl- 2-butynyl, hexyn-1-yl or 1-ethyl-2-butynyl. X选自：烷基酸根、取代烷基酸根、卤素或者其含氧酸根、磷酸根、硫酸根、磺酸根或者硼酸根；The 所述烷基酸根、取代烷基酸根选自C1-C6烷基酸根或者C1-C6卤代烷基酸根；The alkyl acid radicals and substituted alkyl acid radicals are selected from C1-C6 alkyl acid radicals or C1-C6 haloalkylate radicals; 所述卤素或者其含氧酸根选自氟、氯、溴或碘、次氯酸根、亚氯酸根、氯酸根、高氯酸根、次溴酸根、亚溴酸根、溴酸根、高溴酸根、次碘酸根、亚碘酸根、碘酸根或者高碘酸根；The halogen or its oxygen-containing acid radical is selected from fluorine, chlorine, bromine or iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromate, bromate, perbromate, hypoiodide Acid radical, iodate radical, iodate radical or periodate radical; 所述磷酸根选自磷酸一氢根、磷酸二氢根、焦磷酸根、偏磷酸根、次磷酸根、亚磷酸根、多聚磷酸根、磷酸根或者六氟磷酸根；The phosphate radical is selected from monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, phosphate or hexafluorophosphate; 所述硫酸根选自硫负离子、硫酸氢根、硫酸根、亚硫酸氢根、亚硫酸根、焦硫酸根、连二硫酸根、硫代硫酸根、连二亚硫酸根或者过硫酸根；The sulfate is selected from the group consisting of sulfide anion, hydrogen sulfate, sulfate, bisulfite, sulfite, pyrosulfate, dithionate, thiosulfate, dithionite or persulfate; 所述磺酸根选自三氟甲磺酸根、甲磺酸根、苯磺酸根或者对-甲基苯磺酸根；The sulfonate radical is selected from triflate, methanesulfonate, benzenesulfonate or p-toluenesulfonate; 所述硼酸根选自硼酸根或者四氟硼酸根；The borate radical is selected from borate radical or tetrafluoroborate radical; Y选自：卤素；Y is selected from: halogen; 所述的卤素是氟、氯、溴或碘。The halogen is fluorine, chlorine, bromine or iodine. 2.根据权利要求1所述的一种可用于蛋白质半胱氨酸残基可逆化学修饰的探针，其特征在于，R1选自氢、烷氧基、取代氨基或氰基；R2选自氢或烷基；X选自碘、三氟甲磺酸根或者四氟硼酸根；Y选自氯、溴或碘。2. A probe that can be used for reversible chemical modification of protein cysteine residues according to claim 1, characterized in that R 1 is selected from hydrogen, alkoxy, substituted amino or cyano; R 2 is selected from the group consisting of hydrogen, alkoxy, substituted amino or cyano. From hydrogen or alkyl; X is selected from iodine, triflate or tetrafluoroborate; Y is selected from chlorine, bromine or iodine. 3.权利要求1所述的可用于蛋白质半胱氨酸残基可逆化学修饰的探针的制备方法，其特征在于包括以下步骤：3. The preparation method of a probe that can be used for reversible chemical modification of protein cysteine residues according to claim 1, characterized by comprising the following steps: 在一个反应容器中加入化合物Add compounds to a reaction vessel 和烷基化试剂II R2X，所述的烷基化试剂II选自：碘甲烷、三氟甲磺酸甲酯、三氟甲磺酸乙酯、苯磺酸甲酯或者三甲基氧鎓四氟硼酸盐，反应溶剂选自：二氯甲烷、甲苯、乙腈、乙醚或者四氢呋喃，得到可用于蛋白质半胱氨酸残基可逆化学修饰的探针and alkylating reagent II R 2 Onium tetrafluoroborate, the reaction solvent is selected from: dichloromethane, toluene, acetonitrile, diethyl ether or tetrahydrofuran, to obtain a probe that can be used for reversible chemical modification of protein cysteine residues ， , 产物以沉淀的形式直接析出，或添加反应溶剂等体积的乙醚促进产物的析出。The product precipitates directly in the form of a precipitate, or an equal volume of diethyl ether as the reaction solvent is added to promote the precipitation of the product. 4.权利要求1所述的可用于蛋白质半胱氨酸残基可逆化学修饰的探针的含卤素的吡啶盐类化合物或者其衍生物。4. The halogen-containing pyridinium salt compound or derivatives thereof that can be used as a probe for reversible chemical modification of protein cysteine residues according to claim 1. 5.权利要求1所述的可用于蛋白质半胱氨酸残基可逆化学修饰的探针或者其中间体在蛋白质半胱氨酸可逆修饰上的用途。5. The probe according to claim 1 that can be used for reversible chemical modification of protein cysteine residues or the use of its intermediates for reversible modification of protein cysteine. 6.一种蛋白质的可逆修饰方法，其特征在于，加入需要修饰的蛋白质，所述的化学修饰位点为蛋白质半胱氨酸残基；可用于蛋白质半胱氨酸残基化学修饰的探针III投料为蛋白质的0.1当量至200当量；然后使用含巯基的还原剂移除该修饰。6. A method for reversible modification of proteins, characterized in that the protein to be modified is added, and the chemical modification site is a protein cysteine residue; a probe that can be used for chemical modification of a protein cysteine residue III Dosing ranges from 0.1 equiv to 200 equiv of protein; this modification is then removed using a sulfhydryl-containing reducing agent. 7.根据权利要求6所述的一种蛋白质的可逆修饰方法，其特征在于，反应溶剂为水或极性有机溶剂：所述的溶剂选自水、乙腈、甲醇、乙醇、异丙醇、叔丁醇、乙二醇、甘油、三氟乙醇、六氟异丙醇、二甲基亚砜、N,N-二甲基甲酰胺中的任意一种或者它们任意两种的混合溶剂。7. The reversible modification method of a protein according to claim 6, characterized in that the reaction solvent is water or a polar organic solvent: the solvent is selected from the group consisting of water, acetonitrile, methanol, ethanol, isopropyl alcohol, tert. Any one of butanol, ethylene glycol, glycerin, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide, N,N-dimethylformamide or a mixed solvent of any two of them. 8.根据权利要求6所述的一种蛋白质的可逆修饰方法，其特征在于，所述的反应时间为0.1小时至100小时。8. The reversible modification method of a protein according to claim 6, characterized in that the reaction time is 0.1 hour to 100 hours. 9.根据权利要求6所述的一种蛋白质的可逆修饰方法，其特征在于，所述的反应温度为-20至50℃。9. The reversible modification method of a protein according to claim 6, characterized in that the reaction temperature is -20 to 50°C. 10.根据权利要求6所述的一种蛋白质的可逆修饰方法，其特征在于，所述的含巯基的还原剂选自丙硫醇、巯基乙醇、2-巯基吡啶、半胱氨酸或者谷胱甘肽中的任意一种。10. The reversible modification method of a protein according to claim 6, characterized in that the thiol-containing reducing agent is selected from the group consisting of propanethiol, mercaptoethanol, 2-mercaptopyridine, cysteine or glutathione. Any of the glycopeptides.
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