CN112979509B

CN112979509B - Trifluoromethanesulfonyl alkynamide compound and preparation method and application thereof

Info

Publication number: CN112979509B
Application number: CN202110260906.7A
Authority: CN
Inventors: 赵军锋; 王长流
Original assignee: Jiangxi Normal University
Current assignee: Guangzhou Xinpeptide Biopharmaceutical Technology Co ltd
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2022-04-22
Anticipated expiration: 2041-03-10
Also published as: CN112979509A

Abstract

The invention discloses a trifluoromethanesulfonyl alkynamide compound and a preparation method and application thereof. Based on the sulfhydrylation reaction of the alkynylamide and sulfydryl, the selective modification and marking of the sulfydryl in the polypeptide and the protein are further realized. The compound has better stability. But also selectively reacts with sulfhydryl group only, and other active groups on the polypeptide or protein are not affected; the method has the advantages of high reaction rate, no side reaction, simple and mild reaction conditions, easy operation, wide substrate applicability, high atom economy and the like. The polypeptide has good stability under acid-base and oxidation conditions, can stably exist for several months at normal temperature, and provides a new robust approach for bioconjugation and selective modification and labeling of sulfhydryl in polypeptide and/or protein.

Description

Trifluoromethanesulfonyl alkynamide compound and preparation method and application thereof

Technical Field

The invention relates to a sulfhydrylation reaction of alkynylamide, in particular to a trifluoromethanesulfonyl alkynylamide compound, a preparation method thereof, application of the compound in the sulfhydrylation reaction, and selective modification and marking of sulfydryl in polypeptide and/or protein by using the compound. Belongs to the technical field of organic chemical synthesis and chemical modification of polypeptide and/or protein.

Background

The protein is a biological macromolecule with important biological activity formed by connecting natural alpha-amino acids through amido bonds according to a certain sequence, is the material basis of life, and plays an important role in life activities. Proteins often need to be modified to perform their functions, and in order to study the biological functions and activities of proteins, proteins are also modified and labeled. Therefore, it is of great significance to develop and research a method and a tool capable of precisely modifying and labeling proteins, and an important research field today.

There are two main forms of chemical modification of proteins. One is posttranslational modification (PTMs) of proteins, and the precursor proteins, after synthesis, can explosively increase the type of protein through PTMs, giving more complexity to the life process. These modifications include mainly phosphorylation, acetylation, ubiquitination, glycosylation, and the like. PTMs are in fact processes which covalently process proteins by adding functional groups to one or several amino acid residues or by proteolytic cleavage, thereby altering the properties of the protein.

Yet another form is chemical modification, which is based primarily on chemical reactions at amino acid residues. In recent decades, methods for modifying proteins by organic synthesis have been reported more and more, and have greatly promoted the development of the field. Unlike conventional chemical reactions, this type of reaction often needs to be performed under relatively mild conditions because proteins are easily denatured under severe conditions. Chemical modifications of proteins in turn mainly include terminal modifications and side chain modifications. In the terminal modification, methionine (Met) is often synthesized at the first N-terminal as the starting point of protein synthesis, but Met fate is often removed by specific methionine aminopeptidase (MetAPs) or acetylated immediately after synthesis, and N-terminal of the second amino acid which is cleaved to expose is modified subsequently, wherein the modification includes introduction of some fluorescent groups and the like in addition to acetylation. While the C-terminal study is relatively rare. Modification of protein side chains is a well studied field (nat. rev. chem. 2019,3(3), 147. 171.), where cysteine (Cys) is extensively studied by scientists due to its unique properties. The natural abundance of Cys in proteins is low, and because the atomic radius of sulfur in Cys is large and the dissociation energy of S-H is low, nucleophilic reactions, redox reactions and other reactions in which amino acid side chains are difficult to occur easily occur. Common thiol-modifying reagents include halogenated alkanes, maleimides, metal reagents, and the like. Michael addition acceptors are also quite common modifying reagents (curr. opin. chem. biol.2020,58, 28-36.). Despite the extensive research on thiol-modifying agents, several challenges remain: such as the requirement that the reagent has good selectivity, the reagent must be capable of reacting in an aqueous phase, the reaction can be completed efficiently, and the product needs good stability; such as maleimide, are very unstable (nat. biotechnol.2012,30, 184-. Common thiol modifying reagents are shown in FIG. 2.

Disclosure of Invention

Aiming at the defects of the prior art, the invention discloses a trifluoromethanesulfonyl alkyne amide compound which is introduced into an alkyne amide nitrogen atom and has stronger electron-withdrawing capability, and develops a trifluoromethanesulfonyl alkyne amide compound capable of rapidly generating a sulfhydrylation reaction. And a method for selectively modifying and marking sulfydryl in polypeptide and/or protein through the sulfhydrylation reaction of trifluoromethanesulfonyl alkyne amide compounds. The reaction selectivity of the carbon-carbon triple bond in the common alkynylamide to the sulfydryl in the cysteine is extremely high, the reaction condition is quite simple and mild, the selective modification of the polypeptide and the cysteine in the protein can be realized only in an organic solvent and in an alkaline environment, and the product is quite stable. The method has the advantages of high reaction selectivity, no other side reaction, simple and mild conditions, easy operation, wide substrate applicability and high atom economy, but the reaction rate is slow (generally more than 24 h). The novel trifluoromethanesulfonyl alkynylamide compound not only has the advantages of common alkynylamide, but also can rapidly react with a sulfhydryl-containing compound through sulfhydrylation, and cannot react with carboxylic acid, and meanwhile, the stability of the compound to acidic conditions is remarkably improved; but also can selectively react with only sulfhydryl, and other active groups on the polypeptide are not affected. Is an excellent reagent which can realize selective modification of sulfhydryl in polypeptide and/or protein.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

according to a first embodiment of the present invention, there is provided a trifluoromethanesulfonyl alkynamide compound.

A trifluoromethanesulfonyl alkynamide compound having the general structural formula (I):

wherein in the formula (I), R is selected from one or more of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group.

Preferably, the compound is prepared by the following method: firstly, amine compounds with a structural general formula (II) and trifluoromethanesulfonyl chloride react to prepare the trifluoromethylamine compounds with a structural general formula (III). And then reacting the trifluoromethyl amine compound with the general structural formula (III) with a compound (IV) in the presence of alkali to prepare the trifluoromethanesulfonyl alkynylamide compound with the general structural formula (I).

Wherein in the formula (II) and the formula (III), R is selected from one or more of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group.

Preferably, the molar ratio of the amine compound having the general structural formula (II) to trifluoromethanesulfonyl chloride is 1:0.8 to 1.8, preferably 1:1 to 1.5, more preferably 1:1.1 to 1.3.

Preferably, the base is EtONa, EtOLi, Cs₂CO₃、K₂CO₃、Na₂CO₃、Ca(OH)₂One or more of LiOH and DBU.

Preferably, the molar ratio of the compound (IV) to the alkali in the compound (III) is 1:0.5-3:1-10, preferably 1:0.8-2.5:2-8, and more preferably 1:1-2: 3-5.

According to a second embodiment of the present invention, there is provided a process for producing trifluoromethanesulfonyl alkynamide compounds.

A process for the preparation of trifluoromethanesulfonyl alkynamide compounds of general structural formula (I) or a process for the preparation of trifluoromethanesulfonyl alkynamide compounds of general structural formula (I) as described in the first embodiment:

the method comprises the following steps:

1) dissolving amine compounds with a structural general formula (II) and triethylamine in a first solvent, then adding trifluoromethanesulfonyl chloride for reaction, and obtaining the trifluoromethylamine compounds with a structural general formula (III) after the reaction is completed:

2) dissolving a trifluoromethylamine compound with a structural general formula (III) and alkali in a second solvent to obtain a solution A. Then, compound (IV) is dissolved in the first solvent to obtain solution B. And finally, adding the solution B into the solution A for reaction to obtain the trifluoromethanesulfonyl alkynylamide compound with the structural general formula (I):

wherein R is selected from one or more of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group.

Preferably, in step 1), the first solvent is an organic solvent, preferably one or more of dichloromethane, chloroform, and N, N-dimethylformamide.

Preferably, in step 1), the amine compound having the general structural formula (II), triethylamine and trifluoromethanesulfonyl chloride are added in a molar ratio of 1:1.8-5:0.8-1.8, preferably 1:2-4:1-1.5, more preferably 1:2.2-3: 1.1-1.3.

Preferably, in step 2), the second solvent is an organic solvent, preferably N, N-dimethylformamide.

Preferably, in step 2), the base is EtONa, EtOLi, Cs₂CO₃、K₂CO₃、Na₂CO₃、Ca(OH)₂One or more of LiOH and DBU.

Preferably, in the step 2), the molar ratio of the compound of the trifluoromethylamine class with the general structural formula (III), the compound (IV) and the base is 1:0.5-3:1-10, preferably 1:0.8-2.5:2-8, and more preferably 1:1-2: 3-5.

Preferably, step 1) is specifically: firstly, dissolving the amine compound with the general structural formula (II) in a first solvent (preferably dichloromethane), then adding triethylamine, uniformly stirring (stirring for 1-10min, preferably stirring for 3-8min), and then placing the reaction solution in an ice-water bath. And finally adding trifluoromethanesulfonyl chloride for reaction (stirring for 10-100min, preferably for 20-50 min). After the reaction is finished, the compound of the trifluoromethylamine class with the structural general formula (III) is obtained after column chromatography separation.

Preferably, step 2) is specifically: the method comprises the steps of firstly dissolving the trifluoromethylamine compound with the structural general formula (III) in a second solvent (preferably N, N-dimethylformamide), then adding the alkali and uniformly stirring (for example, stirring for 1-10min, preferably for 3-8min) to obtain a solution A. Compound (IV) is dissolved in a first solvent (preferably dichloromethane) to obtain solution B. Finally, the solution A is placed in an ice-water bath, and then (1-8 drops/s, preferably 2-3 drops/s) the solution B is slowly added into the solution A to carry out reaction (stirring reaction is carried out for 0.2-12h, preferably for 0.5-5h, and more preferably for 0.8-3 h). After the reaction is finished, the trifluoromethanesulfonyl alkynylamide compound with the general structural formula (I) is obtained after column chromatography separation.

According to a third embodiment of the present invention, there is provided a use of a trifluoromethanesulfonyl alkynamide compound.

Use of a trifluoromethanesulfonyl alkynylamide compound of general structural formula (I) as described in the first embodiment or of a trifluoromethanesulfonyl alkynylamide compound of general structural formula (I) prepared as described in the second embodiment: the trifluoromethanesulfonyl alkyne amide compound with the structural general formula (I) is used for carrying out a sulfhydrylation reaction with thiophenol and/or thiol, or is used for selectively modifying and marking sulfydryl in peptides and proteins.

Preferably, the trifluoromethanesulfonyl alkyne amide compound with the general structural formula (I) is used for selectively modifying and marking side chain sulfydryl in peptides and proteins.

According to a fourth embodiment of the invention, a method for selectively modifying and labeling sulfydryl in peptides and proteins by using the trifluoromethanesulfonyl alkyne amide compound is provided.

A method for the selective modification and labelling of thiol groups in peptides and proteins using the use of trifluoromethanesulfonyl alkynylamides of general structural formula (I) as described in the first embodiment or using trifluoromethanesulfonyl alkynylamides of general structural formula (I) prepared as described in the second embodiment, which method comprises the steps of:

C) reacting trifluoromethanesulfonyl alkynamide compounds with a general structural formula (I) with sulfhydryl-containing peptides or proteins with a general structural formula (V) in a weakly alkaline reaction system to obtain trifluoromethanesulfonyl-modified thioether compounds with a general structural formula (VI):

wherein R is selected from one or more of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group. R¹Is cysteine group, peptide chain group containing cysteine, protein group containing cysteine, amino and/or carboxyl protected cysteine group, ammoniaOne or more of a group and/or a carboxyl-protected cysteine-containing peptide chain group, an amino-and/or carboxyl-protected cysteine-containing protein group.

Preferably, in step C), the weakly basic reaction system is a mixed system of a more basic buffer and an organic solvent.

Preferably, in step C), the volume ratio of the more basic buffer to the organic solvent is from 1 to 50:1, preferably from 5 to 40:1, more preferably from 10 to 30: 1.

Preferably, in step C), the more basic buffer is selected from one of PBS buffer (phosphate buffered saline), Tris (Tris hydroxymethyl aminomethane) buffer. The organic solvent is one of acetonitrile, N-dimethylformamide and dimethyl sulfoxide.

Preferably, in step C), the molar ratio of the trifluoromethanesulfonyl alkynamide compound of the general structural formula (I) to the thiol-group-containing polypeptide or protein of the general structural formula (V) is 1-10:1, preferably 1.1-4:1, more preferably 1.2-2: 1.

Preferably, step C) is specifically: dissolving trifluoromethanesulfonyl alkynylamide compounds having the general structural formula (I) and sulfhydryl-containing polypeptides or proteins having the general structural formula (V) in a weakly alkaline reaction system, mixing uniformly, and reacting (for example, stirring at 0-50 deg.C for 0-60min, preferably at 30-40 deg.C for 20-50 min). After the reaction is finished, the sulfur ether compound modified by the trifluoromethanesulfonyl group with the general structural formula (VI) is obtained through column chromatography separation.

In the prior art, the conjugate addition of cysteine to michael acceptors is the primary method for selective alkylation of cysteine side chain thiols. As early as 1949, it was reported that N-ethylmaleimide (NEM) can perform a highly efficient specific reaction with a thiol group, and the reaction can be used not only for titration of cysteine-containing peptide chains participating in the reaction, but also in biological studies as an antimitotic agent. The traditional maleimide drugs have the defects of irreversible reaction, modification, limited number of reaction sites and the like, and the succinimide chain part of the addition product is easy to hydrolyze. In 2010, James r. baker group reversibly modified proteins with bromomaleimide, providing three attachment points for protein bio-conjugation. In 2016, Gonc, alo j.l. bernardes proposed a strategy for chemoselective coupling of a carbonyl acrylic acid reagent to cysteine. These reagents react stoichiometrically with thiol groups rapidly with michael addition in a physiological environment. In 2017, Chuanzheng Zhou topic group reported a strategy for obtaining bioconjugates by using the specific reaction of 5-methylene pyrrolidone and sulfhydryl groups. 5-methylene pyrrolidone is easily prepared from primary amine in one step, and compared with common maleimide, the 5-methylene pyrrolidone has better stability under physiological conditions and further improves the selectivity with cysteine. Besides cysteine, other functional groups of the polypeptide and the protein side chain can also participate in Michael addition reaction, so that a certain side reaction exists in precise modification of the polypeptide and the cysteine side chain in the protein, and therefore special functional groups must be designed to realize selective modification of the polypeptide and the cysteine in the protein under mild conditions.

The inventor of the invention researches and discovers that the thiol-containing compound can be subjected to a thiohydrogenation reaction by taking common alkynylamide as a starting material in the presence of alkali, and the selective modification of thiol in polypeptide and protein can be realized through the reaction. The reaction selectivity of the carbon-carbon triple bond in the common alkynylamide to the sulfydryl in the cysteine is extremely high, the reaction condition is quite simple and mild, the selective modification of the polypeptide and the cysteine in the protein can be realized only in an organic solvent and in an alkaline environment, and the product is quite stable. The method has the advantages of high reaction selectivity, simple and mild conditions, easy operation, wide substrate applicability and high atom economy, and meanwhile, the reaction can be used as an effective strategy for building an AviCys structural unit. Meanwhile, a more concise and feasible way with wider application prospect is provided for the selective modification of cysteine in polypeptide or protein.

In the present invention, although a common alkynylamide can undergo a sulfhydrylation reaction under alkaline conditions, if such alkynylamide is applied to a polypeptide side chain cysteine modification, the reaction is found to be very slow (more than 24 hours), and the alkynylamide lacks sufficient stability in an aqueous solution, which is obviously inconvenient for the modification of the polypeptide. On the basis of the earlier research, the invention develops a trifluoromethanesulfonyl alkynylamide compound capable of rapidly generating a sulfhydrylation reaction by improving the structure of the alkynylamide and introducing trifluoromethanesulfonyl with stronger electron-withdrawing capability on the nitrogen atom of the alkynylamide. Compared with other common alkynylamides, the novel trifluoromethanesulfonyl alkynylamide compound not only has the advantages of the common alkynylamides, but also can not react with carboxylic acid, and meanwhile, the stability of the compound to acidic conditions is remarkably improved. But also can selectively react with sulfhydryl groups only, and other active groups on the polypeptide are not affected, thus being an excellent reagent which can realize selective modification on polypeptide and protein cysteine.

In the present invention, the trifluoromethanesulfonyl alkynamide compound is prepared by the following method: firstly, amine compounds with a structural general formula (II) and trifluoromethanesulfonyl chloride react to prepare the trifluoromethylamine compounds with a structural general formula (III). And then reacting the trifluoromethylamine compound with the general structural formula (III) with the high-valence iodoacetylene compound with TMS protection, which has the general structural formula (IV), in the presence of alkali to prepare the trifluoromethanesulfonyl alkynylamide compound with the general structural formula (I). The method specifically comprises the following steps:

the first step is as follows: dissolving amine compounds with a general structural formula (II) and triethylamine in a first solvent (such as dichloromethane), adding trifluoromethanesulfonyl chloride to react, and obtaining the trifluoromethanesulfonic amine compounds with a general structural formula (III):

the second step is that: the solution a is obtained by dissolving a trifluoromethanamine compound having the general structural formula (III) and a base (e.g., cesium carbonate) in a second solvent (e.g., N-dimethylformamide). The TMS protected higher iodoacetylene compound of general structural formula (IV) is then dissolved in a first solvent (e.g., dichloromethane) to obtain solution B. Finally, slowly adding (for example, 1-8 drops/s, preferably 2-3 drops/s) the solution B into the solution A to perform reaction (the reaction temperature is-20-50 ℃, preferably-10-30 ℃, more preferably 0-20 ℃, for example, in an ice water bath, and the reaction time is 0.2-12h, preferably 0.5-5h, more preferably 0.8-3h), and obtaining the trifluoromethanesulfonyl alkynylamide compound with the structural general formula (I) after the reaction is completed:

in the invention, the trifluoromethanesulfonyl alkynylamide compound prepared by the invention not only has the advantages of common alkynylamide, but also can selectively react with only sulfydryl, and other active groups on the polypeptide are not influenced. Is an excellent reagent which can realize selective modification and marking of sulfhydryl in peptide and protein.

In the invention, the trifluoromethanesulfonyl alkyne amide compound with the general structural formula (I) is used for carrying out a sulfhydrylation reaction with thiophenol and/or thiol, or is used for selectively modifying and marking sulfydryl in peptides and proteins. The method specifically comprises the following steps:

reacting a trifluoromethanesulfonyl alkynamide compound with a general structural formula (I) with a sulfhydryl-containing compound (including thiophenol, thiol and sulfhydryl-containing peptide or protein) with a general structural formula (V) in a weakly alkaline reaction system to obtain a trifluoromethanesulfonyl-modified thioether compound with a general structural formula (VI):

wherein R is selected from alkyl, hydroxyethyl, propargyl, and indometacinOne or more of a group of a molecule, a biotin-containing group, a coumarin-containing fluorescent group. R¹Is one or more of C1-C24 alkyl, C1-C24 alkyl substituted by substituent, cysteine group, peptide chain group containing cysteine, protein group containing cysteine, amino and/or carboxyl protected cysteine group, peptide chain group containing cysteine protected by amino and/or carboxyl, and protein group containing cysteine protected by amino and/or carboxyl.

In the invention, based on the sulfhydrylation reaction of alkyne amide, the trifluoromethanesulfonyl alkyne amide compound is adopted to selectively modify and mark sulfydryl in polypeptide and protein. Compared with the reported selective modification method, the reaction selectivity of the carbon-carbon triple bond of the alkynylamide in the trifluoromethanesulfonyl alkynylamide compound on the sulfydryl in the cysteine is extremely high, the reaction condition is quite simple and mild, the selective modification of the sulfydryl in the polypeptide and the protein can be realized only in an organic solvent and in an alkaline environment, the reaction rate is high, and the product is quite stable. Provides a more concise and feasible way with wider application prospect for selectively modifying and marking sulfydryl in peptide and protein.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the trifluoromethanesulfonyl alkynylamide compound is synthesized for the first time, not only has the advantages of common alkynylamide, but also has the advantages of high reaction selectivity, high reaction rate, no other side reaction, simple and mild conditions, easy operation, wide substrate applicability, high atom economy and the like in the selective modification and marking of sulfydryl in peptides and proteins.

2. The sulfhydrylation reaction of the trifluoromethanesulfonyl alkyne amide compound selectively modifies and marks sulfydryl in peptides and proteins. The reaction selectivity of the carbon-carbon triple bond in the alkynylamide to the sulfydryl in the cysteine is extremely high, the reaction condition is quite simple and mild, the reaction can be quickly finished only in an organic solvent in a weakly alkaline environment, and the product is quite stable. Provides a more concise and feasible way with wider application prospect for selectively modifying and marking sulfydryl in peptide and protein.

3. The trifluoromethanesulfonyl alkynamide compound disclosed by the invention has better stability and can stably exist under both acidic and alkaline conditions. Meanwhile, the compound is all trans-addition when reacting with sulfhydryl, and has very good regioselectivity.

Drawings

FIG. 1 is a general structural formula of the trifluoromethanesulfonyl alkynamide compound.

FIG. 2 shows a thiol-modifying reagent commonly used in the prior art.

FIG. 3 is an HPLC chart showing the reaction of alkynylamide with glutathione in twice the amount of preparation example 11 of the present invention using example 5.

FIG. 4 shows the double preparation of the alkynylamide and pentapeptide Ac-Leu-Asp-Phe-Cys-Gly-NH of preparation example 11 according to application example 6 of the invention₂HPLC profile of the reaction.

FIG. 5 is a diagram of the preparation of the alkynylamide and decapeptide H-His-Lys-Asp-Cys-Gln-Ala-Ser-Trp-Arg-Tyr-NH of preparation example 11 in twice the amount of the present invention in application example 7₂HPLC profile of the reaction.

FIG. 6 is an HPLC chart showing the reaction of the alkynylamide with linear oxytocin in four-fold amount in production example 11 according to application example 8 of the present invention.

Detailed Description

The technical solution of the present invention is illustrated below, and the claimed scope of the present invention includes, but is not limited to, the following examples.

Preparation embodiment:

a process for the preparation of trifluoromethanesulfonyl alkynamide compounds having the general structural formula (I):

the method comprises the following steps:

Preferably, step 1) is specifically: the amine compound with the general structural formula (II) is dissolved in a first solvent (preferably dichloromethane), then triethylamine is added and stirred uniformly (for example, stirring for 1-10min, preferably stirring for 3-8min), and then the reaction solution is placed in an ice-water bath. And finally adding trifluoromethanesulfonyl chloride for reaction (stirring for 10-100min, preferably for 20-50 min). After the reaction is finished, the compound of the trifluoromethylamine class with the structural general formula (III) is obtained after column chromatography separation.

Preferably, step 2) is specifically: the method comprises the steps of firstly dissolving the trifluoromethylamine compound with the structural general formula (III) in a second solvent (preferably N, N-dimethylformamide), then adding the alkali and uniformly stirring (for example, stirring for 1-10min, preferably for 3-8min) to obtain a solution A. Compound (IV) is dissolved in a first solvent (preferably dichloromethane) to obtain solution B. Finally, the solution A is placed in an ice-water bath, and then (1-8 drops/s, preferably 2-3 drops/s) the solution B is slowly added into the solution A to carry out reaction (the stirring reaction is carried out for 20-100min, preferably for 20-30 min). After the reaction is finished, the trifluoromethanesulfonyl alkynylamide compound with the general structural formula (I) is obtained after column chromatography separation.

Preparation of example 1

Preparation of 1,1, 1-trifluoromethyl-N-phenethylsulfonamide:

under the ice bath condition, 5mmol of phenethylamine and 10mmol of triethylamine are mixed in a dichloromethane solution for stirring, 5mmol of trifluoromethanesulfonyl chloride is slowly dripped, water is added after the reaction is completed, the water phase is extracted twice by using dichloromethane, the organic phase is combined and washed once by saturated saline solution, the organic phase is separated and dried by anhydrous sodium sulfate, and the organic phase is concentrated to obtain 1,1, 1-trifluoromethyl-N-phenethyl sulfonamide with light yellow liquid and 92% yield. The following are the structural formula of the product, nuclear magnetic resonance experimental data and mass spectrum experimental data:

¹HNMR(400MHz,CDCl₃)δ7.33–7.13(m,5H),4.65(s,1H),3.02(s,2H),2.83(s,2H).

¹³C NMR(100MHz,CDCl₃)δ139.4,129.1,128.1,126.7,121.1,119.0,116.8,114.7,46.4, 46.3,463,46.3,35.6.

HRMS(ESI)m/z calcd.for C₉H₁₁F₃NO₂S⁺[M+H]⁺:254.0457,found:2540.0462。

preparation of example 2

Preparation of N-butyl-1, 1, 1-trifluoromethylsulfonamide:

under the ice bath condition, 5mmol of N-butylamine and 10mmol of triethylamine are mixed in a dichloromethane solution for stirring, 5mmol of trifluoromethanesulfonyl chloride is slowly dripped, water is added after the reaction is completed, the water phase is extracted twice by using dichloromethane, the organic phase is combined and washed once by saturated saline solution, the organic phase is separated, dried by anhydrous sodium sulfate and concentrated to obtain N-butyl-1, 1, 1-trifluoromethyl sulfonamide, light yellow liquid, and the yield is 92%. The following are the structural formula of the product, nuclear magnetic resonance experimental data and mass spectrum experimental data:

¹H NMR(400MHz,CDCl₃)δ4.47(s,1H),2.87(s,2H),1.51(s,2H),1.30(s,2H),0.90(s, 3H).

¹³C NMR(100MHz,CDCl₃)δ121.11,118.97,116.83,114.68,47.27,47.24,47.21,47.18, 31.07,20.45,14.00.

HRMS(ESI)m/z calcd.for C₅H₁₁F₃NO₂S⁺[M+H]⁺:206.0457,found:206.0460。

preparation of example 3

Preparation of tert-butyl (2- ((trifluoromethyl) sulfonamido) ethyl) carbamate:

under the ice bath condition, 5mmol of n-butyl (2-aminoethyl carbamate) and 10mmol of triethylamine are mixed in a dichloromethane solution for stirring, 5mmol of trifluoromethanesulfonyl chloride is slowly dripped, water is added after the reaction is completed, the water phase is extracted twice by using dichloromethane, organic phases are combined and washed once by saturated saline solution, the organic phase is separated, dried by using anhydrous sodium sulfate, and the organic phase is concentrated to obtain tert-butyl (2- ((trifluoromethyl) sulfanylamino) ethyl) carbamate as a light yellow liquid with the yield of 95%. The following are the structural formula of the product, nuclear magnetic resonance experimental data and mass spectrum experimental data:

¹H NMR(400MHz,CDCl₃)δ8.52(s,1H),4.44(s,1H),3.42(s,2H),2.86(s,2H),1.42(s, 9H).

¹³C NMR(100MHz,CDCl₃)δ158.57,121.11,118.97,116.83,114.68,80.89,45.48,45.45, 39.46,28.33.

HRMS(ESI)m/z calcd.for C₈H₁₆F₃N₂O₄S⁺[M+H]⁺:293.0777,found:293.0772。

preparation of example 4

1,1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide:

under the ice bath condition, 5mmol of 2-aminoethanol and 10mmol of triethylamine are mixed in a dichloromethane solution for stirring, 5mmol of trifluoromethanesulfonyl chloride is slowly dripped, water is added after the reaction is completed, a water phase is extracted twice by using dichloromethane, organic phases are combined and washed once by saturated saline solution, the organic phase is separated and dried by anhydrous sodium sulfate, and the organic phase is concentrated to obtain 1,1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide as a light yellow liquid with the yield of 85%. The following are the structural formula of the product, nuclear magnetic resonance experimental data and mass spectrum experimental data:

¹H NMR(400MHz,CDCl₃)δ4.22(s,1H),3.44(d,J＝4.5Hz,2H),2.98(d,J＝4.5Hz,2H), 1.51(s,1H).

¹³C NMR(100MHz,CDCl₃)δ121.11,118.97,116.83,114.68,60.01,46.74,46.71.

HRMS(ESI)m/z calcd.for C₃H₆F₃NO₃S[M+H]⁺:194.0093,found:194.0096。

preparation of example 5

N-cyclohexyl-1, 1, 1-trifluoromethylmethanesulfonamide:

under the ice bath condition, 5mmol of cyclohexylamine and 10mmol of triethylamine are mixed in a dichloromethane solution for stirring, 5mmol of trifluoromethanesulfonyl chloride is slowly dripped, water is added after the reaction is completed, a water phase is extracted twice by using dichloromethane, organic phases are combined and washed once by saturated saline solution, the organic phase is separated, dried by anhydrous sodium sulfate, and concentrated to obtain N-cyclohexyl-1, 1, 1-trifluoromethyl methanesulfonamide as a light yellow liquid with the yield of 80%. The following are the structural formula of the product, nuclear magnetic resonance experimental data and mass spectrum experimental data:

¹H NMR(400MHz,CDCl₃)δ4.45(s,1H),3.13(t,J＝14.9Hz,1H),1.95–1.77(m,2H), 1.73–1.51(m,3H),1.42–1.18(m,2H),1.12(dtd,J＝7.5,5.1,3.1Hz,3H).

¹³C NMR(100MHz,CDCl₃)δ121.56,119.42,117.28,115.13,55.25,55.22,55.19,55.16, 33.10,25.92,24.73.

HRMS(ESI)m/z calcd.for C₇H₁₃F₃NO₂S⁺[M+H]⁺:232.0614,found:232.0621。

preparation of example 6

1,1, 1-trifluoromethyl-N-propargylmethanesulfonamide:

under the ice bath condition, 5mmol of propargylamine and 10mmol of triethylamine are mixed in a dichloromethane solution for stirring, 5mmol of trifluoromethanesulfonyl chloride is slowly added dropwise, water is added after the reaction is completed, the water phase is extracted twice by using dichloromethane, the organic phase is combined and washed once by saturated saline solution, the organic phase is separated, dried by using anhydrous sodium sulfate, and the organic phase is concentrated to obtain 1,1, 1-trifluoromethyl-N-propargyl methanesulfonamide as a light yellow liquid with the yield of 56%. The following are the structural formula of the product, nuclear magnetic resonance experimental data and mass spectrum experimental data:

¹H NMR(400MHz,CDCl₃)δ4.53(s,1H),3.47(dd,J＝14.1,6.0Hz,2H),3.08(t,J＝6.0Hz, 1H).

¹³C NMR(100MHz,CDCl₃)δ121.11,118.97,116.83,114.68,77.75,66.65,33.20,33.17.

HRMS(ESI)m/z calcd.for C₄H₅F₃NO₂S⁺[M+H]⁺:187.9988,found:187.9981。

preparation of example 7

2-oxa-N- (2- ((trifluoromethyl) sulfonamido) ethyl) -2 h-benzopyran-3-carboxamide:

under the ice bath condition, 5mmol of N- (2-aminoethyl) -2-oxa-2-hydrogen-benzopyran-3-formamide and 10mmol of triethylamine are mixed in a dichloromethane solution for stirring, 5mmol of trifluoromethanesulfonyl chloride is slowly added dropwise, water is added after the reaction is completed, a water phase is extracted twice by using dichloromethane, an organic phase is combined and washed once by saturated saline solution, the organic phase is separated, dried by anhydrous sodium sulfate, and concentrated to obtain 2-oxa-N- (2- ((trifluoromethyl) sulfanylamino) ethyl) -2-hydrogen-benzopyran-3-formamide which is white solid and has the yield of 82%. The following are the structural formula of the product, nuclear magnetic resonance experimental data and mass spectrum experimental data:

¹H NMR(400MHz,CDCl₃)δ8.24(s,1H),7.68(dd,J＝14.5,3.1Hz,1H),7.62–7.49(m, 1H),7.42–7.27(m,2H),3.42(t,J＝9.4Hz,2H),2.86(t,J＝9.4Hz,2H).

¹³C NMR(100MHz,CDCl₃)δ164.06,164.01,152.43,134.05,132.91,128.54,125.32, 121.11,120.12,119.91,118.97,117.43,116.83,114.68,45.51,45.48,45.45,45.42,38.65.

HRMS(ESI)m/z calcd.for C₁₃H₁₂F₃N₂O₅S⁺[M+H]⁺:365.0414,found:365.0418。

preparation of example 8

Preparation of N-ethynyl-1, 1, 1-trifluoromethyl-N-phenethylsulfonamide:

in an ice-water bath, 1,1, 1-trifluoromethyl-N-phenethyl sulfonamide prepared in preparation example 1 and 2 times of cesium carbonate are dissolved in N, N-dimethylformamide and uniformly mixed; and dissolving 2.5 times of the compound (IV) in dichloromethane, slowly adding the dichloromethane into the uniformly mixed reaction solution for reaction, detecting by using a TLC (thin layer chromatography) plate, adding ice water into the reaction solution after the reaction is finished, extracting by using ethyl acetate for 3 times, concentrating an organic layer, and carrying out column chromatography separation to obtain pure N-ethynyl-1, 1, 1-trifluoromethyl-N-phenethylsulfonamide, wherein the yield is 95 percent. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ7.32(t,J＝7.2Hz,2H),7.27(d,J＝7.1Hz,1H),7.22(dd,J＝6.1,2.0Hz,2H),3.85–3.68(m,2H),3.16–3.00(m,2H),2.83(s,1H).

¹³C NMR(100MHz,CDCl₃)δ136.08,128.95,128.87,127.28,124.46,121.24,118.02, 114.80,71.76,60.18,54.63,34.36.

HRMS(ESI)m/z calcd.for C₁₁H₁₁F₃NO₂S⁺[M+H]⁺:278.0457,found:278.0464。

preparation of example 9

Preparation of N-butyl-N-ethynyl-1, 1, 1-trifluoromethylsulfonamide:

in an ice-water bath, dissolving the N-butyl-1, 1, 1-trifluoromethyl sulfonamide prepared in the preparation example 2 and 2 times of cesium carbonate in N, N-dimethylformamide and uniformly mixing; and dissolving 2.5 times of the compound (IV) in dichloromethane, slowly adding the dichloromethane into the uniformly mixed reaction solution for reaction, detecting by using a TLC (thin layer chromatography) plate, adding ice water into the reaction solution after the reaction is finished, extracting by using ethyl acetate for 3 times, concentrating an organic layer, and carrying out column chromatography separation to obtain pure N-butyl-N-ethynyl-1, 1, 1-trifluoromethyl sulfonamide, wherein the light yellow liquid is obtained, and the yield is 82%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ3.51(t,J＝7.3Hz,2H),2.70(s,1H),1.82–1.62(m,2H),1.45 –1.26(m,2H),0.89(t,J＝7.4Hz,3H).

¹³C NMR(100MHz,CDCl₃)δ124.50,121.28,118.06,114.84,71.84,59.50,53.32,29.76, 19.11,13.39.

HRMS(ESI)m/z calcd.for C₇H₁₁F₃NO₂S⁺[M+H]⁺:230.0457,found:230.0463。

preparation of example 10

Preparation of tert-butyl (2- ((N-ethynyl-1, 1, 1-trifluoromethyl) sulfonyl) ethyl) carbamate:

in an ice-water bath, firstly, dissolving tert-butyl (2- ((trifluoromethyl) sulfanylamino) ethyl) carbamate prepared in preparation example 3 and 2 times of sodium carbonate in N, N-dimethylformamide, and uniformly mixing; and dissolving 2.5 times of the compound (IV) in dichloromethane, slowly adding the dichloromethane into the uniformly mixed reaction solution for reaction, detecting by using a TLC (thin layer chromatography) plate, adding ice water into the reaction solution after the reaction is finished, extracting by using ethyl acetate for 3 times, concentrating an organic layer, and performing column chromatography separation to obtain pure tert-butyl (2- ((N-ethynyl-1, 1, 1-trifluoromethyl) sulfonyl) ethyl) carbamate as a light yellow liquid with the yield of 85%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹HNMR(400MHz,CDCl₃)δ4.44(s,1H),3.42(s,2H),2.86(s,2H),2.80(s,H),1.42(s,9H).

¹³C NMR(100MHz,CDCl₃)δ158.57,121.11,118.97,116.83,114.68,80.89,71.58, 59.92,45.48,45.45,39.46,28.33.

HRMS(ESI)m/z calcd.for C₁₀H₁₆F₃N₂O₄S+[M+H]⁺:317.0777,found:317.0785。

preparation of example 11

Preparation of N-ethynyl-1, 1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide:

in an ice-water bath, 1,1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide prepared in preparation example 4 and 2 times of potassium carbonate are dissolved in N, N-dimethylformamide and uniformly mixed; and dissolving 2.5 times of the compound (IV) in dichloromethane, slowly adding the dichloromethane into the uniformly mixed reaction solution for reaction, detecting by using a TLC (thin layer chromatography) plate, adding ice water into the reaction solution after the reaction is finished, extracting by using ethyl acetate for 3 times, concentrating an organic layer, and carrying out column chromatography separation to obtain pure N-ethynyl-1, 1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide as a light yellow liquid with the yield of 90%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ3.95(d,J＝4.6Hz,2H),3.73(t,J＝5.2Hz,2H),2.85(s,1H), 2.50(s,1H).

¹³C NMR(101MHz,CDCl₃)δ124.45,121.23,118.01,114.79,71.58,59.92,59.88,58.82, 55.09.

HRMS(ESI)m/z calcd.for C₅H₇F₃NO₃S⁺[M+H]⁺:218.0093,found:218.0099。

preparation of example 12

Preparation of N-cyclohexyl-N-ethynyl-1, 1, 1-trifluoromethylmethanesulfonamide:

under the ice-water bath, dissolving the N-cyclohexyl-1, 1, 1-trifluoromethyl methanesulfonamide prepared in the preparation example 5 and 2 times of lithium hydroxide in N, N-dimethylformamide and uniformly mixing; and dissolving 2.5 times of the compound (IV) in dichloromethane, slowly adding the dichloromethane into the uniformly mixed reaction solution for reaction, detecting by using a TLC (thin layer chromatography) plate, adding ice water into the reaction solution after the reaction is finished, extracting by using ethyl acetate for 3 times, concentrating an organic layer, and carrying out column chromatography separation to obtain pure N-cyclohexyl-N-ethynyl-1, 1, 1-trifluoromethyl methanesulfonamide as a light yellow liquid with the yield of 92%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ3.79(ddd,J＝11.9,8.0,4.1Hz,1H),2.79(s,1H),2.05–1.80 (m,5H),1.77–1.62(m,3H),1.37(q,J＝13.2Hz,2H),1.13(td,J＝13.0,6.6Hz,1H).

¹³C NMR(101MHz,CDCl₃)δ130.23,127.46,121.15,117.94,69.63,62.36,61.04,31.10, 25.25,24.56.

HRMS(ESI)m/z calcd.for C₉H₁₃F₃NO₂S⁺[M+H]⁺:256.0614,found:256.0619。

preparation of example 13

Preparation of N-ethynyl-1, 1, 1-trifluoromethyl-N-propargylmethanesulfonamide:

in an ice-water bath, 1,1, 1-trifluoromethyl-N-propargyl methanesulfonamide prepared in preparation example 6 and 2 times of cesium carbonate are dissolved in N, N-dimethylformamide and uniformly mixed; and dissolving 2.5 times of the compound (IV) in dichloromethane, slowly adding the dichloromethane into the uniformly mixed reaction solution for reaction, detecting by using a TLC (thin layer chromatography) plate, adding ice water into the reaction solution after the reaction is finished, extracting by using ethyl acetate for 3 times, concentrating an organic layer, and carrying out column chromatography separation to obtain pure N-ethynyl-1, 1, 1-trifluoromethyl-N-propargyl methanesulfonamide as a light yellow liquid with the yield of 92%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ4.53(s,1H),3.47(dd,J＝14.1,6.0Hz,2H),3.08(t,J＝6.0Hz, 1H),2.98(s,1H).

¹³C NMR(100MHz,CDCl₃)δ121.11,118.97,116.83,114.68,80.11,60.1277.75,66.65, 33.20,33.17.

HRMS(ESI)m/z calcd.for C₆H₅F₃NO₂S⁺[M+H]⁺:211.9988,found:211.9980。

preparation of example 14

Preparation of N- (2- ((N-ethynyl-1, 1, 1-trifluoromethyl) sulfonyl) ethyl) -2-oxa-2-hydro-benzopyran-3-carboxamide:

in an ice-water bath, 2-oxa-N- (2- ((trifluoromethyl) sulfanylamino) ethyl) -2 h-benzopyran-3-carboxamide prepared in preparation example 7 and 2 times of cesium carbonate are dissolved in N, N-dimethylformamide and mixed uniformly; and dissolving 2.5 times of the compound (IV) in dichloromethane, slowly adding the dichloromethane into the uniformly mixed reaction solution for reaction, detecting by using a TLC (thin layer chromatography) dot plate, adding ice water into the reaction solution after the reaction is finished, extracting by using ethyl acetate for 3 times, concentrating an organic layer, and performing column chromatography separation to obtain pure N- (2- ((N-ethynyl-1, 1, 1-trifluoromethyl) sulfonyl) ethyl) -2-oxa-2-hydrogen-benzopyran-3-formamide which is a white solid and has the yield of 90%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ9.14(s,1H),8.92(s,1H),7.70(t,J＝8.9Hz,2H),7.48–7.36 (m,2H),3.85(dd,J＝10.2,4.6Hz,4H),2.87(s,1H).

¹³C NMR(101MHz,CDCl₃)δ162.37,161.31,154.56,148.81,134.38,129.95,125.41, 121.19,118.53,117.82,116.74,71.57,60.17,52.12,37.94.

HRMS(ESI)m/z calcd.for C₁₅H₁₂F₃N₂O₅S⁺[M+H]⁺:389.0414,found:389.0418。

the application embodiment is as follows:

a method for selectively modifying and marking sulfydryl in peptides and proteins by adopting trifluoromethanesulfonyl alkyne amide compounds with a structural general formula (I), which comprises the following steps:

wherein R is selected from one or more of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group. R¹Is one or more of a cysteine group, a cysteine-containing peptide chain group, a cysteine-containing protein group, an amino and/or carboxyl protected cysteine-containing peptide chain group, an amino and/or carboxyl protected cysteine-containing protein group.

Preferably, in step C), the molar ratio of the trifluoromethanesulfonylalkynamide compound of general structural formula (I) to the thiol-group-containing peptide or protein of general structural formula (V) is 1-10:1, preferably 1.1-4:1, more preferably 1.2-2: 1.

Preferably, step C) is specifically: dissolving trifluoromethanesulfonyl alkynamide compounds with a general structural formula (I) and sulfhydryl-containing peptides or proteins with a general structural formula (V) in a weakly alkaline reaction system, uniformly mixing, and reacting (stirring and reacting at 0-50 ℃ for 0-60min, preferably at 30-40 ℃ for 20-50 min). After the reaction is finished, the sulfur ether compound modified by the trifluoromethanesulfonyl group with the general structural formula (VI) is obtained through column chromatography separation.

Application example 1

Preparation of ethyl-N- ((tert-butoxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N-phenylethyl) sulfonamide) vinyl) -L-cysteine:

to a more basic buffer solution, 0.1mmol of ethyl (tert-butyloxycarbonyl) -L-propionyl-L-cysteine and twice the amount of N-ethynyl-1, 1, 1-trifluoromethyl-N-phenethylsulfonamide prepared in preparation example 8 were added, and after mixing well, acetonitrile at 37 ℃: and 8.0 stirring and reacting in a PBS (PBS) buffer solution of 1:20 for 20min, after the reaction is finished, adding ice water into the reaction solution, extracting the reaction solution for 3 times by using ethyl acetate, concentrating an organic layer, and separating by using column chromatography to obtain pure ethyl-N- ((tert-butoxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N-phenethyl) sulfonamide) vinyl) -L-cysteine, wherein the yield is 95%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ7.42(d,J＝6.3Hz,1H),6.21(d,J＝6.8Hz,1H),6.04(d,J＝ 6.7Hz,1H),5.59–5.26(m,1H),4.95–4.69(m,1H),4.24(dt,J＝12.9,6.5Hz,2H),3.81(d,J＝ 26.6Hz,2H),3.73–3.48(m,2H),3.44–3.00(m,2H),1.45(s,9H),1.34(dd,J＝10.2,6.0Hz, 3H).

¹³C NMR(100MHz,CDCl₃)δ173.87,173.52,157.83,120.59,117.73,117.39,117.35, 117.32,117.29,115.58,113.45,111.30,80.89,61.74,59.54,53.45,50.69,50.66,50.63,50.60, 49.71,32.86,28.33,18.31,14.68.

HRMS(ESI)m/z calcd.for C₂₄H₃₅F₃N₃O₇S₂ ⁺[M+H]⁺:598.1863,found:598.1869。

application example 2

Preparation of ethyl-N- ((tert-butoxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N-hydroxyethyl) sulfonamide) vinyl) -L-cysteine:

adding 0.1mmol of ethyl (tert-butyloxycarbonyl) -L-propionyl-L-cysteine and twice the amount of N-ethynyl-1, 1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide prepared in preparation example 11 into a slightly basic buffer solution, uniformly mixing, stirring and reacting for 20min in an acetonitrile (8.0 PBS) buffer solution (1: 20) at 37 ℃, adding ice water into the reaction solution after the reaction is finished, extracting for 3 times by using ethyl acetate, concentrating an organic layer, and separating by column chromatography to obtain pure ethyl-N- ((tert-butyloxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N-hydroxyethyl) sulfonamide) vinyl) -L-cysteine, white solid, yield 93%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

HRMS(ESI)m/z calcd.for C₁₈H₃₁F₃N₃O₈S₂ ⁺[M+H]⁺:538.1499,found:538.1502。

application example 3

Preparation of ethyl-N- ((tert-butoxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N- (2- (2-oxa-2H-benzopyran-3-carboxamide) ethyl) methyl) sulfonyl) alkenyl) -L-cysteine:

adding 0.1mmol of ethyl (tert-butyloxycarbonyl) -L-propionyl-L-cysteine and twice the amount of N- (2- ((N-ethynyl-1, 1, 1-trifluoromethyl) sulfonyl) ethyl) -2-oxa-2-hydro-benzopyran-3-carboxamide prepared in preparation example 14 into a slightly basic buffer solution, uniformly mixing, stirring and reacting in acetonitrile 8.0PBS buffer solution (1: 20) at 37 ℃ for 20min, after the reaction is finished, adding ice water into the reaction solution, extracting the reaction solution for 3 times by using ethyl acetate, concentrating the organic layer, and separating by column chromatography to obtain pure ethyl-N- ((tert-butyloxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N- (2- (2-oxa-2H-benzo) Pyran-3-carboxamide) ethyl) methyl) sulfonyl) alkenyl) -L-cysteine as a white solid in 94% yield. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ8.23(s,1H),7.67(s,1H),7.54(s,1H),7.34(d,J＝10.0Hz, 2H),6.80(d,J＝6.3Hz,2H),5.99(s,1H),5.21–4.84(m,3H),4.11(s,2H),3.69(d,J＝0.9Hz, 2H),3.54–3.23(m,4H),1.42(s,12H),1.21(s,3H).

¹³C NMR(100MHz,CDCl₃)δ173.87,173.52,164.19,164.14,157.83,153.29,134.37, 133.02,128.62,125.43,120.59,120.14,117.93,117.73,117.50,117.39,117.35,117.32,117.29, 115.58,113.45,111.30,80.89,61.74,53.45,49.71,48.72,48.70,48.66,48.63,38.20,32.86,28.33, 18.31,14.68.

HRMS(ESI)m/z calcd.for C₂₈H₃₆F₃N₄O₁₀S₂ ⁺[M+H]⁺:709.1819,found:709.1828。

application example 4

Preparation of ethyl-N- ((tert-butoxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N- (propargyl) ethyl) methyl) sulfonyl) alkenyl) -L-cysteine:

adding 0.1mmol of ethyl (tert-butyloxycarbonyl) -L-propionyl-L-cysteine and twice the amount of N-ethynyl-1, 1, 1-trifluoromethyl-N-propargylmethanesulfonamide prepared in preparation example 13 into a slightly basic buffer solution, uniformly mixing, stirring and reacting in acetonitrile 8.0PBS buffer solution (1: 20) at 37 ℃ for 20min, adding ice water into the reaction solution after the reaction is finished, extracting with ethyl acetate for 3 times, concentrating the organic layer, and separating by column chromatography to obtain pure ethyl-N- ((tert-butyloxycarbonyl) -L-propionyl) -S- ((Z) -2- ((1,1, 1-trifluoromethyl-N- (propargyl) ethyl) methyl) sulfonyl) alkenyl) -L-cysteine, white solid, yield 92%. The structural formula, nuclear magnetic characterization data and mass spectrum characterization data of the product are as follows:

¹H NMR(400MHz,CDCl₃)δ7.31(s,1H),6.78(s,1H),6.33(s,1H),5.21(s,1H),4.86(s, 1H),4.74(s,1H),4.16(d,J＝7.4Hz,2H),4.10(s,2H),3.69(s,1H),3.44(s,1H),3.07(s,1H), 1.42(s,12H),1.21(s,3H).

¹³C NMR(100MHz,CDCl₃)δ173.87,173.52,157.83,122.39,118.55,114.51,80.89,74.44, 61.74,53.45,49.71,38.79,36.47,32.86,28.33,18.30,14.68.

HRMS(ESI)m/z calcd.for C₁₉H₂₉F₃N₃O₇S₂ ⁺[M+H]⁺:532.1394,found:532.1399。

application example 5

N⁵Preparation of (- (R) -1- (carboxymethyl) amino) -1-oxa-3- (((Z) -2- ((1,1, 1-trifluoromethyl-N- (2-hydroxyethyl) methyl) sulfonamido) vinyl) mercapto) propan-2-oxy) -L-glutamine:

adding 0.1mmol of glutathione and twice the amount of N-ethynyl-1, 1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide prepared in preparation example 11 into a alkalescent buffer solution, uniformly mixing, stirring and reacting for 20min in acetonitrile (8.0 PBS buffer solution) 1:20 at 37 ℃, adding ice water into the reaction solution after the reaction is finished, extracting for 3 times by ethyl acetate, concentrating an organic layer, and carrying out column chromatography separation to obtain pure N⁵- ((R) -1- (carboxymethyl) amino) -1-oxa-3- (((Z) -2- ((1,1, 1-trifluoromethyl-N- (2-hydroxyethyl) methyl) sulfonamido) vinyl) mercapto) propan-2-oxy) -L-glutamine as a white solid in 95% yield. The following are the structural formula of the product and the characterization data of nuclear magnetism, mass spectrum and HPLC:

¹H NMR(400MHz,D₂O)δ6.57(d,J＝6.8Hz,1H),6.05(d,J＝6.7Hz,1H),4.57(dd,J＝ 8.6,5.1Hz,1H),3.99(t,J＝6.6Hz,1H),3.96(s,2H),3.64(dd,J＝7.8,4.1Hz,4H),3.29(dd,J＝ 14.5,5.1Hz,1H),3.08(dd,J＝14.5,8.7Hz,1H),2.54(td,J＝7.5,3.9Hz,2H),2.17(dd,J＝7.6, 3.8Hz,2H).

¹³C NMR(100MHz,D₂O)δ174.49,174.48,173.07,172.16,120.59,117.73,117.39,117.35, 117.32,117.29,115.58,113.45,111.30,59.54,55.84,54.29,50.69,50.66,50.63,50.60,41.39, 32.41,32.24,27.78.

HRMS(ESI)m/z calcd.for C₁₅H₂₄F₃N₄O₉S₂+[M+H]⁺:525.0931,found:525.0937。

application example 6

Pentapeptide Ac-Leu-Asp-Phe-Cys-Gly-NH₂Preparation of modified product:

in alkaline buffer solution, 0.01mmol pentapeptide Ac-Leu-Asp-Phe-Cys-Gly-NH was added₂And twice the amount of N-ethynyl-1, 1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide prepared in preparation example 11, were mixed well, and after stirring the reaction in acetonitrile: 8.0PBS buffer 1:20 at 37 ℃ for 20min, the reaction was terminated and monitored by HPLC to obtain a modified product. The following are mass spectra, HPLC characterization data:

HRMS(ESI)m/z calcd.for C₃₁H₄₅F₃N₇O₁₁S₂ ⁺[M+H]⁺:812.26,found:812.35。

application example 7

Decapeptide H-His-Lys-Asp-Cys-Gln-Ala-Ser-Trp-Arg-Tyr-NH₂Preparation of modified product:

adding 0.01mmol decapeptide H-His-Lys-Asp-Cys-Gln-Ala-Ser-Trp-Arg-Tyr-NH into the alkaline buffer solution₂And twice the amount of N-ethynyl-1, 1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide prepared in preparation example 11, were mixed well, and after stirring the mixture at 37 ℃ in acetonitrile: 8.0PBS buffer (1: 20), the reaction was carried out for 20min, and after completion of the reaction, the decapeptide H-His-Lys-Asp-Cys-Gln-Ala-Ser-Trp-Arg-Tyr-NH was monitored by HPLC₂The modified product of (1). The following are mass spectra, HPLC characterization data:

HRMS(ESI)m/z calcd.for C₆₁H₈₈F₃N₂₀O₁₈S₂ ⁺[M+H]⁺:1509.60,found:1509.35

application example 8

Straight chain oxytocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂Preparation of modified product:

adding 0.01mmol of linear oxytocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH into the slightly alkaline buffer solution₂And four times the amount of N-ethynyl-1, 1, 1-trifluoromethyl-N- (2-hydroxyethyl) methanesulfonamide prepared in preparation example 11, the mixture was uniformly mixed and stirred in acetonitrile at 37 ℃ and 8.0PBS buffer (1: 20) for reaction for 20min, and after the reaction was finished, the mixture was monitored by HPLC to obtain straight-chain oxytocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂The modified product of (1). The following are mass spectra, HPLC characterization data:

HRMS(ESI)m/z calcd.for C₆₁H₈₈F₃N₂₀O₁₈S₂ ⁺[M+H]⁺:1443.46,found:1443.49。

Claims

1. a trifluoromethanesulfonyl alkynamide compound is characterized in that: the compounds are of the general structural formula (I):

wherein in the formula (I), R is selected from one of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group.

2. The trifluoromethanesulfonylalkynamide compound according to claim 1, wherein: the compound is prepared by the following method: firstly, reacting an amine compound with a general structural formula (II) with trifluoromethanesulfonyl chloride to prepare a trifluoromethyl amine compound with a general structural formula (III); then reacting the trifluoromethyl amine compound with the general formula (III) with a compound (IV) in the presence of alkali to prepare the trifluoromethanesulfonyl alkynylamide compound with the general formula (I);

wherein in the formula (II) and the formula (III), R is selected from one of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group.

3. The trifluoromethanesulfonylalkynamide compound according to claim 2, wherein: the mol ratio of the amine compound with the structural general formula (II) to the trifluoromethanesulfonyl chloride is 1: 0.8-1.8; and/or

The alkali is EtONa, EtOLi, Cs₂CO₃、K₂CO₃、Na₂CO₃、Ca(OH)₂One or more of LiOH and DBU; and/or

The molar ratio of the compound with the structural general formula (III) to the compound (IV) to the alkali is 1:0.5-3: 1-10.

4. The trifluoromethanesulfonylalkynamide compound according to claim 3, wherein: the molar ratio of the amine compound with the structural general formula (II) to the trifluoromethanesulfonyl chloride is 1: 1-1.5; the molar ratio of the compound with the structural general formula (III) to the compound (IV) to the alkali is 1:0.8-2.5: 2-8.

5. The trifluoromethanesulfonylalkynamide compound according to claim 3, wherein: the molar ratio of the amine compound with the structural general formula (II) to the trifluoromethanesulfonyl chloride is 1: 1.1-1.3; the molar ratio of the compound with the structural general formula (III) to the compound (IV) to the alkali is 1:1-2: 3-5.

6. A process for the preparation of trifluoromethanesulfonyl alkynamide compounds having the general structural formula (I):

the method comprises the following steps:

2) dissolving a trifluoromethylamine compound with a structural general formula (III) and alkali in a second solvent to obtain a solution A; then dissolving the compound (IV) in a first solvent to obtain a solution B; and finally, adding the solution B into the solution A for reaction to obtain the trifluoromethanesulfonyl alkynylamide compound with the structural general formula (I):

wherein R is selected from one of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group.

7. The method of claim 6, wherein: in step 1), the first solvent is an organic solvent; the molar ratio of the adding amount of the amine compound with the structural general formula (II), triethylamine and trifluoromethanesulfonyl chloride is 1:1.8-5: 0.8-1.8; and/or

In step 2), the second solvent is an organic solvent; the alkali is EtONa, EtOLi, Cs₂CO₃、K₂CO₃、Na₂CO₃、Ca(OH)₂One or more of LiOH and DBU; the molar ratio of the compound with the structural general formula (III) to the compound (IV) to the alkali is 1:0.5-3: 1-10.

8. The method of claim 7, wherein: in the step 1), the first solvent is one or more of dichloromethane, trichloromethane and N, N-dimethylformamide; the molar ratio of the adding amount of the amine compound with the structural general formula (II), triethylamine and trifluoromethanesulfonyl chloride is 1:2-4: 1-1.5;

in step 2), the second solvent is N, N-dimethylformamide; the molar ratio of the compound with the structural general formula (III) to the compound (IV) to the alkali is 1:0.8-2.5: 2-8.

9. The method of claim 7, wherein: in the step 1), the molar ratio of the adding amount of the amine compound with the structural general formula (II), triethylamine and trifluoromethanesulfonyl chloride is 1:2.2-3: 1.1-1.3;

in the step 2), the molar ratio of the compound of the trifluoromethylamine class with the structural general formula (III), the compound (IV) and the alkali is 1:1-2: 3-5.

10. The method according to any one of claims 6-9, wherein: the step 1) is specifically as follows: firstly, dissolving an amine compound with a general structural formula (II) in a first solvent, then adding triethylamine and uniformly stirring, and then placing reaction liquid in an ice-water bath; finally adding trifluoromethanesulfonyl chloride for reaction; after the reaction is finished, obtaining the trifluoromethylamine compound with the structural general formula (III) through column chromatography separation; and/or

The step 2) is specifically as follows: dissolving a trifluoromethylamine compound with a structural general formula (III) in a second solvent, adding alkali, and uniformly stirring to obtain a solution A; dissolving the compound (IV) in a first solvent to obtain a solution B; finally, placing the solution A in an ice-water bath, and then slowly adding the solution B into the solution A for reaction; after the reaction is finished, the trifluoromethanesulfonyl alkynylamide compound with the general structural formula (I) is obtained after column chromatography separation.

11. The method of claim 10, wherein: in the step 1), the stirring is uniformly carried out for 1-10min, and the reaction is carried out for 10-100 min;

in the step 2), the stirring is uniformly carried out for 1-10min, and the reaction is carried out for 0.2-12 h.

12. The method of claim 10, wherein: in the step 1), the stirring is uniformly carried out for 3-8min, and the reaction is carried out for 20-50 min;

in the step 2), the stirring is uniformly carried out for 3-8min, and the reaction is carried out for 0.5-5 h.

13. Use of trifluoromethanesulfonyl alkynylamides of general structural formula (I) according to any one of claims 1 to 5 or prepared by a process according to any one of claims 6 to 12, characterized in that: the trifluoromethanesulfonyl alkyne amide compound with the structural general formula (I) is used for carrying out a sulfhydrylation reaction with thiophenol and/or thiol, or is used for selectively modifying and marking sulfydryl in peptides and proteins.

14. Use according to claim 13, characterized in that: the trifluoromethanesulfonyl alkyne amide compound with the structural general formula (I) is used for selectively modifying and marking side chain sulfydryl in peptides and proteins.

15. A method for selective modification and labelling of thiol groups in peptides and proteins using trifluoromethanesulfonyl alkynylamides of general structural formula (I) as described in any of claims 1 to 5 or prepared by a method as described in any of claims 6 to 12, characterized in that: the method comprises the following steps:

wherein R is selected from one of alkyl, hydroxyethyl, propargyl, an indometacin-containing group, a biotin-containing group and a coumarin-containing fluorescent group; r¹Is one of a cysteine group, a cysteine-containing peptide chain group, a cysteine-containing protein group, an amino and/or carboxyl protected cysteine-containing peptide chain group, an amino and/or carboxyl protected cysteine-containing protein group.

16. The method of claim 15, wherein: the alkalescent reaction system is a mixed system consisting of a slightly alkaline buffer solution and an organic solvent; the alkaline buffer solution is selected from one of PBS buffer solution and Tris buffer solution; the organic solvent is one of acetonitrile, N-dimethylformamide and dimethyl sulfoxide.

17. The method of claim 16, wherein: the volume ratio of the alkalescent buffer solution to the organic solvent is 1-50: 1.

18. The method of claim 16, wherein: the volume ratio of the alkalescent buffer solution to the organic solvent is 5-40: 1.

19. The method of claim 16, wherein: the volume ratio of the alkalescent buffer solution to the organic solvent is 10-30: 1.

20. The method of claim 15, wherein: the molar ratio of the added amount of the trifluoromethanesulfonyl alkyne amide compound with the general structural formula (I) to the added amount of the sulfhydryl-containing peptide or protein with the general structural formula (V) is 1-10: 1.

21. The method of claim 15, wherein: the molar ratio of the added amount of the trifluoromethanesulfonyl alkynamide compound with the general structural formula (I) to the added amount of the sulfhydryl-containing peptide or protein with the general structural formula (V) is 1.1-4: 1.

22. The method of claim 15, wherein: the molar ratio of the added amount of the trifluoromethanesulfonyl alkyne amide compound with the general structural formula (I) to the added amount of the sulfhydryl-containing peptide or protein with the general structural formula (V) is 1.2-2: 1.

23. The method according to any one of claims 15-22, wherein: the step C) is specifically as follows: dissolving trifluoromethanesulfonyl alkynamide compounds with a general structural formula (I) and sulfhydryl-containing polypeptides or proteins with a general structural formula (V) in a weakly alkaline reaction system, uniformly mixing, and reacting; after the reaction is finished, the sulfur ether compound modified by the trifluoromethanesulfonyl group with the general structural formula (VI) is obtained through column chromatography separation.

24. The method of claim 23, wherein: the reaction is specifically as follows: stirring and reacting for 0-60min at 0-50 ℃.

25. The method of claim 23, wherein: the reaction is specifically as follows: stirring and reacting for 20-50min at 30-40 ℃.