Site-directed sulfydryl modified amino acid, polypeptide and protein as well as preparation method and application of amino acid, polypeptide and protein

Abstract

The invention discloses sulfydryl site-directed modified amino acid, polypeptide and protein as well as a preparation method and application thereof, and belongs to the technical field of modification of amino acid, polypeptide and protein. Sulfydryl of the sulfydryl site-directed modified amino acid, polypeptide and protein is substituted by any one group in formulas I-V; wherein the dashed line represents a single bond or none; the A ring in the formula I is selected from I-A-a or I-A-b, the A ring in the formula II is selected from formula II-A, the A ring in the formula III is selected from formula III-A, the A ring in the formula IV is selected from formula IV-A, and the A ring in the formula V is selected from formula V-A; the sulfydryl site-directed modified amino acid, polypeptide and protein contain various bioactive dominant structures such as indolo six-membered nitrogen heterocycle, indoline six-membered nitrogen heterocycle and five-membered azaspiro indoline, and have relatively high medicinal value. Moreover, the preparation method is mild in condition, does not need a catalyst, is compatible with a liquid phase reaction and a polypeptide solid phase synthesis reaction, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of modification of amino acids, polypeptides and proteins, and particularly relates to a thiol-site-specifically modified amino acid, polypeptide and protein, and a preparation method and application thereof. Background Art

[0002] The chemoselective modification of polypeptides and proteins has wide applications and important significance in many fields such as biomedicine, drug research and development, and materials science. The chemoselective modification of specific amino acid residues can introduce chemical groups with special properties, such as fluorophores, affinity tags, drug molecules, etc. into the polypeptide and protein structures, and can also be used to study the folding, conformational changes of polypeptides and proteins, and their interactions with other biomolecules, so as to deeply understand the relationship between the structure and function of proteins; by using chemoselective modification, the active center or allosteric site of an enzyme can also be modified to change the catalytic activity, substrate specificity or regulatory mechanism of the enzyme, thereby contributing to the clarification of the catalytic mechanism and regulatory principle of the enzyme; the chemoselective modification of polypeptide and protein drugs (such as polyethylene glycolylation modification) can increase the water solubility, stability and in vivo circulation time of the drugs, reduce immunogenicity, and improve the efficacy and safety of the drugs; in addition, by introducing pharmacologically active groups onto polypeptide and protein molecules through chemoselective modification, new drugs with novel mechanisms of action and therapeutic effects can be developed, providing new ideas and methods for the design and research and development of new drugs.

[0003] The chemical modification of polypeptides and proteins with radionuclides, fluorescent dyes or magnetic resonance contrast agents, etc. can prepare probes for molecular imaging, realizing the visualization imaging of disease-related biomolecules or cells, and providing a powerful tool for the early diagnosis and treatment monitoring of diseases. By using the chemoselective modification technology of polypeptides and proteins, combining polypeptides or proteins with specific recognition ability with signal conversion elements can construct highly sensitive and highly specific biosensors for detecting various biomolecules in vivo, such as proteins, nucleic acids, small molecule metabolites, etc. The chemoselective modification of polypeptides and proteins on the surface of biomaterials can endow the materials with good biocompatibility, cell affinity and biological activity, promote cell adhesion, proliferation and differentiation, and improve the application performance of the materials in the field of biomedicine. By using the stimulus responsiveness of polypeptides and proteins and combining them with functional materials through chemoselective modification, intelligent responsive biomaterials can be constructed, such as temperature, pH, specific substance concentration and other stimulus-responsive materials, which have broad application prospects in the fields of drug release, tissue engineering, etc.

[0004] Site-directed thiol modification is a technique for precisely chemically modifying specific thiols at the levels of amino acids, polypeptides, and protein molecules. Thiol groups have strong nucleophilicity. In biomolecules, thiol groups often participate in various chemical reactions as active sites, such as redox reactions and nucleophilic substitution reactions. This unique reactivity makes thiol groups an ideal target for chemically modifying biomolecules. Cysteine is the only natural amino acid containing a thiol group. In polypeptides and proteins, the thiol groups of cysteine residues can stabilize the three-dimensional structure of proteins by forming disulfide bonds, playing a key role in protein folding, stability, and function. However, currently, the modified structures of amino acids, polypeptides, and proteins by site-directed thiol modification are simple and have a single biological activity, limiting their further application and development.

[0005] Traditional methods for thiol modification of polypeptides and proteins mostly involve coupling through maleimide chemistry. The main drawback of maleimide coupling is the reversibility of the Michael addition, and its rate highly depends on the pKa of the specific cysteine residue to which it is attached. This reverse Michael reaction may lead to the accidental detachment of the modified group. Moreover, traditional thiol modification methods often lack sufficient selectivity and may act on multiple side chain sites simultaneously during the modification process, resulting in non-specific modification, thus affecting the structure and function of biomolecules. In addition, some traditional methods need to be carried out under the conditions of chemical catalysts, which may cause the denaturation or degradation of biomolecules and lead to the residue of metal catalysts, limiting their application in the fields of biomedicine and so on.

[0006] Photochemical reactions have unique spatiotemporal controllability. By selecting appropriate light wavelengths, intensities, and times, the modification reaction can be precisely initiated within a specific time and space range to achieve site-directed modification of thiols. Photochemical modification can usually be carried out under mild conditions, such as normal temperature, neutral pH, aqueous phase environment, etc., reducing the damage to the structure and function of biomolecules and improving the biocompatibility of the modification. For example, Yoon et al. reported 2+ using [Ru(bpz)3] as a photocatalyst for the visible light modification reaction of polypeptides or proteins based on the reaction between thiols and alkenes (J. Org. Chem., 2014, 79, 1427 - 1436.); Li et al. developed a photoinduced thiol-alkyne reaction for the photoreaction modification of polypeptides in the presence of a photoinitiator (Chem. Sci., 2016, 7, 3325 - 3330). Another commonly reported type of photocatalytic cysteine bioconjugation involves cross-coupling of chemoselective thioarylation, such as He and his colleagues utilized the modified Stadler-Ziegler reaction for site-selective bioconjugation at cysteine residues (Angew. Chem. Int. Ed., 2017, 56, 12702-12707). In addition, researchers at Merck & Co. developed chemoselective arylation modification of cysteine residues on peptides and proteins through visible-light nickel-catalyzed cross-coupling reactions and their applications in the preparation of ADCs (J. Am. Chem. Soc., 2023, 145, 25842-25849).

[0007] However, most of the currently reported methods for thiol-selective photoreaction modification require the addition of catalysts, which often lead to catalyst residues, thereby increasing the application risks and overall process costs in the fields of biomedicine, etc.; in addition, the coupling structures generated by the existing thiol-selective photoreaction modification methods are relatively single and lack chemical diversity, which is not conducive to subsequent screening and applications.

[0008] Therefore, it is of great significance to provide a catalyst-free and chemically divergent thiol-selective photoreaction modification method with good generality and simple process for preparing amino acids, polypeptides, and proteins with various bioactive dominant structures to make their applications more extensive. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide an amino acid, polypeptide, and protein with site-directed thiol modification, and their preparation methods and applications. The amino acid, polypeptide, and protein with site-directed thiol modification contain various bioactive dominant structures such as indolo-hexacyclic azacycles, indolinyl-hexacyclic azacycles, five-membered azaspiroindoline, etc., and have high medicinal value.

[0010] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0011] The present invention provides an amino acid, polypeptide, and protein with site-directed thiol modification, wherein the thiol is replaced by any one of the groups in Formulas I-IV:

[0012]

[0013] Among them, the dotted line represents a single bond or none;

[0014] In Formula I, the A ring is selected from I-A-a or I-A-b, in Formula II, the A ring is selected from Formula II-A, in Formula III, the A ring is selected from Formula III-A, in Formula IV, the A ring is selected from Formula IV-A, and in Formula V, the A ring is selected from Formula V-A;

[0015] In Formula I, when the A ring is selected from I-A-a, R3 is unsubstituted; when the A ring is selected from I-A-b, R2 is unsubstituted;

[0016] When both the bond between C2-N1 and the bond between C2-C3 are single bonds, the bond between the carbons where R8 and R9 are located respectively is a single bond;

[0017] When the bond between C2-N1 is a double bond, R1 and R2 are unsubstituted, and the bond between the carbons where R8 and R9 are located respectively is a single bond;

[0018] When the bond between C2-C3 is a double bond, the bond between the carbons where R8 and R9 are located respectively is a single bond or a double bond, and R2 and R3 are unsubstituted;

[0019] When R2 and R3 are substituted, R2 and R3 are independently selected from hydrogen or C1-C3 alkyl;

[0020] In Formula II, when the bond between C2-N1 is a double bond, R1 is unsubstituted, and R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0021] In Formula III, when the bond between C2-C3 is a single bond, the bond between the carbons where R8 and R9 are located respectively is a single bond; when the bond between C2-C3 is a double bond, the bond between the carbons where R8 and R9 are located respectively is a single bond or a double bond, and R2 is unsubstituted;

[0022] When R2 is substituted, R2 is selected from hydrogen or C1-C3 alkyl;

[0023] R3 is selected from -CHO, -CN, -NO2, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X;

[0024] In Formula IV, when the bond between C2-N1 is a double bond, R1 is unsubstituted; when the bond between C2-C3 is a double bond, R3 is unsubstituted;

[0025] R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0026] When R3 is substituted, R3 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0027] In Formula V, R3 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0028] In Formulas I-V, the said R4-R7 are independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -CO2X, -CONHX or -CON(Me)X, the said R8, R9 are independently selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X, when the said R1 is substituted, R1 is selected from hydrogen or C1-C3 alkyl;

[0029] The total number of -CO2X, -CONHX or -CON(Me)X in the substituents R2-R9 is 0, 1 or 2;

[0030] X in the -CO2X, -CONHX or -CON(Me)X is selected from C1-C6 alkyl or any of the following structures:

[0031]

[0032] wherein, n is selected from integers between 0 and 10, m is selected from 0 or 1, CCG is a click chemical reaction group, and PG is an amino or carboxyl protecting group.

[0033] Preferably, in the amino acids, polypeptides and proteins with site-directed modification of thiol groups, the thiol groups are replaced by any of the following groups:

[0034]

[0035] wherein, R1 is selected from hydrogen or C1-C3 alkyl;

[0036] R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0037] R 3a is selected from hydrogen or C1-C3 alkyl;

[0038] R 3b is selected from -CHO, -CN, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X;

[0039] R 3c is selected from hydrogen, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X;

[0040] R4-R7 are independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -CO2X, -CONHX or -CON(Me)X;

[0041] R8 and R9 are independently selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0042] The total number of -CO2X, -CONHX or -CON(Me)X in the substituents R2-R9 is 0, 1 or 2;

[0043] X in the -CO2X, -CONHX or -CON(Me)X is selected from C1-C6 alkyl or any of the following structures:

[0044]

[0045] Among them, preferably, n is selected from integers between 0 and 10; more preferably, n is selected from integers between 0 and 5; in some specific embodiments of the present invention, n is selected from 0, 1, 2 or 4.

[0046] Preferably, m is selected from 0 or 1, CCG is selected from alkynyl, azide, dibenzocyclooctynyl, bicyclononyne, tetrazine, trans-cyclooctenyl or sulfonyl fluoride group, and PG is selected from allyl (All), 4-methyltrityl (Mtt), allyloxycarbonyl (Alloc), tert-butyl (tBu) or tert-butoxycarbonyl (Boc). In some specific embodiments of the present invention, CCG is preferably alkynyl and PG is preferably Alloc.

[0047] Preferably in the present invention, the thiol group in the site-specifically modified amino acid, polypeptide and protein is replaced by any one of the following groups:

[0048]

[0049] The present invention also provides a method for preparing the above-mentioned site-specifically modified amino acid, polypeptide and protein containing thiol group, comprising the following steps:

[0050] Reacting the amino acid, polypeptide or protein containing thiol group with the indole derivative having an isocyanide group shown in formula VI under nitrogen protection and blue LED illumination to prepare the site-specifically modified amino acid, polypeptide and protein containing thiol group;

[0051] Alternatively, reacting the solid-phase resin of the thiol-containing amino acid fragment with the indole derivative having an isocyanide group shown in formula VI under nitrogen protection and blue LED illumination to prepare the site-specifically modified amino acid and polypeptide containing thiol group;

[0052]

[0053] Wherein, one of R1, R2 or R3 is replaced by group B, or R4 is replaced by group C;

[0054] When R1 is replaced by group B, R3 is selected from -CHO, -CN, -NO2, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X, and R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0055] When R2 is replaced by group B, R1 is selected from hydrogen, C1-C3 alkyl, and R3 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0056] When R3 is substituted by a B group, R1 is selected from hydrogen, C1-C3 alkyl, and R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0057] When R4 is substituted by a C group, R1 is selected from hydrogen or C1-C3 alkyl, R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X, and R3 is selected from hydrogen or C1-C3 alkyl;

[0058] R4-R7 are independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -CO2X, -CONHX or -CON(Me)X;

[0059] R8 and R9 are independently selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X;

[0060] The total number of -CO2X, -CONHX or -CON(Me)X in the substituents R2-R9 is 0, 1 or 2;

[0061] X in the -CO2X, -CONHX or -CON(Me)X is selected from C1-C6 alkyl or any of the following structures:

[0062]

[0063] Wherein, n is selected from integers between 0 and 10, m is selected from 0 or 1, CCG is a click chemical reaction group, and PG is an amino or carboxyl protecting group.

[0064] The more preferred range of n and the preferred ranges of CCG and PG are the same as above and will not be repeated here.

[0065] The wavy lines in the structural formula of the present invention all represent connection positions.

[0066] The general formula of the liquid-phase photoreaction or solid-phase photoreaction in the above preparation method is shown as follows:

[0067]

[0068] The above preparation method adopts a visible-light-driven chemical synthesis strategy, with mild and green conditions, and good tolerance and selectivity for amino acid side-chain functional groups.

[0069] The synthesis route of the indole derivative with an isocyanide group shown in Formula VI in the above preparation method is mature and easy to prepare.

[0070] Preferably, the molar ratio of the mercapto-containing amino acid, polypeptide or protein to the indole derivative with an isocyanide group shown in Formula VI is (5:1)-(1:1000); more preferably (3:1)-(1:100). In some specific embodiments of the present invention, it is preferably 2:1, 1:5 or 1:20.

[0071] The solvent used in the preparation method is compatible with common organic solvents and aqueous environments, and has good scalability.

[0072] Preferably, the solvent for the liquid-phase photoreaction is selected from one or more of DMA, DMF, methanol, acetonitrile, water, and ammonium acetate buffer solution; more preferably, it is a mixed solvent of water and DMA, DMF, methanol or acetonitrile, or a mixed solvent of ammonium acetate buffer solution and DMA, DMF, methanol or acetonitrile.

[0073] The above preparation method is compatible with liquid-phase reactions and conventional solid-phase peptide synthesis reactions, has a wide range of applications, and is suitable for large-scale industrial production.

[0074] Preferably, the temperature of the liquid-phase photoreaction is room temperature, and the time is 0.5-48 h.

[0075] The room temperature is preferably 10°C-30°C.

[0076] Preferably, the solid-phase resin for the mercapto-containing amino acid fragment is selected from 2-chlorotrityl resin or Sieber Amide resin.

[0077] More preferably, when using 2-chlorotrityl resin for solid-phase photoreaction, the cleavage solution is selected from a mixed solution of trifluoroethanol and dichloromethane with a volume ratio of (10:1)-(1:5); the volume ratio of the trifluoroethanol and dichloromethane mixed solution is further preferably 1:1.

[0078] More preferably, when using Sieber Amide resin for solid-phase photoreaction, the cleavage solution is selected from a mixed solution of trifluoroacetic acid and dichloromethane with a trifluoroacetic acid volume fraction of 1%-5%; the volume fraction of trifluoroacetic acid is further preferably 1%.

[0079] Through the above preparation method, the present invention can prepare mercapto-containing amino acids, polypeptides or proteins containing various bioactive dominant structures such as indolo-hexahydropyrimidine, indolin-hexahydropyrimidine, and five-membered nitrogen heterospiroindoline, further enriching the structures of mercapto-containing amino acids, polypeptides or proteins and further broadening their applications.

[0080] In the above preparation method, the amino acids, polypeptides and proteins with site-specific mercapto modification all contain mercapto functional groups before modification.

[0081] The mercapto group-containing amino acids include, but are not limited to, homocysteine, coenzyme A, N-acetylcysteine, penicillamine, cysteamine, thioglycolic acid, mercaptopropionic acid, etc.

[0082] The mercapto group-containing polypeptides include, but are not limited to, polymers coupled by amide bonds, N-alkyl substituted amide bonds or ester bonds from natural amino acids, unnatural amino acids, and hydroxy acids.

[0083] The mercapto group-containing proteins include, but are not limited to, natural proteins containing naked mercapto groups or artificially modified proteins.

[0084] The site-specifically mercapto-modified amino acids, polypeptides and proteins that can be prepared by the above preparation method of the present invention include, but are not limited to, cyclic peptides, polypeptide-small molecule conjugates, polypeptide-radionuclide conjugates, small molecule-small molecule conjugates, protein-small molecule conjugates, polypeptide-polypeptide conjugates, etc.

[0085] The present invention also provides the applications of the above site-specifically mercapto-modified amino acids, polypeptides and proteins or the site-specifically mercapto-modified amino acids, polypeptides and proteins prepared by the above preparation method in biomedicine and drug research and development.

[0086] Compared with the prior art, the mercapto groups of the site-specifically mercapto-modified amino acids, polypeptides and proteins provided by the present invention are substituted by any one of the groups in Formulas I-V; wherein, the dotted line represents a single bond or none; in Formula I, the A ring is selected from I-A-a or I-A-b, in Formula II, the A ring is selected from Formula II-A, in Formula III, the A ring is selected from Formula III-A, in Formula IV, the A ring is selected from Formula IV-A, and in Formula V, the A ring is selected from Formula V-A; the site-specifically mercapto-modified amino acids, polypeptides and proteins contain various bioactive dominant structures such as indolohexazacycles, indolinhexazacycles, five-membered nitrogen heterospiroindolines, etc., and have high medicinal value. The preparation method of the site-specifically mercapto-modified amino acids, polypeptides and proteins has mild conditions and does not require any catalyst, is compatible with liquid-phase reactions and conventional polypeptide solid-phase synthesis reactions, is suitable for large-scale industrial preparation of polypeptides, modified proteins, cyclic peptides, polypeptide-small molecule conjugates, protein-small molecule conjugates, polypeptide-polypeptide conjugates, etc., and has good biosafety and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 LC-MS / MS diagram for confirming the modification site of Comp.6 reacting with ATN-161 in Example 8;

[0088] Figure 2 LC-MS diagram of the crude Comp.29 polypeptide in Example 11;

[0089] Figure 3LC-MS chromatogram after 12 h of solid-phase reaction between Comp.6 and polypeptide 29 in Example 12;

[0090] Figure 4 LC-MS chromatogram after 12 h of solid-phase reaction between Comp.2 and polypeptide 29 in Example 19;

[0091] Figure 5 LC-MS chromatogram after 12 h of solid-phase reaction between Comp.3 and polypeptide 30 in Example 29;

[0092] Figure 6 LC-MS / MS chromatogram for confirming the modification sites of the reaction between Comp.3 and polypeptide 30 in Example 29;

[0093] Figure 7 LC-MS chromatogram of the product Comp.55 obtained by semi-preparative liquid-phase purification in Example 29;

[0094] Figure 8 Mass spectrum of the BSA modification product in Example 31. Detailed implementation manners

[0095] To further illustrate the present invention, the mercapto-site-specific modified amino acids, polypeptides and proteins provided by the present invention, and their preparation methods and applications will be described in detail below in conjunction with examples.

[0096] The following Example 1 is the synthesis of compounds 1-19 (Comp.1-19), Examples 2-7 are the mercapto modifications of cysteine derivatives, Examples 8-29 are the mercapto modifications of cysteine-containing polypeptides, Example 30 is the application of the modification method described in this application in the preparation of PROTAC targeted protein degradation chimeric drug molecules (small molecule-small molecule conjugates), and Example 31 is the mercapto modification of proteins.

[0097] Example 1

[0098] Preparation of indole derivatives (Comp.1-19) of the group

[0099] I. For the preparation of compounds in Comp.1-19 except Comp.4 and Comp.7, the following general methods are used:

[0100] 1. General method for carbamoylation reaction:

[0101]

[0102] Add acetic anhydride to formic acid and reflux for 2 h. Dissolve the amino compound in THF. Under an ice bath, add the amino compound solution dropwise to ethyl formate and stir the reaction at room temperature. After the reaction is monitored by TLC to completion, dilute the reaction solution with DCM, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, rotary evaporate the solvent, and purify by silica gel column chromatography to obtain the formylation product.

[0103] 2. General method for the dehydration reaction of the formylation product:

[0104]

[0105] Dissolve the formylation product in anhydrous dichloromethane, add Et3N (5.0 eq), stir in an ice-salt bath at -20 °C for 10 min, then slowly add POCl3 (1.5 eq) dropwise, react for 0.5 - 1 h, quench with saturated aqueous sodium bicarbonate solution, dilute the reaction solution with DCM, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, rotary evaporate the solvent, and purify by silica gel column chromatography to obtain the isocyanate product.

[0106] 3. General method for the preparation of ethyl ester:

[0107]

[0108] Dissolve the acid in anhydrous ethanol, stir at 0 °C for 10 min. Under an ice bath, add thionyl chloride (1.2 eq) dropwise to the acid solution, reflux for 1 - 2 h, precipitate a solid, stop heating, cool in an ice bath, filter the precipitate by suction, and the solid obtained is the target product.

[0109] 4. General method for amide condensation reaction:

[0110]

[0111] Dissolve the acid (1.0 eq), amine compound (1.5 eq), EDCI (2.0 eq), and DMAP (0.3 eq) in anhydrous DMF and react at room temperature for 30 min - 3 h. After the reaction is completed, rotary evaporate the solvent with an oil pump, dissolve the solid in DCM, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, rotary evaporate the solvent, and purify by silica gel column chromatography to obtain the product.

[0112] 5. General method for indole N-alkylation reaction:

[0113]

[0114] i. Dissolve compound a in anhydrous DMF, add N-Boc-bromoethylamine (2.0 eq) and potassium hydroxide (2.0 eq), react at room temperature for 12 - 24 h. After the reaction is completed, rotary evaporate the solvent with an oil pump, dissolve the solid in DCM, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, rotary evaporate the solvent, and purify by silica gel column chromatography to obtain the product.

[0115] ii. Dissolve compound b in anhydrous DCM, slowly add trifluoroacetic acid dropwise (DCM / TFA, 3 / 1) on ice, react at room temperature for 30 min - 2 h, and rotary evaporate the solvent to obtain the target product c.

[0116] II. Preparation methods of Comp.4 and Comp.7:

[0117] The preparation of Comp.4 is shown as follows:

[0118] Preparation of Comp.4: Dissolve compound a in ethanol / water (1 / 1), add potassium hydroxide (6.0 eq), reflux for 24 h. After the reaction is completed, adjust the pH to 5 with HCl, add ice water until a solid precipitates, filter by suction, and dry the solid to obtain compound b; subsequently, through carbamoylation reaction, amide condensation reaction, and formyl dehydration reaction to obtain Comp.4.

[0119]

[0120] The preparation of Comp.7 is shown as follows:

[0121] Preparation of Comp.7: Compound b is prepared by the general method of indole N-alkylation reaction; dissolve compound b in toluene, add ethyl (triphenylphosphine) acetate (3.0 eq), reflux and react. After the reaction is monitored by TLC to be completed, rotary evaporate the solvent, and purify by silica gel column chromatography to obtain product c; dissolve product c in DCM, add 10% Pd / C, stir and react at room temperature under a hydrogen balloon. After the reaction is completed, filter the reaction solution and rotary evaporate under reduced pressure, and purify by silica gel column chromatography to obtain product d; subsequently, through carbamoylation reaction and formyl dehydration to prepare isocyanide reaction to obtain Comp.7.

[0122]

[0123] Table 1 Compound structures of Comp.1 - 19

[0124]

[0125]

[0126] Characterization data of some compounds are as follows:

[0127] Comp.1: 11H NMR (400 MHz, DMSO-d6) δ 7.55 (dt, J = 7.9, 1.1 Hz, 1H), 7.41 (dt, J = 8.4, 0.9 Hz, 1H), 7.23 (s, 1H), 7.16 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 7.04 (ddd, J = 7.9, 6.9, 1.1 Hz, 1H), 5.05 (dd, J = 6.9, 5.2 Hz, 1H), 4.14 (qd, J = 7.1, 2.0 Hz, 2H), 3.76 (s, 3H), 3.39–3.23 (m, 2H), 1.17 (td, J = 7.1, 4.3 Hz, 3H). 13 13C NMR (101 MHz, DMSO) δ 167.07, 159.46, 136.93, 129.12, 127.78, 121.75, 119.19, 118.95, 110.21, 107.19, 62.56, 57.80, 32.85, 28.71, 14.25.

[0128] Compound 2: 1 1H NMR (400 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.58 (dt, J = 7.9, 0.9 Hz, 1H), 7.39 (dt, J = 8.2, 0.9 Hz, 1H), 7.25–7.20 (m, 2H), 7.15 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 4.53 (dd, J = 8.0, 4.8 Hz, 1H), 4.29–4.08 (m, 2H), 3.48 (dd, J = 14.7, 4.8 Hz, 1H), 3.41–3.30 (m, 1H), 1.23 (t, J = 7.2 Hz, 3H).

[0129] 13 13C NMR (101 MHz, CDCl3) δ 166.55, 160.06, 136.11, 126.81, 123.66, 122.44, 119.85, 118.21, 111.42, 108.79, 62.73, 57.60, 29.48, 13.91.

[0130] Compound 3: 1 1H NMR (400 MHz, DMSO-d6) δ 10.84 (s, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 7.9 Hz, 1H), 7.05– -6.91 (m, 2H), 3.69–3.60 (m, 2H), 3.01 (tt, J = 6.8, 2.1 Hz, 2H), 2.38 (s, 3H).

[0131] 13 C NMR (101 MHz, DMSO) δ 156.14, 135.62, 133.67, 128.31, 120.63, 118.80, 117.78, 110.93, 106.12, 42.48, 24.52, 11.76.

[0132] Comp.5: 1 H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.80 (dd, J=8.1, 1.1 Hz, 1H), 7.68 (dt, J=7.7, 1.0 Hz, 1H), 7.38 (ddd, J=8.4, 7.1, 1.3 Hz, 1H), 7.31 (ddd, J=8.0, 7.1, 1.1 Hz, 1H), 4.61 (t, J=5.7 Hz, 2H), 4.03 (t, J=5.7 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 158.34, 137.75, 135.63, 127.53, 124.18, 122.79, 119.33, 116.23, 112.18, 84.72, 45.54, 41.98.

[0133] Comp.6: 1 H NMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.07–7.99 (m, 1H), 7.73–7.65 (m, 1H), 7.27 (pd, J=7.1, 1.4 Hz, 2H), 4.59 (t, J=5.8 Hz, 2H), 4.02 (t, J=5.8 Hz, 2H), 3.82 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 164.85, 158.13, 136.68, 136.12, 126.56, 123.16, 122.34, 121.20, 111.48, 106.51, 51.27, 45.21, 42.00.

[0134] Comp.7: 11H NMR (400 MHz, Chloroform-d) δ 7.65 (dt, J = 7.8, 1.1 Hz, 1H), 7.33–7.24 (m, 3H), 7.18 (ddd, J = 7.9, 5.9, 2.1 Hz, 1H), 7.00 (s, 1H), 4.40 (t, J = 6.3 Hz, 2H), 4.17 (q, J = 7.2 Hz, 2H), 3.76 (t, J = 6.2 Hz, 2H), 3.16–3.07 (m, 2H), 2.73 (dd, J = 8.6, 6.7 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). 13 13C NMR (101 MHz, CDCl3) δ 173.27, 159.38, 135.89, 128.26, 125.22, 122.30, 119.64, 119.42, 115.14, 108.64, 60.44, 45.19, 41.64, 34.91, 20.44, 14.26.

[0135] Comp.8: 1H NMR (400 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.35 (s, 1H), 4.49–4.41 (m, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.40–3.27 (m, 2H), 1.20 (s, 2H), 1.18 (s, 1H). 13C NMR (101 MHz, Chloroform-d) δ 166.03, 161.31, 136.36, 131.36, 128.18, 122.21, 120.43, 120.06, 110.89, 102.98, 63.25, 56.67, 31.94, 13.95.

[0136] Comp.9: 1 1H NMR (400 MHz, Chloroform-d) δ 8.04 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 2.5 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 6.92 (dd, J = 8.9, 2.3 Hz, 1H), 3.90 (s, 3H), 3.68 (td, J = 7.1, 2.1 Hz, 2H), 3.17 (tt, J = 7.0, 5.0, 3.8 Hz, 2H). 1313C NMR (101 MHz, CDCl3) δ 156.01, 154.25, 131.37, 127.19, 123.36, 112.51, 112.18, 110.79, 100.16, 56.00, 42.28, 25.88.

[0137] Compound 10: 1 1H NMR (400 MHz, Chloroform-d) δ 8.15 (brs, 1H), 7.42 (s, 1H), 7.23–7.14 (m, 1H), 7.10–7.04 (m, 2H), 3.56 (tt, J = 6.9, 1.8 Hz, 2H), 3.01 (tt, J = 7.1, 2.1 Hz, 2H). 13 13C NMR (101 MHz, CDCl3) δ 156.01, 134.61, 127.89, 125.47, 124.14, 122.66, 117.74, 112.53, 110.72, 42.29, 25.57.

[0138] Compound 11: 1 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 7.49–7.35 (m, 2H), 7.13 (t, J = 8.1, 7.2 Hz, 1H), 7.04 (d, J = 7.1, 1.0 Hz, 1H), 6.62 -–6.50 (m, 1H), 5.04 (s, 2H). 13C NMR (101 MHz, DMSO) δ 156.68, 136.36, 126.28, 126.00, 124.55, 121.38, 117.92, 112.27, 99.43, 44.18.

[0139] Compound 14: 11H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.41 (t, J = 5.6 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.1, 0.9 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.09 (t, J = 7.0 Hz, 1H), 7.01 (t, J = 6.9 Hz, 1H), 4.64 (dd, J = 8.3, 5.7 Hz, 1H), 4.14 (d, J = 2.4 Hz, 2H), 3.57 - 3.52 (m, 2H), 3.49 (m, J = 5.7, 2.7 Hz, 2H), 3.41 (m, J = 2.1 Hz, 1H), 3.33 (m, J = 9.9 Hz, 2H), 3.25 (m, J = 10.8, 4.9 Hz, 3H), 3.17 (m, J = 14.4, 8.3 Hz, 1H). 13 13C NMR (101 MHz, DMSO) δ 166.19, 158.19, 136.55, 127.37, 124.89, 121.56, 118.92, 111.91, 108.59, 80.77, 77.61, 69.75, 69.11, 68.89, 58.31, 57.97, 39.48, 29.79.

[0140] Comp.15: 1 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 8.50 (t, J = 5.7 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.13 – 7.05 (m, 1H), 7.04 - 6.94 (m, 1H), 4.67 – 4.57 (m, 1H), 3.27 – 3.11 (m, 4H), 2.85 (t, J = 2.6 Hz, 1H), 2.26 (td, J = 7.1, 2.7 Hz, 2H).

[0141] 13 13C NMR (101 MHz, DMSO) δ 166.23, 158.42, 136.55, 127.34, 124.91, 121.58, 118.93, 118.88, 111.94, 108.53, 82.35, 72.78, 58.40, 38.56, 29.82, 18.80.

[0142] Comp.16: 11H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.42 (t, J = 5.7 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.37 (dt, J = 8.2, 0.9 Hz, 1H), 7.27–7.21 (m, 2H), 7.09 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.00 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 5.96–5.84 (m, 1H), 5.32–5.23 (m, 1H), 5.19–5.11 (m, 1H), 4.63 - 4.41 (m, 3H), 3.31 - 2.95 (m, 7H). 13 13C NMR (101 MHz, DMSO) δ 166.30, 158.35, 156.53, 136.58, 134.17, 127.32, 124.86, 121.59, 118.91, 117.51, 111.95, 108.72, 64.81, 58.58, 29.81.

[0143] Comp.18: 1 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 7.8 Hz, 1H), 8.13–8.06 (m, 2H), 7.63 (d, J = 8.1 Hz, 1H), 7.26 - 7.15 (m, 2H), 4.53 (t, J = 5.5 Hz, 2H), 3.98 (t, J = 5.7 Hz, 2H), 3.43–3.37 (m, 2H), 2.85 (t, J = 2.7 Hz, 1H), 2.44 (td, J = 7.2, 2.7 Hz, 2H). 13 13C NMR (101 MHz, DMSO) δ 164.61, 158.15, 136.41, 131.70, 126.89, 122.68, 121.72, 121.41, 110.91, 110.88, 82.99, 72.55, 45.37, 42.14, 38.34, 19.54.

[0144] Example 2

[0145] Liquid-phase visible-light coupling reaction of Comp.1 with Ac-Cys-OMe (N-acetyl-L-cysteine methyl ester)

[0146]

[0147] (1) Preparation of a scale-up reaction

[0148] Dissolve Comp.1 in DMA (0.2 M), then add 2.0 eq Ac-Cys-OMe. After displacing nitrogen 5 times under ultrasound, irradiate with a 4W blue LED (RLH-18 eight-parallel photoreactor, Beijing Nuozhi Technology Co., Ltd.) and stir at room temperature under nitrogen protection for 12 hours. After the reaction, dilute the reaction solution with ethyl acetate, wash the organic phase 3 times with water, dry over anhydrous sodium sulfate, filter, evaporate the solvent, and purify by silica gel column chromatography to obtain Comp.20a - 20c.

[0149] Comp.20a: 1 H NMR (400 MHz, Chloroform-d) δ 7.63 (dd, J=14.4, 7.1 Hz, 0.46H), 7.54–7.46 (m, 0.59H), 7.10 (td, J=7.7, 1.4 Hz, 1H), 7.00– -6.88 (ddd, J=28.7, 7.4, 1.3 Hz, 0.51H), 6.82 (ddd, J=7.4, 6.0, 1.3 Hz, 0.65H), 6.65 (qd, J=7.4, 1.0 Hz, 1H), 6.47 (dd, J=8.0, 2.3 Hz, 1H), 4.86-–4.71 (m, 1H), 4.70–4.54 (m, 1H), 4.26-–4.12 (m, 2H), 3.73-–3.60 (m, 3H), 3.56-–3.44 (m, 1H), 3.38-–3.12 (m, 3H), 2.71 (m, 3H), 2.70– -2.50 (m, 1H), 2.44-–2.27 (m, 1H), 2.03-–1.94 (m, 3H). 1313C NMR (101 MHz, CDCl3) δ 181.82, 181.68, 181.43, 181.03, 172.75, 172.55, 172.36, 172.22, 171.03, 171.01, 171.00, 170.98, 170.53, 170.50, 152.81, 152.61, 131.15, 131.02, 130.64, 130.58, 129.47, 129.39, 124.05, 123.74, 122.86, 122.77, 118.82, 118.77, 118.50, 118.47, 107.95, 107.92, 107.87, 107.81, 71.17, 71.04, 70.85, 70.69, 66.29, 66.09, 65.66, 63.70, 63.56, 63.42, 63.35, 61.51, 61.49, 61.44, 61.42, 54.37, 54.19, 53.51, 53.47, 52.53, 52.44, 52.38, 43.46, 43.36, 43.28, 43.17, 35.79, 35.76, 35.65, 35.63, 32.17, 32.05, 31.50, 31.47, 22.87, 22.82, 22.75, 22.73, 14.24, 14.21.

[0150] Comp. 20b: 1H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 7.1 Hz, 1H), 7.21–7.09 (m, 2H), 6.80 (t, J = 7.4 Hz, 1H), 6.49 (d, J = 7.8 Hz, 1H), 4.77 (dddd, J = 28.1, 10.3, 6.2, 3.2 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 4.11 (ddd, J = 11.5, 5.8, 3.2 Hz, 1H), 3.94 (dd, J = 9.6, 1.8 Hz, 1H), 3.77 (s, 3H), 3.75–3.49 (m, 2H), 3.29 (dd, J = 14.5, 3.2 Hz, 1H), 2.90 (d, J = 10.4 Hz, 3H), 2.26–2.09 (m, 1H), 2.03 (d, J = 6.5 Hz, 3H), 1.95–1.78 (m, 2H), 1.31 (td, J = 7.1, 1.6 Hz, 3H). 1313C NMR (101 MHz, CDCl3) δ 173.09, 172.49, 171.00, 170.96, 170.84, 170.80, 170.48, 170.12, 152.70, 130.80, 130.40, 128.70, 128.61, 124.71, 124.55, 119.37, 119.24, 107.82, 107.80, 66.45, 66.11, 61.35, 61.28, 58.98, 58.58, 54.94, 54.52, 52.48, 52.44, 38.11, 38.09, 35.79, 35.66, 33.01, 32.36, 30.84, 30.11, 22.72, 22.69, 14.22, 14.20.

[0151] Comp.20c: 1 1H NMR (400 MHz, Chloroform-d) δ 8.22 (t, J = 6.4 Hz, 1H), 7.63 (ddt, J = 8.2, 6.3, 1.0 Hz, 1H), 7.42–7.34 (m, 2H), 7.22–7.17 (m, 1H), 4.82–4.72 (m, 1H), 4.49–4.38 (m, 1H), 4.38–4.30 (m, 2H), 4.02 (d, J = 3.7 Hz, 3H), 3.79 (d, J = 6.8 Hz, 3H), 3.74–3.59 (m, 2H), 3.43–3.23 (m, 2H), 3.07–2.95 (m, 1H), 2.04 (d, J = 15.9 Hz, 3H), 1.39 (td, J = 7.2, 2.1 Hz, 3H). 13 13C NMR (101 MHz, CDCl3) δ 173.20, 172.43, 171.18, 171.07, 170.93, 170.91, 158.56, 158.27, 139.34, 139.27, 129.58, 129.30, 125.53, 125.48, 123.96, 123.90, 120.62, 120.61, 120.37, 120.24, 117.10, 116.36, 110.52, 110.47, 61.74, 61.65, 61.54, 54.96, 54.87, 52.52, 52.46, 32.78, 32.75, 30.27, 29.37, 23.52, 22.77, 22.66, 14.28, 14.24.

[0152] (2) Photocoupling reaction under low concentration conditions of Ac-Cys-OMe

[0153] Dissolve Ac-Cys-OMe in DMA (20 μM), then add Comp.1 (20 eq). After displacing nitrogen 5 times under ultrasound, stir at room temperature under nitrogen protection for 12 hours under irradiation with a 4W blue LED. After the reaction is completed, dilute with ACN and perform LC-HRMS analysis (AB Sciex, TripleTOF 6600; chromatographic column: ACQUITY UPLC BEH C18 1.7 μm).

[0154] Comp.20a / 20b: Product ratio <8%; Calc: C 21 H 28 N3O5S + : 434.1744; Obs: 434.1755

[0155] (Error: 2.5 ppm); Comp.20c: Product ratio >92%; Calc: C 21 H 26 N3O5S + : 432.1588;

[0156] Obs: 432.1622 (Error: 7.9 ppm).

[0157] The above results prove that Comp.20c can be selectively generated under low Cys concentration conditions.

[0158] Example 3

[0159] Liquid-phase visible-light coupling reaction of Comp.2 and Ac-Cys-OMe

[0160]

[0161] (1) Preparation of a preparative-scale reaction

[0162] The specific operation is the same as that in Example 2(1) to obtain compounds Comp.21b and 21c.

[0163] Comp.21b: 11H NMR (400 MHz, DMSO-d6) δ 11.56 (d, J = 8.9 Hz, 1H), 8.57 - 8.47 (m, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.24 (t, J = 7.7 Hz, 1H), 7.10 (s, 1H), 4.57 (dt, J = 15.1, 7.6 Hz, 2H), 4.22–4.13 (m, 2H), 3.79 -–3.69 (m, 1H), 3.66 (d, J = 2.6 Hz, 3H), 3.19 (dd, J = 10.0, 7.1 Hz, 1H), 2.99 (ddd, J = 16.5, 12.5, 10.3 Hz, 1H), 1.87 (s, 3H), 1.26 -–1.21 (m, 3H). 13 13C NMR (101 MHz, DMSO) δ 172.89, 172.74, 171.52, 171.50, 169.95, 169.91, 156.88, 156.50, 137.67, 137.63, 127.41, 125.01, 124.98, 120.45, 114.21, 113.20, 61.89, 61.85, 61.12, 61.06, 52.83, 52.60, 52.56, 52.34, 29.47, 29.31, 22.76, 22.74, 22.64, 22.45, 14.52, 14.47.

[0164] Comp.21b: 1 1H NMR (400 MHz, Chloroform-d) δ 8.88 (d, J = 6.4 Hz, 1H), 8.64 (s, 1H), 8.15 (dd, J = 7.9, 1.2 Hz, 1H), 7.63–-7.58 (m, 2H), 7.37 (ddd, J = 8.1, 4.6, 3.4 Hz, 1H), 4.90 (ddd, J = 9.7, 6.5, 3.2 Hz, 1H), 4.52 (qd, J = 7.1, 2.2 Hz, 2H), 3.87 (dd, J = 14.8, 9.7 Hz, 1H), 3.78 (s, 3H), 3.72 (dd, J = 14.7, 3.3 Hz, 1H), 1.99 (s, 3H), 1.52 (t, J = 7.1 Hz, 3H).

[0165] (2) Photo-coupling reaction under low concentration of Ac-Cys-OMe

[0166] The specific operation is the same as that in Example 2(2).

[0167] Comp.21b / 21d: Product ratio: 87%; Calc: C 20H 26 N3O5S + : 420.1588; Obs: 420.1565 (Error: 5.5 ppm). Comp.20b: Product ratio: 4.58%; Calc: C 20 H 24 N3O5S + : 418.1431; Obs: 418.1440 (Error: 2.2 ppm).

[0168] Comp.20a / c: Product ratio: 13%. The above results prove that Comp.21b / 21d can be selectively generated under low Cys concentration conditions.

[0169] Example 4

[0170] Visible light coupling reaction of Comp.6 and Ac-Cys-OMe

[0171]

[0172] (1) Preparation of a scale reaction

[0173] The specific operation is the same as that in Example 2(1) to obtain compounds Comp.22a - 22c.

[0174] Comp.22a: Product ratio: 82%; Calc: C 19 H 22 N3O5S + : 404.1275; Obs: 404.1289 (Error: 3.5 ppm). 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.39 (t, 1H), 7.28 (t, 1H), 4.60– -4.53 (m, 1H), 4.25– -4.11 (m, 2H), 4.08– -3.94 (m, 2H), 3.87 (s, 2H), 3.65 (s, 3H), 3.41 (s, 1H), 3.22 -–3.15 (m, 1H), 2.04 -–1.94 (m, 1H), 1.84 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 171.70, 169.87, 164.46, 157.56, 134.96, 129.27, 125.70, 125.19, 123.16, 122.15, 111.49, 105.28, 52.55, 51.80, 51.60, 48.70, 38.87, 31.27, 22.73.

[0175] Comp.22b: Product ratio: 10.17%; Calc: C 19 H 24 N3O6S + : 422.1380; Obs: 422.1402 (Error: 5.2 ppm).

[0176] Comp.22c: Product ratio: 7.83%; Calc: C 19 H 24 N3O5S + : 406.1431; Obs: 406.1442 (Error: 2.7 ppm).

[0177] The above results prove that Comp.22a can be selectively generated under preparative-scale reaction conditions.

[0178] Example 5

[0179] Visible light coupling reaction of Comp.7 and Ac-Cys-OMe

[0180]

[0181] Preparative-scale reaction

[0182] The specific operation is the same as that in Example 2(1) to obtain compounds Comp.23a - 23c.

[0183] Comp.23a: Product ratio: 40.63%; Calc: C 22 H 28 N3O5S + : 446.1744; Obs: 446.1736 (Error: 1.8 ppm). 1 H NMR (400 MHz, DMSO-d6) δ8.49 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.29 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H), 7.10 (ddd, J = 8.1, 5.5, 1.0 Hz, 2H), 4.58 (td, J = 8.4, 5.4 Hz, 1H), 4.09 (t, J = 6.2 Hz, 2H), 4.04 (dd, J = 7.1, 3.9 Hz, 2H), 3.93 (t, J = 6.1 Hz, 2H), 3.72–3.67 (m, 1H), 3.65 (s, 3H), 3.25 (dd, J = 13.6, 8.8 Hz, 2H), 2.97–2.88 (m, 1H), 2.65–2.60 (m, 2H), 1.86 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H).13 C NMR (101 MHz, DMSO) δ 172.53, 171.64, 169.88, 156.43, 135.55, 126.88, 124.83, 124.34, 120.74, 120.46, 114.61, 110.48, 60.41, 52.55, 51.76, 49.09, 38.82, 35.80, 29.70, 22.72, 20.70, 14.51.

[0184] Comp.23b: Product ratio: 53.94%; Calc: C 22 H 30 N3O5S + : 448.1901; Obs: 448.1903 (Error: 0.4 ppm).

[0185] Comp.23c: Product ratio: 5.43%; Calc: C 22 H 29 N3O6S + : 464.1850; Obs: 464.1828 (Error: 4.7 ppm).

[0186] The above results prove that 23a / 23b (indicating 23a or 23b) can be selectively generated under preparative-scale reactions.

[0187] Example 6

[0188] Liquid-phase visible-light coupling reaction of Comp.3 and Ac-Cys-OMe

[0189]

[0190] The specific operation is the same as that in Example 2(2).

[0191] Comp.24a: Product ratio: 72.21%; Calc: C 18 H 22 N3O3S + : 360.1376; Obs: 360.1363 (Error: 3.7 ppm). Comp.24b: Product ratio: 8.2%; Calc: C 18 H 24 N3O3S + : 362.1533; Obs: 362.1525 (Error: 2.2 ppm).

[0192] Comp.24c: Product ratio: 19.59%; Calc: C 18 H 24 N3O4S +: 378.1482; Obs: 378.1461 (Error: 5.6 ppm).

[0193] The above results prove that Comp.24a can be selectively generated under low Cys concentration conditions.

[0194] Example 7

[0195] Liquid-phase visible-light coupling reaction of Comp.11 and Ac-Cys-OMe

[0196]

[0197] The specific operation is the same as that in Example 2(2).

[0198] Comp.25a: Product ratio: 16.61%; Calc: C 16 H 18 N3O3S + : 332.1063; Obs: 332.1084 (Error: 6.3 ppm).

[0199] Comp.25b: Product ratio: 10.61%; Calc: C 18 H 20 N3O3S + : 334.1220; Obs: 334.1244 (Error: 7.1 ppm).

[0200] Comp.25c: Product ratio: 72.78%; Calc: C 16 H 20 N3O4S + : 350.1169; Obs: 350.1182 (Error: 3.7 ppm).

[0201] The above results prove that Comp.25c can be selectively generated under low Cys concentration conditions.

[0202] Example 8

[0203] Liquid-phase visible-light coupling reaction of Comp.6 and ATN-161 polypeptide (a β-integrin antagonist polypeptide with anti-tumor activity)

[0204]

[0205] Dissolve Comp.6 in DMA (0.4 M), then add 20.0 eq of ATN-161. After displacing nitrogen 5 times under ultrasound (1 min each time), stir at room temperature for 12 hours under nitrogen protection under irradiation with a 4W blue LED. After the reaction is completed, dilute with ACN and perform LC-HRMS analysis and LC-MS / MS (AB Sciex, TripleTOF 6600) sequencing analysis for the modification site.

[0206]

[0207] Comp.26: Conversion rate: 35%; Retention time of the extracted ion current of the target product: 2.9 - 4.0 min; Calc: C 36 H 46 N 11 O 10 S + : 824.3144; Obs: 824.3077 (error: 8.1 ppm). LC-MS / MS sequencing analysis confirmed that the modification site of the target product is at the cysteine site ( Figure 1 ).

[0208] Example 9

[0209] Liquid-phase visible light coupling reaction of Comp.2 and ATN-161 polypeptide

[0210]

[0211] The specific operation is the same as that in Example 8.

[0212] Comp.27: Conversion rate: 12%; Retention time of the extracted ion current (XIC) of the target product: 3.1 - 4.0 min; Calc: C 37 H 48 N 11 O 10 S + : 838.3301; Obs: 838.3277 (error: 2.9 ppm).

[0213] Example 10

[0214] Liquid-phase visible light coupling reaction of Comp.3 and ATN-161 polypeptide

[0215]

[0216] The specific operation is the same as that in Example 8.

[0217] Comp.28: Conversion rate: 48.6%; Retention time of the extracted ion current (XIC) of the target product: 2.7 - 3.5 min; Calc: C35 H 45 N 11 O8S + : 780.3246; Obs: 780.3192 (Error: 6.9 ppm).

[0218] Example 11

[0219] Solid-phase synthesis of cysteine-containing polypeptides

[0220]

[0221] a. Fmoc deprotection: Add Fmoc-Ala-2-ChlorotritylResin (Gil Biochemical) to the solid-phase synthesis tube, and swell it with an appropriate amount of DMF at room temperature for 30 min. Drain the reaction solvent, add a DMF mixed solution of 1% HOBT (w / v) and 2% DBU (v / v) to the solid-phase synthesis tube, vortex for 2 min, drain the reaction solvent, and repeat 3 times. Wash the resin 9 times alternately with DMF, DCM, and DMF.

[0222] b. 1) Amino acid coupling: Dissolve Fmoc-AA-OH (5.0 eq), COMU (5.0 eq), and TMP (7.5 eq) in DMF, vortex for 2 min, then transfer to the polypeptide reaction tube, and react on a rotary shaker at room temperature for 30 min. Drain the reaction solvent, and wash the resin 9 times alternately with DMF, DCM, and DMF.

[0223] 2) Fmoc deprotection: Add a DMF mixed solution of 1% HOBT (w / v) and 2% DBU (v / v) to the solid-phase synthesis tube, vortex for 2 min, drain the reaction solvent, and repeat 3 times. Wash the resin 9 times alternately with DMF, DCM, and DMF.

[0224] Repeat the above two steps to extend the peptide segment. Among them, AA represents an amino acid, and the above general formula is not a limitation on the polypeptide length but only used to indicate the polypeptide structure. AA can each independently be any amino acid.

[0225] c. Deprotection of the cysteine STmp protecting group: Dissolve 0.1 M N-methylmorpholine (NMM) in DTT / DMF (5%: 95%), vortex for 5 min, drain the reaction solvent, and repeat 3 times. Wash the resin 9 times alternately with DMF, DCM, and DMF. Take 5 mg of the resin and dry it overnight in a vacuum drying oven. Carry out a cleavage reaction with 400 μL of a TFE / DCM (v / v, 1 / 1) mixed solution for 1 h, collect the cleavage solution, dilute it with ACN, and perform LC-HRMS analysis to obtain a cysteine-containing polypeptide with the sequence shown in Table 1.

[0226] Table 1. Sequences of cysteine-containing polypeptides

[0227] Comp. Sequence 29 Ac-Ala-Trp-Phe-Cys-Val-Gly-Ala-OH 30 Ac-Phe-Asn(trt)-Gly-Cys-Ala-OH 31 Ac-Phe-Val-Gln(trt)-Cys-Glu(Oall)-Ala-OH 32 Ac-Ala-Pro-Met-Cys-Glu(Oall)-Ala-OH 33 Ac-Cys-Tle-Tyr(Oall)-Trp-Gly-Ala-OH 34 Ac-Phe-Asp(Oall)-Trp-Gly-Cys-Pro-HoPhe-Val-Leu-Ala-OH 35 Ac-Cys-Gly-Phe-Pro-Val-Ala-Gly(ally)-Gly-Ala-OH 36 Ac-Cys-Gly-Phe-Pro-Val-Ala-Glu(Oall)-Gly-Ala-OH 37 <![CDATA[Ac-Cys-Gly-Phe-Pro-Val-Ala-Lys(N3)-Gly-Ala-OH]]>

[0228] Example 12

[0229] Solid-phase visible light coupling reaction of Comp.6 and polypeptide 29

[0230]

[0231] The 2-Cl resin (7.72 μmol) linked with polypeptide 29 (crude peptide LC-MS: Figure 2 , UV retention time: 3.7 min) was swollen in DMA for 30 min. After pumping out DMA, 500 μL of DMA and Comp.6 (5.0 eq) were added. After displacing nitrogen 5 times under ultrasonic treatment, it was stirred at room temperature for 12 hours under nitrogen protection under irradiation of an 8W blue light LED. After the reaction, the resin was washed 9 times alternately with DCM, DMF, and DCM, and then dried overnight in a vacuum drying oven. 5 mg of the resin was cut and reacted with 400 μL of a 1 / 1 mixed solution of TFE / DCM for 1 h, the cutting solution was collected, diluted with ACN, and analyzed by LC-HRMS ( Figure 3 ).

[0232] Comp.38: Conversion rate: 31.4%, purity of the reaction solution: 26%; UV (254 nm) retention time of the target product: 5.1 - 5.5 min; Calc: C 51 H 61 N 10 O 11 S + : 1021.4237; Obs: C 51 H 61 N 10 O 11 S + : 1021.4152 (error: 8.3 ppm).

[0233] Example 13

[0234] Solid-phase visible light coupling reaction of Comp.15 and polypeptide 29

[0235]

[0236] The specific operation is the same as that in Example 12.

[0237] Comp.39a: Conversion rate: 6.2%; UV (254 nm) retention time of the target product: 5.2 - 5.4 min; Calc: C 54 H 62 N 11 O 10 S +: 1056.4396; Obs: 1056.4344 (Error: 4.9 ppm).

[0238] Comp.39b: Conversion rate: 41.3%; Retention time of the target product at UV (254 nm): 4.5 - 5.9 min; Calc: C 54 H 64 N 11 O 10 S + : 1058.4553; Obs: 1058.4496 (Error: 5.4 ppm).

[0239] Example 14

[0240] Solid-phase visible light coupling reaction of Comp.8 and polypeptide 29

[0241]

[0242] The specific operation is the same as that in Example 12.

[0243] Comp.40a: Conversion rate: 39.5%; Retention time of the target product at UV (254 nm): 3.5 - 3.8 min; Calc: C 52 H 63 N 10 O 11 S + : 1035.4393; Obs: 1035.4319 (Error: 7.1 ppm).

[0244] Comp.40b: Conversion rate: 18.9%; Retention time of the target product at UV (254 nm): 5.1 - 5.3 min; Calc: C 52 H 61 N 10 O 11 S + : 1033.4237; Obs: 1033.4136 (Error: 9.8 ppm).

[0245] Comp.40c: Conversion rate: 16.1%; Retention time of the target product at UV (254 nm): 5.5 - 5.7 min; Calc: C 52 H 65 N 10 O 11 S + : 1037.4550; Obs: 1037.4469 (Error: 7.8 ppm).

[0246] Example 15

[0247] Solid-phase visible-light coupling reaction of Comp.7 and polypeptide 29

[0248]

[0249] The specific operation is the same as that in Example 12.

[0250] Comp.41: Conversion rate: 45.8%; Retention time of the target product at UV (254 nm): 4.8 - 5.0 min; Calc: C 54 H 67 N 10 O 11 S + : 1063.4706; Obs: 1063.4622 (Error: 7.9 ppm).

[0251] Example 16

[0252] Solid-phase visible-light coupling reaction of Comp.9 and polypeptide 29

[0253]

[0254] The specific operation is the same as that in Example 12.

[0255] Comp.42a: Conversion rate: 46.9%; Retention time of the target product at UV (254 nm): 3.5 - 3.8 min; Calc: C 50 H 61 N 10 O 10 S + : 993.4287; Obs: 993.4215 (Error: 7.2 ppm).

[0256] Comp.42b: Conversion rate: 29.4%; Retention time of the target product at UV (254 nm): 4.0 - 4.2 min; Calc: C 50 H 59 N 10 O 10 S + : 991.4131; Obs: 991.4073 (Error: 5.6 ppm).

[0257] Example 17

[0258] Solid-phase visible-light coupling reaction of Comp.18 and polypeptide 29

[0259]

[0260] The specific operation is the same as that in Example 12.

[0261] Comp.43: Conversion rate: 76%; Retention time of the target product at UV (254 nm): 4.3 - 4.5 min; Calc: C 54 H 64 N 11 O 10 S + : 1058.4553; Obs: 1058.4441 (Error: 10.5 ppm).

[0262] Example 18

[0263] Solid-phase visible light coupling reaction of Comp.17 and polypeptide 29

[0264]

[0265] The specific operation is the same as that in Example 12.

[0266] Comp.44a: Conversion rate: 82.5%; Retention time of the target product at UV (254 nm): 4.3 - 4.6 min; Calc: C 54 H 66 N 11 O 10 S + : 1060.4709; Obs: 1060.4650 (Error: 5.5 ppm).

[0267] Comp.44b: Conversion rate: 4.7%; Retention time of the target product at UV (254 nm): 5.5 - 5.6 min; Calc: C 54 H 64 N 11 O 10 S + : 1058.4553; Obs: 1058.4615 (Error: 5.9 ppm).

[0268] Example 19

[0269] Solid-phase visible light coupling reaction of Comp.2 and polypeptide 29

[0270]

[0271] The specific operation is the same as that in Example 12.

[0272] Comp.45a: Conversion rate: 9.2%; Retention time of the target product at UV (254 nm): 6.1 - 6.2 min; Calc: C 52 H 61 N 10 O 11 S +: 1033.4237; Obs: 1033.4115 (Error: 11.8 ppm) ( Figure 4 ).

[0273] Comp.45b: Conversion rate: 66.2%; Retention time of the target product at UV (254 nm): 4.8 - 5.0 min; Calc: C 52 H 63 N 10 O 11 S + : 1035.4393; Obs: 1035.4234 (Error: 15.4 ppm) ( Figure 4 ).

[0274] The 2-Cl resin linked with the polypeptide sequence was recovered, compound 2 was added, and the photoreaction was continued for 12 h. The post-treatment was the same as that in Example 12.

[0275] Comp.45a: Conversion rate: 9.2%; Retention time of the target product at UV (254 nm): 6.1 - 6.2 min; Calc: C 52 H 61 N 10 O 11 S + : 1033.4237; Obs: 1033.4115 (Error: 11.8 ppm).

[0276] Comp.45b: Conversion rate: 84.9%; Retention time of the target product at UV (254 nm): 4.8 - 5.0 min; Calc: C 52 H 63 N 10 O 11 S + : 1035.4393; Obs: 1035.4187 (Error: 19.8 ppm).

[0277] Example 20

[0278] Solid-phase visible light coupling reaction of Comp.3 and polypeptide 29

[0279]

[0280] The specific operation was the same as that in Example 12.

[0281] Comp.46: Conversion rate: 73%; Retention time of the target product at UV (254 nm): 4.1 - 4.3 min; Calc: C 50 H 61 N 10 O9S + : 977.4338; Obs: C50 H 61 N 10 O9S + : 977.4258 (Error: 8.2 ppm).

[0282] Example 21

[0283] Solid-phase visible light coupling reaction of Comp.15 and polypeptide 30

[0284]

[0285] The specific operation is the same as that in Example 12.

[0286] Comp.47a: Conversion rate: 1.26%; Retention time of the target product in UV (254 nm): 7.3 - 7.5 min; Calc: C 58 H 60 N9O9S + : 1058.4229; Obs: 1058.3968 (Error: 24.7 ppm).

[0287] Comp.47b: Conversion rate: 89.94%, Retention time of the target product in UV (254 nm): 7.8 - 8.1 min; Calc: C 58 H 58 N9O9S + : 1056.4073; Obs: 1056.3910 (Error: 15.4 ppm).

[0288] Example 22

[0289] Solid-phase visible light coupling reaction of Comp.2 and polypeptide 31

[0290]

[0291] The specific operation is the same as that in Example 12.

[0292] Comp.48a: Conversion rate: 9.6%; Retention time of the target product in UV (254 nm): 10.0 - 10.1, 10.4 - 10.6 min; Calc: C 68 H 76 N9O 13 S + : 1258.5278; Obs: 1258.5218 (Error: 4.7 ppm)

[0293] Comp.48b: Conversion rate: 62.4%; Retention time of the target product in UV (254 nm): 8.6 - 9.0, 9.0 - 9.3 min; Calc: C 68 H78 N9O 13 S + : 1260.5434; Obs: 1260.5344 (Error: 7.1 ppm)

[0294] The 2-Cl resin linked with the polypeptide sequence was recovered, and the photoreaction was continued for 12 h. The post-treatment was the same as that in Example 12.

[0295] Comp.48a: Conversion rate: 18%; Retention time of the target product UV (254 nm): 10.0 - 10.1, 10.4 - 10.6 min; Calc: C 68 H 76 N9O 13 S + : 1258.5278; Obs: 1258.5218 (Error: 4.7 ppm).

[0296] Comp.48b: Conversion rate: 82%; Retention time of the target product UV (254 nm): 8.6 - 9.0, 9.0 - 9.3 min; Calc: C 68 H 78 N9O 13 S + : 1260.5434; Obs: 1260.5344 (Error: 7.1 ppm)

[0297] Example 23

[0298] Solid-phase visible light coupling reaction of Comp.2 and polypeptide 32

[0299]

[0300] The specific operation was the same as that in Example 12.

[0301] Comp.49a: Conversion rate: 85%; Retention time of the target product UV (254 nm): 3.1 - 3.5 min; Calc: C 43 H 59 N8O 13 S2 +: 959.3638; Obs: 959.3643 (Error: 0.5 ppm).

[0302] Comp.49b: Conversion rate: 15%; Retention time of the target product UV (254 nm): 3.8 - 4.1 min; Calc: C 43 H 59 N8O 12 S2 + : 943.3688; Obs: 943.3636 (Error: 5.5 ppm).

[0303] Example 24

[0304] Solid-phase visible-light coupling reaction of Comp.2 and polypeptide 33

[0305]

[0306] The specific operation is the same as that in Example 12.

[0307] Comp.50a: Conversion rate: 7.6%; Retention time of the target product at UV (254 nm): 8.5 - 8.6 min; Calc: C 53 H 62 N9O 11 S + : 1032.4284; Obs: 1032.4207 (Error: 7.5 ppm).

[0308] Comp.50b: Conversion rate: 75.7%; Retention time of the target product at UV (254 nm): 6.8 - 7.1 min; Calc: C 53 H 64 N9O 11 S + : 1034.4441; Obs: 1034.4371 (Error: 6.8 ppm).

[0309] Example 25

[0310] Solid-phase visible-light coupling reaction of Comp.2 and polypeptide 34

[0311]

[0312] The specific operation is the same as that in Example 12.

[0313] Comp.51a: Conversion rate: 2.61%; Retention time of the target product at UV (254 nm): 8.6 - 8.8 min; Calc: C 77 H 94 N 13 O 16 S + : 1488.6657; Obs: 1488.6608 (Error: 3.3 ppm).

[0314] Comp.51b: Conversion rate: 47.39%; Retention time of the target product at UV (254 nm): 6.8 - 7.3 min; Calc: C 77 H 96 N 13 O 16 S +: 1490.6813; Obs: 1490.6711 (Error: 6.8 ppm).

[0315] Example 26

[0316] Solid-phase visible light coupling reaction of Comp.17 and polypeptide 35

[0317]

[0318] The specific operation is the same as that in Example 12.

[0319] Comp.52a: Conversion rate: 80.4%; Retention time of the target product in UV (254 nm): 5.0 - 5.4 min; Calc: C 55 H 73 N 12 O 12 S + : 1125.5186; Obs: 1125.5018 (Error: 14.9 ppm).

[0320] Comp.52b: Conversion rate: 19.6%; Retention time of the target product in UV (254 nm): 5.5 - 5.7 min; Calc: C 55 H 71 N 12 O 12 S + : 1123.5030; Obs: 1123.4899 (Error: 11.7 ppm).

[0321] Example 27

[0322] Solid-phase visible light coupling reaction of Comp.2 and polypeptide 36

[0323]

[0324] The specific operation is the same as that in Example 12.

[0325] Comp.53a: Conversion rate: 34%; Retention time of the target product in UV (254 nm): 6.2 - 6.4 min; Calc: C 56 H 72 N 11 O 15 S + : 1170.4925; Obs: 1170.4802 (Error: 10.5 ppm).

[0326] Comp.53b: Conversion rate: 32%; Retention time of the target product in UV (254 nm): 5.7 - 5.8 min; Calc: C 56 H74 N 11 O 15 S + : 1172.5081; Obs: 1172.4861 (Error: 18.8 ppm).

[0327] Example 28

[0328] Solid-phase visible light coupling reaction of Comp.18 and polypeptide 37

[0329]

[0330] The specific operation is the same as that in Example 12.

[0331] Comp 54: Conversion rate: 47.6%; Retention time of the target product UV (254 nm): 5.1 - 5.4 min; Calc: C 56 H 74 N 15 O 12 S + : 1180.5357; Obs: 1180.5268 (Error: 7.5 ppm).

[0332] Example 29

[0333] Solid-phase visible light coupling reaction of Comp.3 and polypeptide 30

[0334]

[0335] (1) Small-scale reaction: The specific operation is the same as that in Example 12.

[0336] Comp.55: Conversion rate: 95.6%; Retention time of the target product UV (254 nm): 5.7 - 6.0 min; Calc: C 54 H 57 N8O8S + : 977.4015; Obs: 977.3930 (Error: 8.7 ppm); LC-MS / MS sequencing analysis confirmed that the modification site of the target product is located at the cysteine site ( Figures 5 - 6 )

[0337]

[0338] (2) Preparation-scale solid-phase visible light coupling reaction

[0339] The 2-Cl resin (30, 77.2 μmol) linked with a polypeptide sequence was swollen in DMA for 30 min. After pumping out DMA, 2 ml of DMA and Comp.3 (5.0 eq) were added, and the subsequent operations were the same as those in Example 12. The cutting and recovery of the filtrate were repeated 3 times. The filtrate was concentrated and purified by semi-preparative liquid chromatography (equipment model: UV2000, Beijing Aonuo Technology Co., Ltd.) to obtain 12 mg of pure product. The LC-MS of the pure product is shown in Figure 7 。

[0340] Comp.53: UV (254 nm) retention time: 6.2 - 6.7 min; Calc: C 54 H 57 N8O8S + : 977.4015; Obs: 977.3943 (error: 7.4 ppm).

[0341] Example 30

[0342] Synthesis and application of a targeted Aurora A PROTAC (proteolysis targeting chimera) compound

[0343]

[0344] i. Solid-phase synthesis of cysteine- and MLN-8237 fragment-containing compound on Fmoc-Ala-2-ChlorotritylResin was carried out. The specific operation was the same as that in Example 10. 2 mg of resin was cut and reacted with 200 μL of a TFE / DCM (v / v, 1 / 1) mixed solution for 1 h, and the cutting solution was collected to obtain Comp.56. After dilution with ACN, LC-HRMS analysis was performed.

[0345] Comp.56: Conversion rate: 98%; Target product UV (254 nm) retention time: 5.9 - 6.1 min; Calc: C 33 H 31 ClFN6O6S + : 693.1693; Obs: 693.1687 (error: 0.9 ppm).

[0346] ii. Solid-phase photoreaction: The specific operation was the same as that in Example 12. 2 mg of resin was cut and reacted with 200 μL of a TFE / DCM (v / v, 1 / 1) mixed solution for 1 h, and the cutting solution was collected to obtain Comp.55. After dilution with ACN, LC-HRMS analysis was performed.

[0347] Comp.57: Conversion rate: 46.9%; Target product UV (254 nm) retention time: 7.0 - 7.2 min; Calc: C 49 H 44 ClFN9O7S+ : 956.2751; Obs: 956.2625 (Error: 13.1 ppm).

[0348] iii. Solid-phase click reaction: Take 2 mg of the resin after the above solid-phase photoreaction, swell it in DMA for 30 min, then incubate CuSO4 and TBTA at room temperature for 5 min and add them to the resin. Finally, add pomalidomide-carbon tri-azide (CAS No.: 2357108-05-3) and TCEP successively (refer to Table 2).

[0349] After reacting on a rotary shaker at room temperature for 4 h, dry the reaction solvent by suction and wash the resin 9 times alternately with DCM, DMF, and DCM. Place it in a vacuum drying oven and dry overnight. Take 2 mg of the resin and cut and react it with 200 μL of TFE / DCM (v / v, 1 / 1) mixed solution for 1 h, collect the cutting solution, obtain Comp.58, and perform LC-HRMS analysis after dilution with ACN. Comp.58: Calc: C 62 H 56 ClFN 15 O9S + : 1240.3773; Obs: 1240.3755 (Error: 1.5 ppm).

[0350] Table 2 Raw material table for alkyne-azide click reaction

[0351]

[0352] Example 31

[0353] Liquid-phase visible light coupling reaction of Comp.3 and BSA

[0354]

[0355] Dissolve BSA (purchased from MCE) in ammonium acetate buffer (pH 7.0, 20 mM) to prepare a 20 μM solution, and add Comp.3 (100.0 eq) to the solution. Replace nitrogen 5 times under ultrasound (10 s / time), and under blue light LED irradiation, stir at room temperature under nitrogen protection for 3 hours. After the reaction, perform LC-HRMS analysis (AB Sciex, TripleTOF 6600; chromatographic column: ACQUITY UPLC Protein BEH C4.

[0356] Figure 8 It is the LC-MS diagram of the chemically modified product of bovine serum albumin BSA. The reaction conversion rate is 53.3%. We obtained the expected protein modification product, and the molecular weight of the product is in good agreement with the prediction.

[0357] Calc: 66607.9 Da; Obs: 66606.5 (error: 21 ppm). Figure 8 The *marked part in the middle* is the impurity in the BSA raw material.

[0358] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A cysteine site-specific modified amino acid, polypeptide, and protein, characterized in that, Its mercapto group is substituted by any one of the groups in Formula I-IV: Wherein, the dashed line represents a single bond or none; In Formula I, the A ring is selected from I-A-a or I-A-b, in Formula II, the A ring is selected from Formula II-A, in Formula III, the A ring is selected from Formula III-A, in Formula IV, the A ring is selected from Formula IV-A, and in Formula V, the A ring is selected from Formula V-A; In Formula I, when the A ring is selected from I-A-a, R3 is unsubstituted; when the A ring is selected from I-A-b, R2 is unsubstituted; When both C2-N1 and C2-C3 are single bonds, the carbon atoms where R8 and R9 are located respectively are connected by a single bond; When C2-N1 is a double bond, R1 and R2 are unsubstituted, and the carbon atoms where R8 and R9 are located respectively are connected by a single bond; When C2-C3 is a double bond, the carbon atoms where R8 and R9 are located respectively are connected by a single bond or a double bond, and R2 and R3 are unsubstituted; When R2 and R3 are substituted, R2 and R3 are independently selected from hydrogen or C1-C3 alkyl; In Formula II, when C2-N1 is a double bond, R1 is unsubstituted, and R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; In Formula III, when C2-C3 is a single bond, the carbon atoms where R8 and R9 are located respectively are connected by a single bond; When C2-C3 is a double bond, the carbon atoms where R8 and R9 are located respectively are connected by a single bond or a double bond, and R2 is unsubstituted; When R2 is substituted, R2 is selected from hydrogen or C1-C3 alkyl; R3 is selected from -CHO, -CN, -NO2, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X; In Formula IV, when C2-N1 is a double bond, R1 is unsubstituted; when C2-C3 is a double bond, R3 is unsubstituted; R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; When R3 is substituted, R3 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; In Formula V, R3 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; In Formulas I-V, the R4-R7 are independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -CO2X, -CONHX or -CON(Me)X, the R8 and R9 are independently selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X, and when R1 is substituted, R1 is selected from hydrogen or C1-C3 alkyl; The total number of -CO2X, -CONHX or -CON(Me)X in the substituents R2-R9 is 0, 1 or 2; X in the -CO2X, -CONHX or -CON(Me)X is selected from C1-C6 alkyl or any one of the following structures: Wherein, n is an integer between 0 and 10, m is selected from 0 or 1, CCG is a click chemical reaction group, and PG is an amino or carboxyl protecting group.

2. The mercapto-site-modified amino acids, polypeptides and proteins according to claim 1, characterized in that, The mercapto group in the amino acid, polypeptide and protein with site-specific modification of mercapto group is substituted by any one of the following groups: Wherein, R1 is selected from hydrogen or C1-C3 alkyl; R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; R 3a selected from hydrogen or C1-C3 alkyl; R 3b selected from -CHO, -CN, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X; R 3c selected from hydrogen, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X; R4-R7 are independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -CO2X, -CONHX or -CON(Me)X; R8 and R9 are independently selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; The total number of -CO2X, -CONHX or -CON(Me)X in the substituents R2-R9 is 0, 1 or 2; X in the -CO2X, -CONHX or -CON(Me)X is selected from C1-C6 alkyl or any of the following structures: wherein, n is selected from an integer between 0 and 10, m is selected from 0 or 1, CCG is selected from alkynyl, azide, dibenzocyclooctynyl, bicyclononyne, tetrazine, trans-cyclooctenyl or sulfonyl fluoride group, and PG is selected from allyl, 4-methyltrityl, allyloxycarbonyl, tert-butyl or tert-butoxycarbonyl.

3. The mercapto-site-specifically modified amino acids, polypeptides and proteins according to claim 1, characterized in that, The mercapto group in the mercapto site-specific modified amino acids, polypeptides and proteins is replaced by any of the following groups:

4. The preparation method of the amino acid, polypeptide and protein with site-specific thiol modification according to any one of claims 1 to 3, characterized in that, Comprising the following steps: Reacting the mercapto-containing amino acid, polypeptide or protein with the indole derivative having an isocyanide group shown in Formula VI under nitrogen protection and blue LED illumination to prepare the mercapto site-specific modified amino acids, polypeptides and proteins; Alternatively, reacting the solid-phase resin of the mercapto-containing amino acid fragment with the indole derivative having an isocyanide group shown in Formula VI under nitrogen protection and blue LED illumination to prepare the mercapto site-specific modified amino acids and polypeptides; Wherein, one of R1, R2 or R3 is replaced by group B, or R4 is replaced by group C; When R1 is replaced by group B, R3 is selected from -CHO, -CN, -NO2, -CH2CH2CO2X, -CO2X, -CONHX or -CON(Me)X, and R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; When R2 is replaced by group B, R1 is selected from hydrogen, C1-C3 alkyl, and R3 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; When R3 is replaced by group B, R1 is selected from hydrogen, C1-C3 alkyl, and R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; When R4 is replaced by group C, R1 is selected from hydrogen or C1-C3 alkyl, R2 is selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X, and R3 is selected from hydrogen or C1-C3 alkyl; R4-R7 are independently selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, -CO2X, -CONHX or -CON(Me)X; R8 and R9 are independently selected from hydrogen, C1-C3 alkyl, -CO2X, -CONHX or -CON(Me)X; The total number of -CO2X, -CONHX or -CON(Me)X in the substituents R2-R9 is 0, 1 or 2; X in -CO2X, -CONHX or -CON(Me)X is selected from C1-C6 alkyl or any of the following structures: Wherein, n is selected from integers between 0 and 10, m is selected from 0 or 1, CCG is a click chemical reaction group, and PG is an amino or carboxyl protecting group.

5. The preparation method according to claim 4, characterized in that The molar ratio of the mercapto-containing amino acid, polypeptide or protein to the indole derivative having an isocyanide group shown in Formula VI is (5:1)-(1:1000).

6. The preparation method according to claim 4, characterized in that, The solvent for the liquid-phase photoreaction is selected from one or more of DMA, DMF, methanol, acetonitrile, water, and ammonium acetate buffer solution.

7. The preparation method according to claim 4, characterized in that, The temperature of the liquid-phase photoreaction is room temperature, and the time is 0.5-48 h.

8. The preparation method according to claim 4, characterized in that, The solid-phase resin of the mercapto-containing amino acid fragment is selected from 2-chlorotrityl resin or Sieber Amide resin.

9. The preparation method according to claim 8, wherein, When using 2-chlorotrityl resin for solid-phase photoreaction, the cutting solution is selected from a mixed solution of trifluoroethanol and dichloromethane with a volume ratio of (10:1)-(1:5); When using Sieber Amide resin for solid-phase photoreaction, the cutting solution is selected from a mixed solution of trifluoroacetic acid and dichloromethane with a trifluoroacetic acid volume fraction of 1%-5%.

10. Use of the site-specifically modified mercapto amino acid, polypeptide and protein according to any one of claims 1-3 or the site-specifically modified mercapto amino acid, polypeptide and protein prepared by the preparation method according to any one of claims 4-9 in biomedicine and drug research and development.

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