CN111087450B

CN111087450B - Histidine-containing polypeptide compound modified by visible light-promoted C-H alkylation and preparation method thereof

Info

Publication number: CN111087450B
Application number: CN201910758444.4A
Authority: CN
Inventors: 王平; 陈小平; 叶发荣
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2022-03-08
Anticipated expiration: 2039-08-16
Also published as: CN111087450A

Abstract

The present invention relates to histidine-containing polypeptides modified by visible light-promoted C-H alkylation and methods for their preparation. The preparation method comprises the following steps: the compound of formula I, the compound of formula II and acid are subjected to intermolecular Minisci alkylation reaction under the irradiation of visible light to generate the compound of formula III modified by an imidazole ring of a histidine residue in polypeptide or protein. The method realizes the high-selectivity post-transcriptional modification (PTM) on the C-2 position of the histidine residue imidazole ring for the first time, simplifies the modification method of unnatural amino acids in polypeptides and proteins, overcomes the limitation that the amino acids and functional groups existing in the traditional polypeptide and protein modification are incompatible, and widens the new method for post-transcriptional modification of polypeptides or proteins. The method provides an efficient and novel method for establishing a novel drug molecule library and screening high-flux active drugs for protein and polypeptide drugs through post-transcriptional modification.

Description

Histidine-containing polypeptide compound modified by visible light-promoted C-H alkylation and preparation method thereof

Technical Field

The invention belongs to the field of chemical synthesis of polypeptides, and particularly relates to a C-H alkylation modified histidine-containing polypeptide promoted by visible light and a preparation method thereof.

Background

Polypeptides, as a very important class of biomolecules, play an essential role in living systems. Natural or synthetic polypeptides have been successfully used as medicinal molecules and are an indispensable role in the development of new drugs. Compared with the full synthesis, the site selective modification of the existing polypeptide can simplify the synthesis steps, thereby leading the activity research of the polypeptide to be more direct and efficient. Many effective methods of polypeptide modification have been developed, but most are limited to some nucleophilic residues, such as cysteine (Cys) and lysine (Lys). The elimination of this constraint would greatly expand the means of polypeptide functionalization. A method for directly modifying a class of C-H bonds commonly found in polypeptide molecules is described herein, which provides another means for selectively modifying a polypeptide at different active sites. In contrast to the extensive research on the functionalization of small molecules with C-H bonds that has been developed, the modification of C-H bonds for polypeptides remains in the initial stage^1,2. Of which mostThe major challenge is to get rid of the interference of various functional groups in polypeptide chain to the reaction, so as to achieve the goal of chemoselective highly efficient modification of a specific site (as shown in the following figure).

An electron-deficient imidazole heterocycle existing in a histidine (His) side chain enables the histidine (His) side chain to be a very important protein amino acid, and has no replaceable action on protein functions such as metal ion coordination, hydrogen bond interaction between a donor/receptor, proton transfer, nucleophilic catalysis and the like. Histidine is an important site for modification of a class of proteins because of its importance and low abundance (about 2.2%). However, histidine is less nucleophilic than cysteine and lysine, and also lacks other unique properties that make selective modification of proteins at histidine sites exceptionally difficult, and a formidable challenge in protein or polypeptide labeling chemistry. However, the enzymatic process of Selective Adenylyl Methionination (SAM) of histidine C2 is naturally available, providing a very important method of post-translational modification of proteins for biosynthesis of forskolin (Zhang, Y, et al, Dianthamide biosyntheses requisites an organic radial produced by an iro-sulfate enzyme. Nature 465,891 (2010)). Although the mechanism of the biosynthetic process is not fully understood, we hope to achieve the construction of such bonds by free radical mediated Minisci C-H functionalization of the electron deficient aromatic ring.

The key to the selective C-H bond functionalization of histidine is the development of an orthogonal activation method for specific C-H bonds. Furthermore, such conversion reactions must be well compatible with the functional groups on the different polypeptide fragments. Removing interference from other nucleophilic groups and avoiding oxidation side-products such as Cys, Met, His, Trp and TyrThe reaction takes place (Li, S, et al chemical industries of protein pharmaceuticals: mechanisms of oxidation and protocols for stabilization. Biotechnology and Bioengineering 48,490-500 (1995)). Unlike phenylalanine, which has an electrically neutral residue, and electron-rich tyrosine and tryptophan, histidine is the only amino acid in such amino acid residues with aromatic ring side chains that has an electron-deficient heterocyclic imidazole. Meanwhile, nitrogen atoms on the imidazole ring can be protonated under physiological conditions, so that C-2 on the imidazole ring becomes a special electrophilic site on a polypeptide side chain or a skeleton. We hypothesized that the electrophilicity of the imidazole ring can be used in the Minisci radical-mediated C-H bond alkylation reaction to achieve chemoselective modification of histidine. In the Minisci reaction, an electrophilic alkyl radical can add to the C2 position of the imidazole ring, followed by a one-electron transfer, and deprotonation/re-aromatization to give the alkylated product. However, current studies on the histidine Minisci response have not been effectively developed. In recent years, significant progress has been made in the study of the C-H functionalization of electron deficient heterocycloarenes by C-centered radicals formed under oxidative conditions, such as the well-known Minisci reaction, which provides a new approach to the alkylation modification of heterocycloarenes (Duncton, M.A.J.Minisci reactions: versatil CH-functionalization for medical chemists.Med.chem.Comm.,2, 1135-) 1161(2011). We hypothesized whether the electrophilicity of the electron-deficient imidazole ring in histidine would also allow for Minisci free radical mediated C-H bond alkylation. Nucleophilic alkyl free radical can be added to C2 position of imidazole ring, and then single electron transfer is carried out, and alkylation product is obtained through deprotonation/re-aromatization, thereby realizing selective chemical modification of histidine. However, there is currently no report of a Minisci response selective for histidine residues in polypeptides or proteins under mild conditions. It is worth mentioning that Jain et al report a C-H alkylation reaction on a simple histidine derivative using the carboxylic acid free from histidine as a free radical donor at high temperature (NH)₄)₂S₂O₈The product 1-a (Jain, R, et al., regiospecific alkylation) was obtained as an oxidizing agent in moderate yield under relatively vigorous conditionsof histidine and histamine at C-2.Tetrahedron 53,2365-2370(1997).)。

Therefore, how to carry out efficient and high-selectivity C-H alkylation on histidine residues in an unprotected peptide chain under a relatively mild condition by utilizing a Minisci reaction is a technical problem to be solved at present.

Disclosure of Invention

The invention aims to provide a histidine-containing polypeptide modified by visible light-promoted C-H alkylation and a preparation method thereof. We report a C-H alkylation method for efficiently and selectively modifying C2 position of histidine imidazole ring under visible light irradiation and inert atmosphere conditions by using a compound (1, 4-dihydropyridine derivative, DHP-R) as an alkylation reagent. The method can be carried out under very mild conditions, and has good universality for a series of polypeptides and DHP-R reagents. Studies of the mechanism show that DHP-R agents not only act as a source of alkyl radicals in the reaction, but also act as an oxidizing agent to accept hydrogen atoms. Therefore, no additional oxidant is required for the reaction, so that side reactions due to oxidation are largely avoided.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing histidine-containing polypeptide compounds modified by C-H alkylation promoted by visible light,

adding a compound shown in a formula I, a compound shown in a formula II and acid into a solvent, carrying out Minisci C-H functionalization reaction under the action of inert environment and photocatalysis, and carrying out reversed phase preparation on a crude product after ether precipitation to obtain a compound shown in a formula III;

the compound of the formula I is a peptide chain or protein containing histidine; the histidine (His) of the compound of formula I is present at different positions (C-terminal, N-terminal and in the middle of the peptide chain), so there are no special requirements on the position, and the longest is 76 amino acids for the length of the polypeptide; AA refers to short hand of amino acids.

The reaction site of the Minisci C-H functionalization reaction is C2 site of an imidazole ring of histidine; r in the compound of the formula II is open-chain or cyclic secondary and tertiary alkyl containing a hydroxyl group, a carboxyl ether bond, a double bond, a triple bond, azido, alkynyl, a sulfonic group, amino, halogen and a polyethylene glycol chain with amphipathy; the acid is organic acid or inorganic acid, and the addition amount of the acid is more than or equal to zero.

As a further improvement of the above preparation process, the specific alkyl groups represented by R in the compound of formula II are as follows:

as a further improvement of the above preparation method, the acid is selected from one of trifluoroacetic acid (TFA), formic acid, acetic acid, hydrochloric acid, sulfuric acid or sulfonic acid.

As a further improvement of the above production method, the solvent is selected from one of an organic solvent or a mixed solvent of an organic solvent and water; the organic solvent is methanol, ethanol, acetonitrile, tetrahydrofuran, 1, 4-dioxane, 2,2, 2-trifluoroethanol or hexafluoroisopropanol. The ratio of organic solvent to water in the mixed solvent is preferably 1:1 to 10:1, wherein the organic solvent is mainly used for dissolving the compound of formula II and the water is used for dissolving the polypeptide.

As a further improvement of the preparation method, the compound in the formula I is selected from one of short peptide, pharmaceutical peptide (salasin, angiotensin II, gonadorelin, leuprorelin, exenatide, lixisenatide, teckotide, secretin), cyclic peptide (Bremelanotide), glycopeptide or protein in any amino acid combination; the inert environment is an inert environment of an argon atmosphere.

The glycopeptide is specifically the following compound:

as a further improvement of the preparation method, the reaction temperature of the Minisci C-H functionalization reaction is 10-50 ℃, and the reaction time is 1-12H.

As a further improvement of the above preparation process, the compound of formula I is reacted in a solvent at a concentration of 5. mu.M-0.2 mM; the reaction concentration of the compound shown in the formula II is 15 mu M-20 mM; the reaction concentration of the acid is 0. mu.M-20 mM. In the reaction, acid can be added to accelerate the reaction and improve the conversion rate.

As a further improvement of the preparation method, under the photocatalytic condition, the wavelength range of the selected visible light is 380-500nm, and the total light intensity can be 3-40W.

As a further improvement of the preparation method, the structural general formula of the compound is shown as a formula III:

the reaction principle of the invention is as follows:

firstly, under the irradiation of visible light, the substrate DHP-R is subjected to homogeneous C-C cleavage to generate alkyl R & lt & gt and a DHP free radical intermediate A. A can be oxidized by SET (single electron transfer) or Proton Coupled Electron Transfer (PCET) with another molecule of DHP-R to form a cation B and a radical C. And B loses hydrogen to obtain an aromatized pyridine product 5. The nucleophilic alkyl radical R.reacts with the His substrate E activated by the proton to obtain the radical cation intermediate F. By hydrogen transfer (HAT) or single electron transfer/proton(SET/H⁺) Transfer oxidizes F to give the final alkylated product G and tetrahydropyridine D. An alternative route by which the reaction may take place is that between two molecules of DHP-R under light a pair of radical anions H and a pair of radical cations I are generated, these intermediates acting in a subsequent step similarly to the R.donor and electron/hydrogen acceptor. In summary, the DHP-R reagent acts as both an R.donor and an oxidant (as an acceptor for an electron or hydrogen atom) in our light-promoted C-H alkylation reaction of His.

Advantageous effects

1. The modification method breaks through the limitation of His modification in the original polypeptide, and no literature report directly carries out post-transcriptional modification on amino acid His residues in the polypeptide, expands selective chemical modification of the amino acid residues in the polypeptide, and provides a new scheme for polypeptide drug modification and establishment of an active drug molecule library. Secondly, the method has good compatibility to various functional groups, and the used substrate molecules (the compound shown in the formula I) including the polypeptide or the protein can realize high-efficiency and high-selectivity chemical modification without protection. Both traditional C-H activation and Minisci reaction require metal catalysis or the participation of an oxidizing agent, and the strong coordination of N, O, S atoms in the polypeptide and the presence of easily oxidized amino acids (such as Cys, His, Met, Trp and Tyr) in the sequence limit the application of traditional methods in polypeptide chemical modification. The method can realize the high-region selective chemical modification of the free radical precursor DHP to the polypeptide histidine residue only by the action of external visible light under the mild condition of the action of a proper amount of protonic acid. The experimental result shows that the method does not influence the secondary structure of the protein. Therefore, when the method is applied to polypeptide drug modification, the activity reduction caused by the change of the secondary structure of the protein can be avoided.

2. The modified polypeptide or protein has diversity and can be expanded to oligopeptides, polypeptides and proteins; the position of histidine residues in the polypeptide or protein is not influenced, and the histidine residues can extend to the C end, the N end and the middle of a peptide chain; the C-2 modification of the imidazole ring of the histidine residue is highly selective and is not affected in the presence of other natural amino acids. Secondly, the method has good functional group compatibility to the compound of the formula II, and comprises hydroxyl, carboxyl ether bond, double bond, triple bond, azido, alkynyl, sulfonic group, amino, halogen and polyethylene glycol chain with amphipathy. The method realizes the high-selectivity post-transcriptional modification (PTM) on the C-2 position of the histidine residue imidazole ring for the first time, simplifies the modification method of unnatural amino acids in polypeptides and proteins, overcomes the limitation that the amino acids and functional groups existing in the traditional polypeptide and protein modification are incompatible, and widens the new method for post-transcriptional modification of polypeptides or proteins. The method provides an efficient and novel method for establishing a novel drug molecule library and screening high-flux active drugs for protein and polypeptide drugs through post-transcriptional modification.

3. The preparation method of the invention has mild conditions and can realize selective modification on complex polypeptide. The method can be carried out under very mild conditions, and has good universality on a series of polypeptides and DHP reagents. Studies on the mechanism show that DHP agents not only act as a source of alkyl radicals in the reaction, but also act as an oxidizing agent to accept hydrogen atoms. Therefore, the reaction does not need additional oxidant, thereby avoiding side reaction caused by oxidation to a great extent, and the used raw materials are cheap and have wide and easily available sources.

4. The invention has wide temperature application range, and the reaction yield does not have obvious change within the range of 10-45 ℃. On the premise of not influencing the polypeptide structure, the temperature controllability is larger, and the temperature influence caused by the heating of the light source is avoided.

5. The invention has simple and convenient operation, economy and environmental protection. In order to improve the conversion rate and yield, the reaction can be circulated for many times by the same method, after each reaction is finished, the reaction liquid only needs to be precipitated by ether and then centrifuged, the obtained solid can be put into the reaction again by the same method after being simply dried, and only reversed-phase purification needs to be carried out finally. As a result, it was found that the ether treatment in the intermediate step did not affect the overall reaction yield.

6. The invention has important effects on the compatibility of functional groups and the convenience of introducing the functional groups, and further modification and modification of subsequent polypeptide or protein, such as the introduction of alkynyl or azide, and can realize antibody drug coupling through a click chemical reaction. Provides more space for the application of the polypeptide or protein.

Drawings

FIG. 1 is a schematic diagram of a preparation experiment of the present invention;

FIG. 2 is an HPLC chromatogram of crude product 6-a (percentage is the integrated area ratio of 230nm UV absorption);

FIG. 3 is a product 6-a HPLC chromatogram;

FIG. 4 is a mass spectrum of product 6-a ESI;

FIG. 5 is a diagram of product 6-a by tandem mass spectrometry (LCMS/MS);

FIG. 6 is the hydrogen spectrum (500MHz, CD) of product 6-a₃OD)；

FIG. 7 shows the 6-a carbon spectrum (125MHz, CD) of the product₃OD)；

FIG. 8 shows the product 6-a two-dimensional heteronuclear correlation Spectrum (HSQC) (500MHz, CD)₃OD)；

FIG. 9 shows the 15-g hydrogen spectrum (400MHz, CD) of the product₃OD)；

FIG. 10 shows a general reaction scheme for preparing polypeptide compounds according to the present invention.

Detailed Description

The invention will be further illustrated with reference to specific embodiments.

1. Solid phase synthesis of compounds of formula I:

1) synthesis of raw material peptide with carboxylic acid at C terminal:

A.2-Cl-trt resin was added to a polypeptide synthesis tube, and an appropriate amount of dry DCM (10mL/g) was added and nitrogen was bubbled through for swelling for 5 minutes at room temperature. It was filtered off with suction and washed twice with DCM and DMF (10mL/g) in succession. A solution of Fmoc-AA-OH (4equiv.) and DIPEA (8equiv.) in DMF (0.2M) was added and nitrogen bubbled at room temperature for 4 hours. Suction filtration, sequential use of DMF, DCM, MeOH and Et₂O (10mL/g) wash. After the resin is dried under reduced pressure, a small amount of dried resin is accurately taken to determine the loading capacity of the amino acid. A solution of DMF/DIPEA/MeOH (17/2/1, v/v/v) (10mL/g) was added and the mixture was bubbled with nitrogen at room temperature for 1 hour. Suction filtering, sequentially using DMF,DCM and DMF were washed twice (10 mL/g).

A25. mu. mol loading of the resin was weighed and a 20% piperidine/DMF solution (10mL/g) was added to the solid phase synthesis tube and nitrogen bubbled for 10 minutes at room temperature. Suction was applied, and 20% piperidine/DMF solution was added again and nitrogen bubbled for 10 minutes. Suction filtration was performed, and washing was performed twice with DMF, DCM and DMF (10mL/g) in this order.

b. A DMF solution (0.2M) of Fmoc-AA-OH (4equiv.), HATU (3.8equiv.), and DIPEA (8equiv.) was added to the polypeptide reaction tube and nitrogen bubbled at room temperature for 30 minutes. Suction filtration was performed, and washing was performed twice with DMF, DCM and DMF (10mL/g) in this order.

A20% piperidine/DMF solution (10mL/g) was added to the solid phase synthesis tube and nitrogen bubbled at room temperature for 10 minutes. Suction was applied, and 20% piperidine/DMF solution was added again and nitrogen bubbled for 10 minutes. Suction filtration was performed, and washing was performed twice with DMF, DCM and DMF (10mL/g) in this order.

The above two steps of operation are repeated.

c. After Fmoc removal of the resin, DMF, DCM, MeOH and Et are sequentially added₂O (10mL/g) and dried under reduced pressure. Addition of TFA/H₂O/Phenol/TIPES (88/5/5/2, v/v/v/v) shear fluid (10mL/g), shaking in a shaker at room temperature for 2 hours. Suction filtration was carried out, the filtrate was concentrated to 1/10 volumes with a nitrogen stream, diethyl ether was added, centrifugation was carried out, and the supernatant was discarded. The solid was again dispersed in ether and centrifuged. The crude product after drying under reduced pressure was further prepared in reverse phase and lyophilized to give compound of formula I (7-13).

2) Synthesis of raw material peptide with C terminal as amide

rink-Amide-AM resin was added to a polypeptide synthesis tube, and an appropriate amount of dry DCM (10mL/g) was added and nitrogen was bubbled for swelling for 5 minutes at room temperature. It was filtered off with suction and washed twice with DCM and DMF (10mL/g) in succession. 20% piperidine/DMF solution (10mL/g) was added and nitrogen bubbled at room temperature for 10 min. Suction was applied, and 20% piperidine/DMF solution was added again and nitrogen bubbled for 10 minutes. Suction filtration was performed, and washing was performed twice with DMF, DCM and DMF (10mL/g) in this order.

A solution of Fmoc-AA-OH (4equiv.) and DIPEA (8equiv.) in DMF (0.2M) was added and nitrogen bubbled at room temperature for 30 minutes. Suction filtration, sequential use of DMF, DCM, MeOH and Et₂O (10mL/g) wash. After the resin is dried under reduced pressure, a small amount of dried resin is accurately taken to determine the loading capacity of the amino acid. Addition of Ac₂O/pyridine/DMF solution (6/10/84, v/v/v) (10 mL/g). After shaking in a shaker at room temperature for 2 hours, it was filtered off with suction and washed twice with DMF, DCM and DMF in succession (10 mL/g).

b, C, the operation is the same as the synthesis of the raw material peptide of which the C terminal is carboxylic acid. And carrying out reverse phase preparation and freeze-drying to obtain the compound (18-20) of the formula I.

3) Other compounds of formula I, salasin (6), angiotensin ii (14), leuprorelin (15), gonadorelin (16), tikkopeptide (17), ubiquitin (21) and brireum dane (22), were purchased from gill biochemistry and used without any treatment. The bleomycin a2 mixture was purchased from alatin chemistry and used for subsequent reactions after reverse phase preparative purification.

2. Synthesis of Compound of formula II (DHP-R)

To a round bottom flask containing ethylene glycol (2.5M), in order, were weighed aldehyde (1equiv.), ethyl acetoacetate (1equiv.), ethyl 3-amino-2-butenoate (1equiv.), and tetrabutylammonium hydrogen sulfate (12 mol%). Under nitrogen, the mixture was heated to 85 ℃ and reacted at this temperature for 4 hours. After the completion of the reaction monitored by TLC, the reaction solution was cooled to room temperature, diluted with ethyl acetate and water, and extracted 2 times with ethyl acetate. The organic layers were combined, washed successively with water and saturated sodium chloride, and dried over sodium sulfate. And after concentration, the crude product is purified by recrystallization or column chromatography to obtain a compound (DHP- (a-n)) shown in the formula II (wherein DHP-a, b, e, f, h and l are known compounds).

3. Feasibility exploration of different radical precursors for simple histidine Minisci reactions

1) Based on Jain reported literature, the feasibility of the cyclohexyl radical formed by decarboxylation of cyclohexanecarboxylic acid at high temperature under catalysis of silver was attempted for the simple histidine Minisci reaction.

To a round bottom flask were added compound 1(0.21g,0.8mmol,1equiv.), compound 2(0.31g,2.4mmol,3equiv.), AgNO in that order₃(80.6mg,0.48mmol,0.6equiv.) and 16mL H₂SO₄(10%) solution. Heated to 90 ℃ and (NH) dissolved in 15mL of water was slowly added dropwise to the reaction mixture₄)₂S₂O₄(0.55g,2.4mmol,3equiv.) solution. Stirring at room temperature for 15 min, adding the reaction solution into ice water, adjusting pH to 7-8 with ammonia water, extracting with ethyl acetate (2 × 20mL), mixing organic layers, washing with saturated NaCl solution, adding a little Na₂SO₄Drying, filtering and concentrating to obtain a crude product. The crude product was purified by column chromatography (1/15MeOH/DCM, v/v) to give compound 1-a (108.3mg,0.31mmol, 39% isolated yield) (Jain, R, et al.

2) Based on the reported literature of Macmillan, feasibility attempts of simple histidine Minisci reactions with phthalimide cyclohexanecarboxylate as a radical precursor under the action of iridium catalysts were made.

Compound 3(20.2mg,0.1mmol,1equiv.), compound 5(82.0mg,0.3mmol,3equiv.) and [ Ir (dF (CF)₃)ppy)₂(dtbbpy)]PF₆(1.12mg, 1.0. mu. mol,1 mol%) was charged to a 4mL reaction tube, followed by 1mL TFE. After bubbling nitrogen for 15 min, TFA (22. mu.L, 0.3mmol,3equiv.) was added. Blue light at 35 ℃ (blue LED, 1)0w,450nm) for 3 hours. Cooled to room temperature and quenched by addition of TEA (69. mu.L, 0.5mmol,10 equiv.). The reaction was concentrated and purified by column chromatography (1/15MeOH/DCM, v/v) to give compound 4-a (6.2mg, 21.2. mu. mol, 21% isolated yield).

3) Based on the Molander reported literature, DHP was used as a free and precursor feasibility test for simple histidine Minisci reaction.

4(20.2mg, 0.1mmol,1equiv.), DHP-a (100.5mg, 0.3mmol,3equiv.), Na were sequentially added to a 4mL reaction tube₂S₂O₈(71.4mg, 0.3mmol,3equiv.), TFA (22. mu.L, 0.3mmol,3equiv.), and MeCN/H₂O (1mL, 2/1, v/v). Stirring vigorously under nitrogen, reacting at 35 ℃ for 12h, and quenching with TEA (42. mu.L, 0.3mmol, 6 equiv.). The reaction was concentrated and purified by column chromatography (1/15MeOH/DCM, v/v) to give compound 4-a (14.4mg, 49.2. mu. mol, 49% isolated yield) as a viscous liquid.

4. Preparation method of visible light-promoted C-H alkylation modified histidine-containing polypeptide or protein

Examples 1 to 10

Screening of reaction solvent:

polypeptide 6 (2.0. mu. mol,1equiv.), DHP-a (20.0. mu. mol,10equiv.), oxygen-depleted TFA (1.49. mu.L, 20.0. mu. mol,10equiv.) and oxygen-depleted solvent (0.2mL, 10mM) were added to a 1mL dry microtube containing a magnetic stirrer in a glove box, sealed and removed from the argon atmosphere glove box. The reaction was prepared as shown in FIG. 1 by irradiating 2 10W blue LED lamps (sample 3cm from lamp, temperature kept at about 35 ℃ by cooling fan) for 3 hours, adding glacial ethyl ether to the reaction solution, centrifuging, carefully discarding the upper layer, drying the precipitate under reduced pressure, and performing LCMS analysis. The second and third cycles were carried out in the same manner as for example 5. Examples 1-10 differ only in the reaction solvent, as shown in Table 1, and the structural analysis of product 6-a is shown in FIGS. 2-8.

TABLE 1 screening of reaction solvents

a: LCMs Yield; b: circulating for 2 times; c: the cycle was 3 times.

From the above results, it was found that the reaction was carried out in all solvents, and the reaction was most preferable when the solvent was TFE (2, 2, 2-trifluoroethanol).

Screening of the acids used in the reactions of examples 11-16:

polypeptide 6 (2.0. mu. mol,1equiv.), DHP-a (20.0. mu. mol,10equiv.), deoxygenated acid (20.0. mu. mol,10equiv.) and deoxygenated TFE (0.2mL, 10mM) were added to a 1mL dry microtube equipped with a magnetic stirrer in a glove box, sealed and removed from the argon atmosphere glove box. 2 10W blue LED lamps (sample 3cm from lamp, cooling fan to keep the temperature at about 35 ℃) were used for 3 hours of irradiation. Ice ether was added to the reaction solution, the upper layer liquid was carefully discarded after centrifugation, and the precipitate was dried under reduced pressure and subjected to LCMS analysis. Examples 11-16 differ only in the acid used in the reaction, as shown in Table 2.

TABLE 2 screening of acids in the reactions

a：LCMS Yield

From the above results, it can be seen that the reaction can be carried out using different protonic acids, and TFA is the best choice compared to example 5.

Examples 17 to 20

Screening of reagent equivalents in the reaction:

polypeptide 6 (2.0. mu. mol,1equiv.), DHP-a, deoxygenated TFA and deoxygenated TFE (0.2mL, 10mM) were added to a 1mL dry microtube containing a magnetic stirrer in a glove box, sealed and removed from the argon atmosphere glove box. 2 10W blue LED lamps (sample 3cm from lamp, cooling fan to keep the temperature at about 35 ℃) were used for 3 hours of irradiation. Ice ether was added to the reaction solution, the upper layer liquid was carefully discarded after centrifugation, and the precipitate was dried under reduced pressure and subjected to LCMS analysis. Examples 17-20 differ only in the equivalents of DHP-a and TFA used in the reaction, as shown in Table 3.

TABLE 3 screening of DHP-a and TFA equivalents in the reactions

a：LCMs Yleld

From the above results, it can be seen that the use of different equivalents of DHP-a and TFA has different degrees of influence on the reaction. Both the increase and decrease in the equivalents of DHP-a and TFA are reaction disadvantages compared to example 5, with 10 equivalents of DHP-a and 10 equivalents of TFA being the best choice.

Examples 21 to 22

Screening of reaction concentration:

polypeptide 6 (2.0. mu. mol,1equiv.), DHP-a (20.0. mu. mol,10equiv.), oxygen-depleted TFA (1.49. mu.L, 20.0. mu. mol,10equiv.) and oxygen-depleted TFE were added to a 1mL dry microtube containing a magnetic stirrer in a glove box, sealed and removed from the argon atmosphere glove box. 2 10W blue LED lamps (sample 3cm from lamp, cooling fan to keep the temperature at about 35 ℃) were used for 3 hours of irradiation. Ice ether was added to the reaction solution, the upper layer liquid was carefully discarded after centrifugation, and the precipitate was dried under reduced pressure and subjected to LCMS analysis. Examples 21-22 differ only in the reaction concentrations, as shown in Table 4.

TABLE 4 screening of reaction concentrations

a：LCMS Yield

From the above results, it was found that the reaction concentration had little influence on the reaction. In contrast, the reaction yield at a high concentration was slightly lower than that at a low concentration in example 5, and the reaction concentration was set to 10 mM.

Examples 23 to 26

Control experiment of acid, light, temperature and oxygen in the reaction: the procedure was substantially the same as that of examples 1 to 10, except that the acid, light, temperature and oxygen were different in the experiment, as shown in Table 5.

TABLE 5 control of reaction-related conditions

a：LCMS Yield

From the above results, it is not necessarily required that the reaction is accelerated by the light energy. The acid can promote the reaction, but is not required. The temperature also has certain influence on the reaction, and proper heating is beneficial to the reaction. Whereas in the absence of acid and light, no reaction takes place. The presence of oxygen can lead to oxidation of the feedstock and products. Therefore, the reaction is ensured to be carried out smoothly in the inert gas environment under the participation of light and acid.

Through the process of the condition screening, the optimal preparation method can be determined as follows: the compound of formula I (100.0. mu. mol,1equiv.), the compound of formula II (1.0mmol, 10equiv.), oxygen-depleted TFA (75. mu.L, 1.0mmol, 10equiv.), and oxygen-depleted TFE (10mL, 10mM) were added to a 20mL dry reaction flask equipped with a magnetic stirrer. Argon was bubbled to remove oxygen for 2 minutes, sealed and irradiated with 2 10W blue LED lamps (sample 3cm from lamp, cooling fan to maintain temperature around 35 ℃) for 3 hours. The nitrogen stream was concentrated to 1/10, 10mL of glacial ethyl ether were added, and the supernatant liquid was carefully discarded after centrifugation. The precipitate was dried under reduced pressure and repeated twice under the same reaction conditions as above. The precipitate from the three reactions was dried under reduced pressure and analyzed by LCMS, further purified by reverse phase high performance liquid chromatography and lyophilized to give the compound of formula III.

Examples 28 to 33

Examples 28-33 compounds of formula III were prepared under the same general preparative conditions described above using different compounds of formula I, formula II:

1) example 28 Synthesis of Saralasin (6) with functional groups of a Compound of formula II containing different functional groups (DHP- (a-n))

And (3) adaptability expansion:

2) example 29 optional short peptides (7-13) with DHP-a for universal expansion of different amino acid residues:

3) example 30 substrate Cross-propagation of other pharmaceutical peptides (14-20) with Compounds of formula II containing different functional groups (DHP- (a-n)): the structural analysis of the product 15-g is shown in FIG. 9;

4) example 31 protein ubiquitin (21) reacts with DHP-g:

5) example 32 reaction of the cyclic peptide Bremelanotide (22) with DHP-a, g:

6) example 33 reaction of the glycopeptide bleomycin A2(23) with DHP-g:

in the reaction of the example 33 of the invention, no acid is needed, and the reaction time is 2 hours each time, and the cycle is three times.

Example 34

Click chemistry reaction of 6-k with intermediate rhodamine B derivative 24:

rhodamine B derivative 24(3.69mg, 7.2. mu. mol,20equiv.) was weighed and added to a solution containing MeOH (25. mu.L) and CHCl₃(25. mu.L) in a 1mL microteaction flask mixed with solvent. 6-k (0.50mg, 0.36. mu. mol,1equiv.) was dissolved in 50. mu.L of water and added to the above solution, and CuSO was added with vigorous stirring₄·5H₂O (5.0. mu.g, 0.02. mu. mol,5 mol%) and sodium ascorbate (7.9. mu.g, 0.04. mu. mol,10 mol%). The mixture was stirred vigorously at room temperature for 12 hours under nitrogen. The solid insolubles were removed by filtration and the filtrate was made up in reverse phase to give a pink solid 25(0.48mg, 0.24. mu. mol, 68% isolated yield).

5. Characterization of Compound Properties and Structure

1) Characterization data for part of the Compounds of formula I

Compound 7 of formula I (11.9mg, 7.5. mu. mol, 30% isolated yield).

Normalized Mass (theoretical molecular weight) ("theoretical molecular weight")M+2H]²⁺:623.8；[M+3H]³⁺416.2; mass Found (actual molecular weight) (ESI +) [ M +2H ]]²⁺:623.5；[M+3H]³⁺:416.2.

Compound 8 of formula I (11.9mg, 6.5. mu. mol, 26% isolated yield).

Calculated Mass[M+2H]²⁺:692.3；[M+3H]³⁺:461.9；Mass Found(ESI+)[M+2H]²⁺:692.3；[M+3H]³⁺:462.0.

Compound 9 of formula I (10.0mg, 6.5. mu. mol, 26% isolated yield).

Calculated Mass[M+H]⁺:1085.6；[M+2H]²⁺:543.8；Mass Found(ESI+)[M+H]⁺:1086.9；[M+2H]²⁺:544.3.

Compound 10 of formula I (8.89mg, 5.8. mu. mol, 23% isolated yield).

Calculated Mass[M+2H]²⁺:538.8；Mass Found(ESI+)[M+2H]²⁺:539.2.

Compound 11 of formula I (8.62mg, 6.2. mu. mol, 31% isolated yield).

Calculated Mass[M+H]⁺:1039.5；[M+2H]²⁺:520.3；Mass Found(ESI+)[M+H]⁺:1039.7；[M+2H]²⁺:520.6.

Compound 12 of formula I (9.52mg, 6.0. mu. mol, 24% isolated yield).

Calculated Mass[M+H]⁺:1132.5；[M+2H]²⁺:566.8；Mass Found(ESI+)[M+H]⁺:1132.7；[M+2H]²⁺:567.2.

Compound 13 of formula I (10.4mg, 6.3. mu. mol, 25% isolated yield).

Calculated Mass[M+2H]²⁺:602.7；[M+3H]³⁺:402.1；Mass Found(ESI+)；[M+2H]²⁺:602.8；[M+3H]³⁺:402.2.

Secretin 18(26.3mg, 7.5. mu. mol, 15% isolated yield).

Calculated Mass[M+3H]³⁺:1019.5；[M+4H]⁴⁺:764.9；[M+5H]⁵⁺:612.1；Mass Found(ESI+)[M+3H]³⁺:1019.8；[M+4H]⁴⁺:765.1；[M+5H]⁵⁺:612.4.

Exenatide 19(29.8mg, 6.3. mu. mol, 13% isolated yield).

Calculated Mass[M+3H]³⁺:1396.8；[M+4H]⁴⁺:1047.9；[M+5H]⁵⁺:838.5；[2M+5H]⁵⁺:1676.0；Mass Found(ESI+)；[M+3H]³⁺:1396.8；[M+4H]⁴⁺:1047.8；[M+5H]⁵⁺:838.5；[2M+5H]⁵⁺:1676.1.

Lixisenatide 20(30.6mg, 5.0. mu. mol, 10% isolated yield).

Calculated Mass[M+4H]⁴⁺:1215.7；[M+5H]⁵⁺:972.7；[M+6H]⁶⁺:810.9；[M+7H]⁷⁺:695.2；[M+8H]⁸⁺:608.5Mass Found(ESI+)；[M+4H]⁴⁺:1215.8；[M+5H]⁵⁺:972.8；[M+6H]⁶⁺:810.9；[M+7H]⁷⁺:695.2.

2) Characterization data for a part of the compounds of the formula II

DHP-c

¹H NMR(400MHz,CDCl₃)δ5.87(s,1H),4.26–4.06(m,4H),4.01(d,J＝4.4Hz,1H),3.56(t,J＝6.6Hz,2H),2.26(s,6H),1.56(dtt,J＝17.0,13.3,6.7Hz,2H),1.41(dt,J＝12.6,6.2Hz,1H),1.36–1.17(m,7H),1.08–0.91(m,1H),0.72(d,J＝6.8Hz,3H)；¹³C NMR(100MHz,CDCl₃)δ169.1,168.6,145.0,145.0,102.2,101.0,63.4,59.8,59.8,41.0,37.5,30.8,28.7,19.5,19.5,15.3,14.5,14.5；HRMS(ESI):Calcd for C₁₈H₃₀NO₅[M+H]⁺:340.2124,found:340.2130.

DHP-d

¹H NMR(400MHz,CDCl₃)δ5.80(s,1H),4.27–4.07(m,4H),4.01(d,J＝4.3Hz,1H),2.36(t,J＝7.8Hz,2H),2.29(s,6H),1.67–1.58(dt,J＝13.9,7.4Hz,1H),1.50–1.39(m,1H),1.36–1.22(m,8H),0.74(d,J＝6.8Hz,3H)；¹³C NMR(100MHz,CDCl₃)179.7,168.9,168.5,145.5,145.3,101.9,100.7,59.9,59.9,40.7,37.09,32.5,27.8,19.6,19.6,15.2,14.5,14.4；HRMS(ESI):Calcd for C₁₈H₂₇NO₆[M+H]⁺:354.1917,found:354.1922.

DHP-g

¹H NMR(500MHz,DMSO-d⁶)δ8.68(s,1H),4.10(dq,J＝10.9,7.1Hz,2H),4.03(dq,J＝10.9,7.1Hz,2H),3.73(s,1H),2.24(s,6H),1.81(s,3H),1.55(d,J＝11.8Hz,3H),1.46(d,J＝11.5Hz,3H),1.27(s,6H),1.19(t,J＝7.1Hz,6H)；¹³C NMR(125MHz,DMSO-d⁶)δ168.6,145.5,96.95,58.8,42.4,41.4,37.4,36.8,27.8,18.2,14.4；HRMS(ESI):Calcd for C₂₃H₃₃NO₄[M+H]⁺:388.2488,found:388.2494.

DHP-i

¹H NMR for the major diastereomer(500MHz,CDCl₃)δ5.61(d,J＝3.7Hz,1H),4.29–4.08(m,4H),3.95(d,J＝5.7Hz,1H),3.35(d,J＝6.4Hz,2H),2.32(s,6H),1.73(d,J＝11.0Hz,2H),1.61(d,J＝11.2Hz,2H),1.41–1.24(m,8H),1.23–1.15(m,1H),0.98(ddd,J＝25.5,12.8,3.2Hz,3H),0.89(S,9H),0.78(td,J＝12.6,2.8Hz,2H)；¹³C NMR for the major diastereomer(125MHz,CDCl₃)δ168.8,144.6,102.1,69.0,59.7,46.0,40.7,38.3,29.9,28.24,26.2,26.1,19.6,18.5,14.5,-5.2.

DHP-j

¹H NMR showed a 4:1mixture of diastereomers.¹H NMR for the major diastereomer(400MHz,CDCl₃)δ7.79–7.69(m,2H),7.34–7.31(m,2H)5.80(s,1H),4.26–4.07(m,4H),3.94–3.89(m,1H),3.74(d,J＝6.3Hz,2H),2.43(s,Hz,3H),2.27(s,6H),1.79–1.45(m,5H),1.30–1.22(m,7H),1.19–1.06(m,1H),0.92(ddd,J＝15.1,12.7,2.8Hz,2H),0.76(ddd,J＝15.1,12.7,2.8Hz,2H)；¹³C NMR for the major diastereomer(100MHz,CDCl₃)δ168.63,168.5,144.9,144.7,144.7,133.1,129.9,129.9,127.9,127.9,101.6,59.7,59.7,45.3,38.0,37.3,29.2,27.68,2.72,19.6,19.5,14.5；HRMS(ESI):Calcd for C₂₇H₃₇NO₇S[M+H]⁺:520.2369,found:520.2383.

DHP-k

¹H NMR showed a 4:1mixture of diastereomers.¹H NMR for the major diastereomer(500MHz,CDCl₃)δ5.72(d,J＝7.4Hz,1H),4.30–4.06(m,4H),3.96(d,J＝5.6Hz,1H),3.07(d,J＝6.6Hz,2H),2.18(s,6H),1.73(dd,J＝16.9,8.4Hz,2H),1.61(dd,J＝23.2,8.4Hz,2H),1.53–1.39(m,1H),1.29(dt,J＝11.5,7.0Hz,1H),,1.00(qd,J＝12.8,2.9Hz,2H),0.85(qd,J＝12.7,2.8Hz,2H)；¹³C NMR for the major diastereomer(125MHz,CDCl₃)δ168.7,168.6,144.8,144.6,101.8,59.8,59.8,58.1,45.5,38.2,38.2,30.7,28.0,27.4,197,19.6,14.5；HRMS(ESI):Calcd for C₂₀H₃₀N₄O₄[M+H]⁺:391.2345,found:391.2352.

DHP-m

¹H NMR for the major diastereomer(400MHz,CDCl₃)δ5.60(s,1H),4.27-4.21(m,2H),4.20–4.12(m,2H),4.11(d,J＝2.4Hz,2H),3.95(d,J＝5.7Hz,1H),3.28(d,J＝6.4Hz,2H),2.40(t,J＝2.4Hz,1H),2.32(s,6H),1.76(d,J＝10.6Hz,2H),1.61(d,J＝7.9Hz,2H),1.57–1.41(m,1H),1.31(t,J＝7.1Hz,6H),1.28–1.14(m,1H),0.99(qd,J＝12.7,3.0Hz,2H),0.83(qd,J＝12.7,2.8Hz,2H)；¹³C NMR for the major diastereomer(100MHz,DMSO-d⁶)δ167.8,145.7,99.5,80.6,76.8,74.8,58.8,57.4,45.5,37.5,37.4,29.5,27.6,18.2,14.3；HRMS(ESI):Calcd for C₂₃H₃₃NO₅[M+H]⁺:404.2437,found:404.2444.

DHP-n

¹H NMR showed a 6:1mixture of diastereomers.¹H NMR for the major diastereomer(400MHz,CDCl₃)δ6.97(t,J＝5.8Hz,1H),5.99(s,1H),4.35–4.06(m,4H),3.97(s,2H),3.94(d,J＝5.6Hz,1H),3.74–3.60(m,10H),3.60–3.48(m,2H),3.38(s,3H),3.07(t,J＝6.4Hz,2H),2.30(s,6H),1.71(d,J＝11.0Hz,2H),1.60(d,J＝11.1Hz,2H),1.47–1.35(m,1H),1.33–1.24(m,6H),1.23–1.12(m,1H),0.97(qd,J＝12.6,2.5Hz,2H),0.81(qd,J＝25.1,12.6,2.5Hz,2H)；¹³CNMR for the major diastereomer(100MHz,CDCl₃)δ169.9,168.7,144.9,101.6,72.0,71.1,70.7,70.6,70.6,70.4,59.7,59.7,59.1,45.6,45.1,38.1,38.1,30.9,28.1,19.5,19.4,14.5；HRMS(ESI):Calcd for C₂₉H₄₈N₂O₉[M+H]⁺:569.3438,found:569.3454.

3) Characterization data for the Compounds of formula III

3-a

¹H NMR(400MHz,CD₃OD)δ6.73(s,1H),4.65(dd,J＝8.7,5.4Hz,1H),3.69(s,3H),3.10–3.03(m,1H),2.92(dd,J＝14.9,8.7Hz,1H),2.70(ddt,J＝12.0,7.7,3.7Hz,1H),1.98–1.95(m,2H),1.94(s,4H),1.83(dt,J＝12.4,3.2Hz,2H),1.78–1.70(m,1H),1.57–1.45(m,2H),1.40(dd,J＝7.8,4.9Hz,1H),1.37–1.25(m,2H)；¹³C NMR(100MHz,CD₃OD)δ172.8,172.5,153.5,132.3,116.8,53.3,52.1,38.3,32.3,32.2,26.5,26.3,21.6.HRMS(ESI):Calcd for C₁₅H₂₃N₃O₃[M+H]⁺:294.1812,found:294.1804.

6-a

¹H NMR(500 MHz,CD₃OD)δ7.14(s,1H),6.95(d,J＝8.5 Hz,2H),6.57(d,J＝8.5 Hz,2H),4.55–4.49(m,1H),4.40(dd,J＝8.1,5.4 Hz,1H),4.32(dd,J＝7.8,6.0 Hz,1H),4.28(q,J＝7.4 Hz,1H),4.09(d,J＝7.5 Hz,1H),3.98(d,J＝7.7 Hz,1H),3.79–3.71(m,3H),3.42(dt,J＝8.2,6.5 Hz,1H),3.13–3.01(m,4H),2.94–2.84(m,2H),2.71(dd,J＝13.9,8.5 Hz,1H),2.62(s,3H),2.25–2.21(m,1H),2.00–1.84(m,7H),1.82–1.74(m,2H),1.73–1.65(m,2H),1.64–1.56(m,1H),1.55–1.42(m,4H),1.40–1.30(m,5H),1.28–1.13(m,1H),0.83–0.77(m,12H).¹³C NMR(125MHz,CD₃OD)δ174.2,173.2,171.9,171.9,171.5,168.9,165.1,157.2,155.8,151.7,130.0,127.5,127.3,117.3,114.8,59.9,58.7,54.6,53.0,50.2,49.1,48.5,48.3,48.2,40.5,36.7,35.9,32.2,30.9,30.5,30.3,29.5,28.9,26.1,25.2,25.0,24.7,24.7,18.4,18.3,17.5,17.4,15.8；Calculated Mass[M+H]⁺:994.6；[M+2H]²⁺:497.8；Mass Found(ESI+)；[M+H]⁺:994.9；[M+2H]²⁺:498.2.

6-b

¹H NMR(500 MHz,CD₃OD)δ7.27(s,1H),7.07(d,J＝8.4 Hz,2H),6.68(d,J＝8.4 Hz,2H),4.64(dd,J＝8.2,6.4 Hz,1H),4.51(dd,J＝8.1,5.3 Hz,1H),4.44(dd,J＝7.9,5.9 Hz,1H),4.37(q,J＝7.3 Hz,1H),4.21(d,J＝7.5 Hz,1H),4.10(dd,J＝7.7,5.8 Hz,1H),3.87(s,3H),3.60–3.45(m,1H),3.25–3.09(m,4H),3.01(dd,J＝13.9,6.1 Hz,1H),2.84(dd,J＝13.9,8.5 Hz,1H),2.74(s,3H),2.41–2.23(m,1H),2.11–1.92(m,5H),1.86–1.75(m,2H),1.76–1.67(m,2H),1.62(dd,J＝14.2,6.9 Hz,3H),1.51–1.34(m,6H),0.92(dd,J＝8.9,6.9 Hz,12H).¹³C NMR(125 MHz,CD₃OD)δ174.3,173.1,171.9,171.9,171.9,171.5,168.9,165.2,157.3,155.8,152.6,130.0,127.5,117.3,114.8,59.9,58.8,58.7,54.6,53.0,50.2,49.1,48.4,40.5,36.7,32.2,30.9,30.5,29.5,28.9,26.7,26.2,24.7,24.6,19.3,18.4,18.3,17.6,17.4,15.8；Calculated Mass[M+H]⁺:954.5；[M+2H]²⁺:477.8；Mass Found(ESI+)；[M+H]⁺:954.8；[M+2H]²⁺:478.2.

6-c

¹H NMR(500 MHz,CD₃OD)δ7.29(d,J＝5.5 Hz,1H),7.08(d,J＝8.3 Hz,2H),6.69(d,J＝8.5Hz,2H),4.64(ddd,J＝8.6,6.4,2.5 Hz,1H),4.52(dd,J＝8.1,5.2 Hz,1H),4.44(dd,J＝8.0,5.8Hz,1H),4.38(q,J＝7.4 Hz,1H),4.22(d,J＝7.5 Hz,1H),4.09(d,J＝7.8 Hz,1H),3.92–3.85(m,3H),3.59–3.50(m,3H),3.29–3.13(m,5H),3.01(ddd,J＝13.8,6.2,2.0 Hz,1H),2.84(dd,J＝13.9,8.4 Hz,1H),2.74(s,3H),2.41–2.31(m,1H),2.11–1.95(m,5H),1.88–1.77(m,3H),1.76–1.68(m,1H),1.66–1.58(m,2H),1.58–1.50(m,1H),1.49–1.38(m,6H),1.36–1.28(m,1H),0.99–0.88(m,12H).¹³C NMR(125 MHz,CD₃OD)δ174.3,173.2,171.9,171.9,171.5,169.0,165.1,157.3,155.8,152.0,130.0,127.5,117.4,114.8,60.8,60.7,59.9,58.7,58.6,54.6,53.0,50.3,49.1,48.4,48.2,40.5,36.7,32.2,32.1,31.6,30.9,30.6,29.6 29.5,28.9,26.3,24.7,24.6,18.4,18.3,17.6,17.5,17.4,15.8；Calculated Mass[M+H]⁺:999.2；[M+2H]²⁺:500.1；Mass Found(ESI+)；[M+H]⁺:998.9；[M+2H]²⁺:500.2.

6-d

Calculated Mass[M+H]⁺:1012.5；[M+2H]²⁺:506.8；Mass Found(ESI+)；[M+H]⁺:1012.7；[M+2H]²⁺:507.1.

6-e

Calculated Mass[M+H]⁺:968.6；[M+2H]²⁺:484.8；Mass Found(ESI+)；[M+H]⁺:968.7；[M+2H]²⁺:485.2.

6-g

Calculated Mass[M+H]⁺:1047.3；[M+2H]²⁺:524.2；Mass Found(ESI+)；[M+H]⁺:1046.9；[M+2H]²⁺:524.2.

6-h

Calculated Mass[M+H]⁺:996.6；[M+2H]²⁺:498.8；Mass Found(ESI+)；[M+H]⁺:996.7；[M+2H]²⁺:499.1.

6-i

Calculated Mass[M+2H]⁺:1024.6；[M+2H]²⁺:512.8；Mass Found(ESI+)[M+2H]⁺:1024.5；[M+2H]²⁺:512.9.

6-j

Calculated Mass[M+2H]²⁺:590.2；Mass Found(ESI+)；[M+2H]²⁺:590.3.

6-k

¹H NMR(500 MHz,CD₃OD)δ7.27(s,1H),7.07(d,J＝8.5 Hz,2H),6.69(d,J＝8.4 Hz,2H),4.68–4.60(m,1H),4.51(dt,J＝10.3,5.1 Hz,1H),4.44(dd,J＝8.0,5.8 Hz,1H),4.37(dt,J＝7.4,5.3Hz,1H),4.21(d,J＝7.5 Hz,1H),4.09(d,J＝7.8 Hz,1H),3.89–3.81(m,3H),3.58–3.53(m,1H),3.25(d,J＝7.5 Hz,2H),3.22–3.14(m,4H),3.07–2.95(m,2H),2.83(dd,J＝13.9,8.5 Hz,1H),2.74(s,3H),2.42–2.23(m,1H),2.17–1.86(m,10H),1.83–1.52(m,8H),1.51–1.38(m,3H),1.34–1.16(m,2H),0.96–0.80(m,12H).¹³C NMR(125 MHz,CD₃OD)δ174.2,173.2,172.0,171.9,171.5,168.9,165.2,161.7 161.4,157.2,155.8,151.2,143.6,130.0,127.5,127.4,117.4,114.8,59.9,58.7,56.9,54.6,53.0,50.2,49.1,48.4,40.5,36.9,36.7,35.6,32.2,30.9,30.5,29.6,29.5,29.0,28.9,26.1,24.7,18.4,18.3,17.5,17.4,15.8；Calculated Mass[M+2H]²⁺:525.6；Mass Found(ESI+)[M+2H]²⁺:525.6.

6-l

¹H NMR(400 MHz,CD₃OD)δ7.24(d,J＝2.9 Hz,1H),7.05(d,J＝8.5 Hz,2H),6.66(d,J＝8.5Hz,2H),5.86–5.68(m,2H),4.67–4.57(m,1H),4.49(dd,J＝7.8,4.6 Hz,1H),4.46–4.37(m,1H),4.35(q,J＝7.4 Hz,1H),4.18(d,J＝7.5 Hz,1H),4.07(dt,J＝7.8,3.9 Hz,1H),3.84(s,3H),3.60–3.51(m,1H),3.21–3.12(m,4H),2.98(dd,J＝13.9,6.0 Hz,1H),2.82(dt,J＝13.9,7.1 Hz,1H),2.71(s,3H),2.50–2.36(m,1H),2.37–2.26(m,2H),2.22(dd,J＝15.7,2.6 Hz,2H),2.13–1.91(m,7H),1.89–1.64(m,3H),1.62–1.57(m,2H),1.41(d,J＝7.4 Hz,3H),0.89(t,J＝7.0 Hz,12H).¹³C NMR(100 MHz,CD₃OD)δ175.6,174.6,173.3,173.3,172.9,170.2,166.6,158.6,157.7,157.2,152.6,131.4,128.9,128.9,128.2,128.1,125.2,125.1,118.9,116.2,61.3,60.2,60.1,55.9,54.4,51.6,50.5,41.9,38.1,33.6,32.3,31.9,30.9,30.3,30.2 30.0,27.8,27.5,27.4,26.1,26.0,25.4,19.8 19.7,19.0,18.8,17.2,17.2；Calculated Mass[M+H]⁺:992.6；[M+2H]²⁺:496.8；Mass Found(ESI+)；[M+H]⁺:992.8；[M+2H]²⁺:497.0.

6-m

Calculated Mass[M+2H]²⁺:532.2；Mass Found(ESI+)；[M+2H]²⁺:531.9.25

Calculated Mass[M+H]²⁺:765.4；[M+2H]³⁺:510.6；Mass Found(ESI+)[M+H]²⁺:765.6；[M+2H]³⁺:510.8.

6-n

Calculated Mass[M+H]⁺:1228.5；[M+2H]²⁺:614.8；[M+3H]³⁺:410.2；Mass Found(ESI+)；[M+H]⁺:1228.1；[M+2H]²⁺:614.9；[M+3H]³⁺:410.3.

8-a

Calculated Mass[M+2H]²⁺:733.4；[M+3H]³⁺:489.2；Mass Found(ESI+)[M+2H]²⁺:733.5；[M+3H]³⁺:489.4.

9-a

Calculated Mass[M+H]⁺:1169.4；[M+2H]²⁺:585.2；Mass Found(ESI+)；[M+H]⁺:1169.1；[M+2H]²⁺:585.3.

10-a

Calculated Mass[M+2H]²⁺:538.8；Mass Found(ESI+)[M+2H]²⁺:539.2.

11-a

Calculated Mass[M+H]⁺:1122.3；[M+2H]²⁺:561.6；Mass Found(ESI+)；[M+H]⁺:1121.9；[M+2H]²⁺:561.8.

13-a

Calculated Mass[M+2H]²⁺:643.8；[M+3H]³⁺:429.5；Mass Found(ESI+)[M+2H]²⁺:643.7；[M+3H]³⁺:429.6.

14-a

Calculated Mass[M+H]⁺:1129.3；[M+2H]²⁺:565.2；Mass Found(ESI+)[M+H]⁺:1128.9；[M+2H]²⁺:565.2.

14-c

Calculated Mass[M+H]⁺:1133.3；[M+2H]²⁺:567.2；Mass Found(ESI+)[M+H]⁺:1132.8；[M+2H]²⁺:567.2.

14-e

Calculated Mass[M+H]⁺:1103.3；[M+2H]²⁺:552.2；Mass Found(ESI+)[M+H]⁺:1103.0；[M+2H]²⁺:552.2.

14-k

Calculated Mass[M+H]⁺:1184.4；[M+2H]²⁺:592.7；Mass Found(ESI+)[M+H]⁺:1184.0；[M+2H]²⁺:592.8.

15-a

Calculated Mass[M+2H]²⁺:646.8；Mass Found(ESI+)；[M+2H]²⁺:646.9.

15-b

Calculated Mass[M+H]⁺:1252.4；[M+2H]²⁺:626.8；Mass Found(ESI+)；[M+H]⁺:1252.0；[M+2H]²⁺:626.9.

15-d

Calculated Mass[M+2H]²⁺:655.8；Mass Found(ESI+)[M+2H]²⁺:655.8.

15-g

¹H NMR(400 MHz,CD₃OD)δ7.56(d,J＝7.9 Hz,1H),7.34(d,J＝8.1 Hz,1H),7.16–7.05(m,3H),6.99(dd,J＝11.4,4.6 Hz,3H),6.69(d,J＝8.5 Hz,2H),4.72(dd,J＝8.1,5.6 Hz,1H),4.68–4.62(m,2H),4.49–4.46(m,2H),4.35–4.25(m,2H),4.14(dd,J＝9.9,5.4 Hz,1H),4.08(dd,J＝8.5,4.7 Hz,1H),4.87–4.81(m,1H),3.74(dd,J＝11.2,5.5 Hz,1H),3.57–3.66(m,2H),3.24–3.04(m,6H),3.03–2.82(m,3H),2.36–2.17(m,3H),2.18–2.08(m,4H),2.08–1.98(m,7H),1.96–1.77(m,10H),1.77–1.53(m,7H),1.52–1.35(m,2H),1.20–1.00(m,4H),0.90(d,J＝6.3 Hz,3H),0.82(dd,J＝9.9,6.4 Hz,6H),0.77(d,J＝6.5 Hz,3H).¹³C NMR(100 MHz,CD₃OD)δ178.0,173.6,173.6,173.1,172.7,172.5,172.4,170.6,170.6,157.2,156.1,154.4,136.6,129.9,128.6,127.4,127.1,123.5,121.2,118.6,117.9,116.2,115.0,111.1,109.2,64.0,61.9,60.4,56.3,55.9,55.4,54.4,52.4,52.1,51.9,50.6,40.8,39.6,39.4,36.2,35.5,34.6,33.9,29.5,29.0,28.0,27.7,27.3,26.5,25.2 24.6,24.5,24.4,24.0,22.2,21.9,20.5,20.2,13.4；Calculated Mass[M+2H]²⁺:672.8；Mass Found(ESI+)[M+2H]²⁺:673.0.

15-h

Calculated Mass[M+2H]²⁺:647.8；Mass Found(ESI+)[M+2H]²⁺:647.8.

15-i

Calculated Mass[M+H]⁺:1322.5；[M+2H]²⁺:661.8；Mass Found(ESI+)[M+H]⁺:1322.0；[M+2H]²⁺:661.8.

15-j

Calculated Mass[M+2H]²⁺:738.8；Mass Found(ESI+)[M+2H]²⁺:739.0.

15-k

Calculated Mass[M+H]⁺:1347.6；[M+2H]²⁺:674.3；Mass Found(ESI+)；[M+H]⁺:1347.1；[M+2H]²⁺:674.5.

16-a

Calculated Mass[M+H]⁺:1265.4；[M+2H]²⁺:633.2；Mass Found(ESI+)[M+H]⁺:1265.0；[M+2H]²⁺:633.3.

16-b

Calculated Mass[M+H]⁺:1224.6；[M+2H]²⁺:612.8；Mass Found(ESI+)[M+H]⁺:1224.6；[M+2H]²⁺:613.2.

16-h

Calculated Mass[M+2H]²⁺:634.2；[M+H]⁺:1266.4；Mass Found(ESI+)；[M+2H]²⁺:634.3；[M+H]⁺:1266.9.

16-i

Calculated Mass[M+H]⁺:1294.6；[M+2H]²⁺:647.9；Mass Found(ESI+)[M+H]⁺:1294.9；[M+2H]²⁺:648.3.

16-j

Calculated Mass[M+2H]²⁺:725.4；Mass Found(ESI+)[M+2H]²⁺:725.5.

16-k

Calculated Mass[M+2H]²⁺:660.8；Mass Found(ESI+)；[M+2H]²⁺:660.9.

16-l

Calculated Mass；[M+2H]²⁺:632.2；Mass Found(ESI+)；[M+2H]²⁺:632.3.

16-m

Calculated Mass[M+2H]²⁺:667.3；Mass Found(ESI+)[M+2H]²⁺:667.4.

16-n

Calculated Mass[M+2H]²⁺:749.9；Mass Found(ESI+)；[M+2H]²⁺:750.0.

17-a

Calculated Mass[M+3H]³⁺:1006.2；[M+4H]⁴⁺:754.9；[M+5H]⁵⁺:604.1；[M+6H]⁶⁺:503.6；Mass Found(ESI+)[M+3H]³⁺:1006.4；[M+4H]⁴⁺:755.1；[M+5H]⁵⁺:604.3；[M+6H]⁶⁺:503.8.

17-g

Calculated Mass[M+3H]³⁺:1023.5；[M+4H]⁴⁺:767.9；[M+5H]⁵⁺:614.5；[M+6H]⁶⁺:512.3；Mass Found(ESI+)[M+3H]³⁺:1023.7；[M+4H]⁴⁺:768.0；[M+5H]⁵⁺:614.7；[M+6H]⁶⁺:512.4.

17-k

Calculated Mass[M+3H]³⁺:1024.5；[M+4H]⁴⁺:768.7；[M+5H]⁵⁺:615.1；[M+6H]⁶⁺:512.8；Mass Found(ESI+)[M+3H]³⁺:1024.7；[M+4H]⁴⁺:768.9；[M+5H]⁵⁺:615.3；[M+6H]⁶⁺:513.0.

18-g

Calculated Mass[M+3H]³⁺:1064.2；[M+4H]⁴⁺:798.4；[M+5H]⁵⁺:638.9；Mass Found(ESI+)[M+3H]³⁺:1064.3；[M+4H]⁴⁺:798.6；[M+5H]⁵⁺:639.1.

18-k

Calculated Mass[M+3H]³⁺:1065.2；[M+4H]⁴⁺:799.2；[M+5H]⁵⁺:639.5；Mass Found(ESI+)[M+3H]³⁺:1065.6；[M+4H]⁴⁺:799.6；[M+5H]⁵⁺:639.9.

19-a

Calculated Mass[M+5H]⁵⁺:854.9；[M+4H]⁴⁺:1068.4；[M+3H]³⁺:1424.2；Mass Found(ESI+)[M+5H]⁵⁺:854.9；[M+4H]⁴⁺:1068.3；[M+3H]³⁺:1424.

20-g

Calculated Mass[M+3H]³⁺:1665.2；[M+4H]⁴⁺:1249.2；[M+5H]⁵⁺:999.9；[M+6H]⁶⁺:833.1；[M+7H]⁷⁺:714.2；Mass Found(ESI+)[M+3H]³⁺:1665.6；[M+4H]⁴⁺:1249.4；[M+5H]⁵⁺:999.7；[M+6H]⁶⁺:833.3；[M+7H]⁷⁺:714.4.

21-g

Calculated Mass[M+6H]⁶⁺:1450.8；[M+7H]⁷⁺:1243.7；[M+8H]⁸⁺:1088.4；[M+9H]⁹⁺:967.6；M+10H]¹⁰⁺:870.9；[M+11H]¹¹⁺:791.8；[M+12H]¹²⁺:725.9；Mass Found(ESI+)[M+6H]⁶⁺:1451.2；[M+7H]⁷⁺:1243.9；[M+8H]⁸⁺:1088.6；[M+9H]⁹⁺:967.7；M+10H]¹⁰⁺:871.1；[M+11H]¹¹⁺:792.0；[M+12H]¹²⁺:726.1.

22-a

Calculated Mass[M+2H]²⁺:554.7；Mass Found(ESI+)[M+2H]²⁺:554.7.

22-g

Calculated Mass[M+2H]²⁺:580.7；Mass Found(ESI+)；[M+2H]²⁺:580.8.

23-g

Calculated Mass[M+H]²⁺:775.4,[M+2H]³⁺:517.3；Mass Found(ESI+)；[M+H]²⁺:775.4,[M+2H]³⁺:。

Claims

1. A method for preparing histidine-containing polypeptide compounds modified by visible light-promoted C-H alkylation, comprising the steps of:

adding a compound shown in a formula I, a compound shown in a formula II and acid into a solvent, and carrying out Minisci C-H functionalization reaction under the action of inert environment and photocatalysis to obtain a compound shown in a formula III;

under the photocatalysis condition, the wavelength range of the selected visible light is 380-500nm, and the total light intensity range is 3-40W.

The compound of the formula I is a peptide chain or protein containing histidine;

the reaction site of the Minisci C-H functionalization reaction is C2 site of an imidazole ring of histidine;

r in the compound of the formula II is open-chain or cyclic secondary and tertiary alkyl containing a polyethylene glycol chain with amphipathy, wherein the polyethylene glycol chain comprises hydroxyl, carboxyl, ether bond, double bond, triple bond, azido, alkynyl, sulfonic group, amino, halogen and the like;

the addition amount of the acid is more than or equal to zero;

the specific alkyl groups represented by R in the compounds of formula II are as follows:

2. the method for preparing a histidine-containing polypeptide compound modified by visible light-promoted C-H alkylation according to claim 1, wherein the acid is one selected from trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid and sulfonic acid.

3. The method for preparing histidine-containing polypeptide compounds modified by visible light-promoted C-H alkylation according to claim 1, wherein the solvent is selected from one of organic solvents or mixed solvents of organic solvents and water; the organic solvent is methanol, ethanol, acetonitrile, tetrahydrofuran, 1, 4-dioxane, 2,2, 2-trifluoroethanol or hexafluoroisopropanol.

4. The method for preparing histidine-containing polypeptide compounds modified by visible light-promoted C-H alkylation according to claim 1, wherein the inert environment is an inert environment of nitrogen atmosphere;

the compound of the formula I is selected from one of any amino acid combination short peptide, medicinal peptide, glycopeptide, cyclic peptide or protein;

the random amino acid combination short peptide is specifically the following compounds:

the drug peptide is specifically the following compound:

the protein is specifically the following compounds:

cyclic peptides are in particular the following compounds:

glycopeptides are in particular the following compounds:

5. the method for preparing histidine-containing polypeptide compounds modified by visible light-promoted C-H alkylation according to claim 1, wherein the reaction temperature of the Minisci C-H functionalization reaction is 10-50 ℃ and the reaction time is 1-12H.

6. The process for preparing histidine-containing polypeptide compounds modified by visible light-promoted C-H alkylation according to claim 1, wherein the reaction concentration of the compound of formula I in the solvent is 5 μ M to 0.2 mM; the reaction concentration of the compound shown in the formula II is 15 mu M-20 mM; the reaction concentration of the acid is 0. mu.M-20 mM.