Biological coupling method of phosphine-catalyzed sulfhydryl compound

Abstract

The invention discloses a phosphine-catalyzed sulfhydryl compound biological coupling method which comprises the following steps: adding an allyl compound, a sulfhydryl-containing compound and a phosphine catalyst into a reaction solvent, and stirring at room temperature to react for 10 minutes to 4 hours; and after the reaction is monitored to be complete by TLC, filtering the reaction liquid, removing the solvent, and purifying to obtain the sulfydryl-specific bioconjugate compound. According to the method disclosed by the invention, rapid (k2 = 1.49 * 106M <-1 > s <-1 >) and high-yield coupling reaction is realized under the conditions of room temperature and biological compatibility, the yield of reaction products is high, the selectivity is strong, the specific reaction of sulfydryl is realized, and the compatibility of the reaction products with various mercaptans and allyl esters is proved, so that the formation of multifunctional products is promoted, and the method is suitable for industrial production. The obtained cysteine conjugate compound shows strong stability under various conditions, so that the method becomes a promising alternative scheme in the prior art, and a solution is provided for key challenges in ADCs connector development.

Description

Technical Field

[0001] The present invention belongs to the cross - field of synthetic chemistry and chemical biology, and particularly relates to a method for phosphine - catalyzed bioconjugation of thiol compounds. Background Art

[0002] Cysteine is the most common target amino acid in the current ADC clinical development. Seven out of eleven FDA - approved ADCs utilize the cysteine conjugation strategy. Because the nucleophilicity of cysteine is different from other amino acids, promoting chemoselective conjugation, it has become an attractive target. In addition, according to Pearson's "hard and soft, acids and bases (HSAB)" principle, the nucleophilicity of the thiol group is a soft nucleophilicity compared to the ε - amine of lysine or the amino group with a higher charge density at the N - terminus. This theory holds that soft nucleophilic reagents are thermodynamically more inclined to bind with soft electrophilic reagents, and the same is true for hard nucleophilic reagents and hard electrophilic reagents. In fact, a series of Michael acceptors (soft electrophilic reagents) can selectively bind to the cysteine residues of proteins. Moreover, under neutral conditions, the thiol group of cysteine (pKa = 8) is more acidic than other common protein nucleophiles, such as the ε - amino group of lysine (pKa = 10). Secondly, compared with lysine, DAR is relatively scarce, so DAR can be better controlled in ADC production.

[0003] In short, the direct modification of cysteine is mainly represented by two typical chemical pathways, namely nucleophilic substitution of the leaving group by the thiol of cysteine, such as in the case of α - halo - carbonyl derivatives (e.g., 2 - iodoacetic acid and related variants), and Michael addition of the thiol group to a Michael acceptor, such as an α,β - unsaturated system. Another less common strategy is metal - catalyzed cysteine modification, among which the most well - known work involves cross - metathesis of allyl sulfides using a ruthenium catalyst reported by the Davis group, and thiol - diene coupling using a gold catalyst reported by the Che group. In recent years, some notable efforts to successfully target cysteine residues can be summarized as cross - metathesis of allyl sulfides, conjugation of thiol groups with allyl selenosulfate, electron - deficient alkynes or bromomaleimides, dithiomaleimide derivatives, and native disulfide polyethylene glycolylation using bis - cysteine alkylating reagents. However, most strategies are limited by factors such as reaction rate, chemoselectivity, and product stability. Therefore, it is still very difficult to find a general method. Although the cross - reactivity of classical reagents (including α - halo - acyl analogs and maleimides) with other amino acids (such as histidine and lysine residues) may affect selectivity, their reversibility / irreversibility is a newly discovered problem. Therefore, there is an urgent need to find promising labeling strategies that can selectively and irreversibly bind to cysteine. Summary of the Invention

[0004] To address the drawbacks and deficiencies of the existing technology, the objective of the present invention is to provide a method for the bio-conjugation of thiol compounds catalyzed by phosphine.

[0005] The present invention is implemented as follows. A method for the bio-conjugation of thiol compounds catalyzed by phosphine, the method comprising the following steps:

[0006] (1) Add an allyl-activated compound, a thiol compound, and a phosphine catalyst to a reaction solvent, and stir the reaction at room temperature for 10 min to 4 h;

[0007] Among them, the chemical structural formula of the allyl-activated compound is:

[0008]

[0009] Among them, LG is a leaving group, selected from any one of OAc, OBoc, Br, Cl, OH, OTs, OP(O)Et3, and OP(O)Me3;

[0010] EWG is COR 3 , CN, and COOR 4 Any one of them; R 3 Is selected from any one of hydrogen, alkyl, and amino; R 4 Is selected from alkyl, aryl, heteroaryl, 4-methyl-7-propoxy-2H-chromene-2-one, 1-((2R, 4S, 5S)-5-(hydroxymethyl)-4-(4-propyl-1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H, 3H)-dione, 5-((3aS, 4S, 6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-N-(2-(4-propyl-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)pentaamide, 1-isopropyl-2,4-dimethylcyclohexyl, 5-ethyl-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyranyl, (R)-2,5,6,7,8-pentamethyl-2-(4S, 8S)-4,8,11-trimethyldodecyl)chromanyl, and (3S, 8S, 9S, 10R, 13R, 14S, 17R)-3,10,13-trimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrenyl) any electron-withdrawing group;

[0011] The chemical structural formula of the thiol compound is:

[0012] R2 -SH;

[0013] (2) After monitoring the reaction to completion by TLC, filter the reaction solution obtained in step (1), remove the solvent, and purify to obtain a cysteine conjugate compound with the following chemical structural formula:

[0014]

[0015] wherein, R 1 is selected from any one of hydrogen, phenyl, aryl, fused-ring aryl, and heteroaryl;

[0016] R 2 is selected from alkyl, heteroaryl, fused-ring aryl, N-acetyl-L-cysteine ethyl ester, N-acetyl-L-cysteine methyl ester, methimazole, captopril, tiopronin, D-penicillamine, D-glucose, glutathione, maytansine, reduced lanreotide, reduced octreotide, reduced oxytocin, thiol-containing cysteine, dipeptide, polypeptide, protein, and any one of methyl, methoxy, chloro, fluoro, and bromo groups on the benzene ring.

[0017] Preferably, in step (1), the reaction solvent is a PBS phosphate buffer solution with a pH of 4 - 10, or a mixed solution of the PBS phosphate buffer solution and an organic solvent, and the organic solvent is selected from any one of dichloromethane, acetonitrile, methanol, ethanol, dimethyl sulfoxide, and N,N-dimethylformamide.

[0018] Preferably, in step (1), the phosphine catalyst is a homogeneous phosphine catalyst or a heterogeneous phosphine catalyst.

[0019] Preferably, the homogeneous phosphine catalyst is selected from at least one of triphenylphosphine, tributylphosphine, trimethylphosphine, triethylphosphine, tricyclohexylphosphine, diphenylmethylphosphine, dimethylphenylphosphine, tris(o-methylphenyl)phosphine, 1,2-bis(diphenylphosphino)benzene, tris(2-furyl)phosphine, 1,2-bis(diphenylphosphino)ethane, 1,3-bis(diphenylphosphino)propane, ethyldiphenylphosphine, tris(p-tolyl)phosphine, bis(2-diphenylphosphinophenyl)ether, triisopropylideneacetonylphosphine, 2-bis(cyclohexylphosphino)-2',4',6'-triisopropylbiphenyl, 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene, 4,5-bis(diphenylphosphino)-9,9-dimethyloxanthrene, 2-(di-tert-butylphosphino)biphenyl, ethyldiphenylphosphine, (R)-(+)-2,2'-bis(diphenylphosphino)-1,1'-binaphthalene, 1,4-bis(diphenylphosphino)butane, 2-bis(cyclohexylphosphino)-2',6'-diisopropoxybiphenyl, and tris(4-methoxyphenyl)phosphine.

[0020] Preferably, the heterogeneous phosphine catalyst is selected from at least one of NCPS-PPh3, EOF-17, POL-PPh3, PDVB-PPh3, and POPs-3.

[0021] Preferably, in step (1), the molar volume ratio of the active allyl compound, the thiol-containing compound, the phosphine catalyst and the reaction solvent is 0.24-1.2 mmol: 0.2-1.0 mmol: 0.01-0.05 mmol: 2-10 mL.

[0022] Preferably, in step (1), the reaction system is reacted in an air atmosphere or an inert gas atmosphere.

[0023] Preferably, the alkyl group is selected from a C1-C15 chain alkyl group or a C1-C15 cycloalkyl group;

[0024] The normal aryl group is selected from any one of a methyl group, a methoxy group, a nitro group, a tert-butyl group, a fluoro group, a chloro group, a bromo group, and a trimethylsilylethynyl group on the benzene ring;

[0025] The fused ring aromatic group is selected from naphthyl or anthracenyl;

[0026] The heteroaryl group is selected from any one of furan, indole, pyridine and thiophene.

[0027] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0028] (1) The method of the present invention achieves an efficient and high-yield coupling reaction at room temperature and under biocompatible conditions. The yield of the reaction product is high and the selectivity is strong. The specific reaction of the sulfhydryl group is achieved and the compatibility of the reaction product with a variety of thiols and allyl esters is demonstrated, thereby promoting the formation of multifunctional products. The obtained cysteine conjugate compounds show strong stability under various conditions, making this method a promising alternative to current technologies and providing a solution to the key challenges in the development of ADCs linkers.

[0029] (2) The present method uses PBS (phosphate buffered saline) as the reaction solvent, which has significant advantages over traditional organic solvents. First, PBS is safe, inexpensive, and pollution-free, which conforms to the core concept of modern green chemistry. The PBS-based reaction system not only significantly simplifies chemical synthesis steps and improves synthesis efficiency, but also provides important support for the sustainable development of future chemistry.

[0030] (3) The method of the present invention has an extremely high yield and is applicable to a variety of thiols, polypeptides, proteins, and allyl acetate substrates, generating structurally diverse coupling products. These coupling products exhibit excellent stability under various conditions, making this strategy a powerful alternative to the prior art and providing a new idea for solving the key bottleneck in the design of antibody-drug conjugates (ADCs).

[0031] (4) The allyl ester used in the method of the present invention is allyl acetate with simple synthesis and good conversion rate. The applicable substrate range is wide, such as various substituted phenyl groups, alkyl groups, etc., and it has the characteristic of low preparation cost. In addition, the preparation method of the present invention has simple steps, is convenient to operate, environmentally friendly, has excellent stereoselectivity, and is tolerant to a wide range of functional groups. Moreover, the reaction rate of the method of the present invention is fast, has high selectivity for cysteine, and the reaction is irreversible.

[0032] (5) The cysteine conjugate prepared by the present invention can utilize its allyl acetate and thiol moieties to be used as organic synthesis building blocks for various derivatizations. It is also an important backbone widely present in natural products, biological, and pharmaceutical molecules, and has potential biological and pharmaceutical activities.

[0033] (6) The phosphine catalyst in the method of the present invention can be selected as a heterogeneous catalyst, which has the advantages of simple synthesis and wide applicable reaction range. It successfully replaces traditional metal catalysts and homogeneous phosphine catalysts, showing high atom economy and environmental friendliness. Based on its heterogeneous catalyst characteristics, the catalyst can be efficiently recovered and reused after the reaction, providing important support for large-scale industrial applications. In addition, this catalytic system can efficiently catalyze the coupling reaction of cysteine, providing a new strategy for the green bio-modification of polypeptides and proteins, and also showing broad application potential in the field of bioconjugation of natural product derivatives and pharmaceutical molecules. Description of the Drawings

[0034] Figure 1 is the 1H NMR spectrum of compound 3 in Example 1 of the present invention;

[0035] Figure 2 is the 13C NMR spectrum of compound 3 in Example 1 of the present invention;

[0036] Figure 3 is the 13C NMR spectrum of compound 5 in Example 2 of the present invention;

[0037] Figure 4 is the 13C NMR spectrum of compound 5 in Example 2 of the present invention;

[0038] Figure 5 is the 13C NMR spectrum of compound 7 in Example 3 of the present invention;

[0039] Figure 6 It is the carbon-13 NMR spectrum of Compound 7 in Example 3 of the present invention;

[0040] Figure 7 It is the carbon-13 NMR spectrum of Compound 10 in Example 4 of the present invention;

[0041] Figure 8 It is the carbon-13 NMR spectrum of Compound 10 in Example 4 of the present invention;

[0042] Figure 9 It is the carbon-13 NMR spectrum of Compound 12 in Example 5 of the present invention;

[0043] Figure 10 It is the carbon-13 NMR spectrum of Compound 12 in Example 5 of the present invention;

[0044] Figure 11 It is the LC / MS spectrum of reduced lanreotide modified with Compound 10 in Example 6 of the present invention;

[0045] Figure 12 It is the MS / MS spectrum of reduced lanreotide modified with Compound 10 in Example 6 of the present invention;

[0046] Figure 13 It is the HPLC spectrum of the purity of reduced lanreotide modified with Compound 10 in Example 6 of the present invention;

[0047] Figure 14 It is the LC / MS spectrum of reduced octreotide modified with Compound 10 in Example 7 of the present invention;

[0048] Figure 15 It is the MS / MS spectrum of reduced octreotide modified with Compound 10 in Example 7 of the present invention;

[0049] Figure 16 It is the HPLC spectrum of the purity of reduced octreotide modified with Compound 10 in Example 7 of the present invention;

[0050] Figure 17 It is the ESI-MS spectrum of BSA without modification by Compound 10 in Example 8 of the present invention;

[0051] Figure 18 It is the graph of the relative abundance values of the components of BSA without modification by Compound 10 in Example 8 of the present invention;

[0052] Figure 19 It is the ESI-MS spectrum of BSA modified with Compound 10 in Example 8 of the present invention;

[0053] Figure 20It is the relative abundance value diagram of the components of BSA modified by compound 10 in Example 8 of the present invention. Detailed implementation mode

[0054] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] Example 1

[0056] (1) In a 10 mL glass tube, add 0.24 mmol of an active allyl compound (1-1 or 1-2 or 1-3 or 1-4), 0.2 mmol of N-acetyl-L-cysteine ethyl ester, and 0.01 mmol of triphenylphosphine. Add 2 mL of PBS (pH = 7), and stir and react at room temperature for 10 min. The reaction equation is:

[0057]

[0058] (2) After monitoring the reaction to completion by TLC, filter the reaction solution with an F-caliber sintered glass funnel, remove the solvent with a rotary evaporator under vacuum, and separate the product by thin-layer chromatography. The developing agent is a petroleum ether / ethyl acetate system (1 / 1). The product is a purple-red thick liquid compound 3, and the yield is 99%.

[0059] Characterize compound 3, and the data are as follows:

[0060] 1 H NMR (400 MHz, Chloroform-d) δ 7.76 (s, 1H), 7.51–7.36 (m, 5H), 6.83 (d, J = 8.1 Hz, 1H), 4.88 (dt, J = 8.0, 4.7 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 3.75–3.64 (m, 2H), 3.16–3.01 (m, 2H), 2.06 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 170.7, 170.1, 167.8, 141.7, 134.6, 129.5, 129.3, 128.8, 128.4, 61.8, 52.5, 52.4, 35.4, 30.2, 23.1, 14.2. IR (KBr): 3005, 1714, 1435, 1275, 1203, 750 cm -1 .HRMS (ESI) m / z: [M+H]+ Calcd. for: C 18 H24 NO5S 366.1375; Found 366.1378.

[0061] The NMR spectrum is as Figures 1 - 2 shown. The above characterization results indicate that Compound 3 is methyl (R,Z)-2-(((2-acetamido-3-ethoxy-3-oxopropyl)thio)methyl)-3-phenylacrylate.

[0062] Example 2

[0063] (1) In a 10 mL glass tube, 0.24 mmol of 2-cyanoallyl acetate, 0.2 mmol of ethyl N-acetyl-L-cysteinate, and 0.01 mmol of triphenylphosphine were added. 2 mL of PBS (pH = 7) was added, and the mixture was stirred at room temperature for 20 min. The reaction equation is:

[0064]

[0065] (2) After monitoring the completion of the reaction by TLC, the reaction solution was filtered through a sintered glass funnel with an F frit, and the solvent was removed using a rotary evaporator under vacuum. The product was separated by thin layer chromatography, and the eluent was a dichloromethane / methanol system (20 / 1). The product was a yellow oily compound 5 with a yield of 92%.

[0066] Compound 5 was characterized, and the data are as follows:

[0067] 1 H NMR (400 MHz, Chloroform-d) δ 6.40 (d, J = 7.28 Hz, 1H), 5.97 (t, J = 0.71 Hz, 1H), 5.87 (t, J = 1.22 Hz, 1H), 4.86–4.73 (m, 1H), 4.26 (q, J = 7.12 Hz, 2H), 3.32 (s, 2H), 3.06–2.85 (m, 2H), 2.07 (s, 3H), 1.32 (t, J = 7.13 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 170.5, 170.0, 132.1, 119.8, 117.5, 62.2, 51.7, 35.6, 33.1, 23.1, 14.1. IR (KBr): 2968, 1738, 1661, 1509, 1373, 1275, 1267, 1198, 1031, 764, 750 cm -1 . HRMS (ESI) m / z: [M+K]+ Calcd. for: C 11 H 17 N2O3S 257.0960; Found 257.0964.

[0068] The nuclear magnetic spectrum is as follows Figures 3 - 4 As shown above, the above characterization results indicate that Compound 5 is ethyl N-acetyl-S-(2-cyanoallyl)-L-cysteinate.

[0069] Example 3

[0070] (1) In a 10 mL glass tube, 0.24 mmol of ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yl)oxy)acrylate, 0.2 mmol of ethyl N-acetyl-L-cysteinate, and 0.01 mmol of POL-PPh3 were added. 1.8 mL of PBS (pH = 7) and 0.2 mL of ethanol were added, and the mixture was stirred at room temperature for 30 min. The reaction equation is as follows:

[0071]

[0072] (2) After monitoring the reaction to completion by TLC, the reaction solution was filtered through a glass funnel with a F frit, and the solvent was removed using a rotary evaporator under vacuum. The product was separated by thin-layer chromatography, and the eluent was a dichloromethane / methanol system (20 / 1). The product was a white solid 7 with a yield of 93%.

[0073] The characterization data of Compound 7 are as follows:

[0074] Mp: 106 - 108 °C. 1 1H NMR (400 MHz, Chloroform-d) δ 7.48 (d, J = 8.8 Hz,

[0075] 1H), 6.85 (dd, J = 8.8, 2.5 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 6.51 (d, J = 7.7 Hz, 1H), 6.22 (d, J = 0.9 Hz, 1H), 6.11 (d, J = 1.3 Hz, 1H), 5.69 (d, J = 1.0 Hz, 1H), 4.80–4.71 (m, 1H), 4.56–4.49 (m, 2H), 4.29–4.23 (m, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.36 (d, J = 0.9 Hz, 2H), 2.87 (qd, 2H), 2.36 (d, J = 1.2 Hz, 3H), 2.01 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H). 1313C NMR (101 MHz, Chloroform-d) δ 170.8, 170.1, 165.8, 161.5, 161.3, 155.2, 152.7, 136.0, 127.6, 125.8, 114.1, 112.6, 112.3, 101.7, 66.3, 63.1, 62.0, 52.0, 33.6, 33.3, 23.2, 18.8, 14.2. IR (KBr): 3447, 2993, 2831, 2717, 1770, 1597, 1363, 1275, 1259, 1131, 750 cm -1 . HRMS (ESI) m / z: [M+H] + Calcd. for: C 23 H 28 NO8S 478.1536; Found 478.1532.

[0076] The NMR spectrum is as follows Figures 5 - 6 shown above. The above characterization results indicate that Compound 7 is 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethyl (R)-2-((2-acetamido-3-ethoxy-3-oxopropyl)thio)methyl) acrylate.

[0077] Example 4

[0078] (1) In a 10 mL glass tube, 0.24 mmol of 2-(acetyloxymethyl) methyl acrylate, 0.2 mmol of glutathione, and 0.01 mmol of triphenylphosphine were added. Then 2 mL of PBS (pH = 7) was added, and the mixture was stirred at room temperature for 30 min. The reaction equation is as follows:

[0079]

[0080] (2) After monitoring the completion of the reaction by TLC, the reaction solution was filtered through a F-frit funnel, and the solvent was removed by a rotary evaporator under vacuum. The product was separated by thin-layer chromatography, and the developing solvent was a tert-butanol / acetic acid / water system (3 / 1 / 1). The product was a yellow solid compound 10 with a yield of 84%.

[0081] The characterization data of Compound 10 are as follows:

[0082] Mp: 180 - 182 °C. 11H NMR (400 MHz, D2O-d) δ 6.07 (s, 1H), 5.65 (s, 1H), 4.37 (dd, J = 8.8, 5.0 Hz, 1H), 3.59 (s, 3H), 3.56 (d, J = 7.3 Hz, 3H), 3.25 (s, 2H), 2.81 (dd, J = 14.2, 5.0 Hz, 1H), 2.62 (dd, J = 14.2, 8.8 Hz, 1H), 2.33 (td, J = 7.5, 3.6 Hz, 2H), 1.96 (q, J = 7.5 Hz, 2H). 13 13C NMR (101 MHz, D2O-d) δ 176.2, 174.8, 171.8, 168.5, 135.7, 128.3, 54.0, 52.9, 52.5, 43.3, 32.3, 31.3, 26.2. IR (KBr): 3268, 2952, 1722, 1645, 1543, 1397, 1204, 1135 cm -1 . HRMS (ESI) m / z: [M+H]+ Calcd. for: C 15 H 24 N3O8S 406.1284; Found 406.1275.

[0083] The NMR spectra are as Figures 7 - 8 shown. The above characterization results indicate that Compound 10 is N5-(1-((carboxymethyl)amino)-3-((2-(methoxycarbonyl)allyl)thio)-1-oxopropan-2-yl)glutamine.

[0084] Example 5

[0085] (1) In a 10 mL glass tube, 0.24 mmol of 2-(1-(2-(2-(2-(5-(3aS, 4S, 6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl 2-(acetoxymethyl)acrylate, 0.2 mmol of N-acetyl-L-cysteine ethyl ester, and 0.01 mmol of POL-PPh3 were added. 1.8 mL of PBS (pH = 7) and 0.2 mL of ethanol were added, and the mixture was stirred at room temperature for 30 min. The reaction equation is as follows:

[0086]

[0087] (2) After monitoring the completion of the reaction by TLC, the reaction solution was filtered through a sintered glass funnel with an F frit, and the solvent was removed using a rotary evaporator under vacuum. The product was separated by thin-layer chromatography, and the eluent was a dichloromethane / methanol system (10 / 1). The product was a pale yellow oily liquid 12 with a yield of 95%.

[0088] Compound 12 was characterized, and the data are as follows:

[0089] 1 H NMR (400 MHz, Chloroform-d) δ 7.61 (s, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.92 (t, J = 5.7 Hz, 1H), 6.61 (s, 1H), 6.21 (s, 1H), 5.86 (s, 1H), 5.69 (s, 1H), 4.82–4.72 (m, 1H), 4.56–4.44 (m, 5H), 4.37–4.27 (m, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.88 (t, J = 5.1 Hz, 2H), 3.63–3.58 (m, 8H), 3.55 (t, J = 5.3 Hz, 2H), 3.43 (t, J = 5.4 Hz, 2H), 3.38 (s, 2H), 3.13 (t, J = 6.9 Hz, 3H), 2.96–2.83 (m, 3H), 2.74 (d, J = 12.8 Hz, 1H), 2.21 (t, J = 7.5 Hz, 2H), 2.05 (s, 3H), 1.75–1.60 (m, 4H), 1.44 (q, J = 7.5 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 173.2, 170.8, 170.1, 165.6, 163.9, 143.5, 136.0, 126.8, 122.6, 70.2, 70.2, 70.1, 69.8, 69.7, 69.3, 63.7, 61.7, 61.6, 60.0, 55.5, 51.7, 49.9, 40.3, 38.9, 35.7, 33.2, 32.9, 28.0, 27.9, 25.4, 25.2, 22.8, 13.9. IR (KBr): 3282, 3005, 2989, 2319, 1726, 1460, 1276, 1261, 1161, 897, 750 cm -1 . HRMS (ESI) m / z: [M+H] + Calcd. for: C 33 H 54 N7O 10 S2 772.3३74; Found 772.3३79.

[0090] The NMR spectra are as Figures 9 - 10As shown above, the above characterization results indicate that Compound 12 is 2-(1-(2-(2-(2-(5-(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentaamido)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)ethyl 2-(2-(S)-2-acetamido-3-ethoxy-3-oxo-3-oxopropyl)thio)methyl)acrylate.

[0091] Example 6

[0092] (1) 5.0 equivalents of solid TCEP-HCl were added to 1 mL (2 mM) of a lanreotide solution (2.2 mg, 2.0 μmol, 1.0 equivalent) in PBS (phosphate buffered saline) buffer at pH 6.0 containing 10% dimethyl sulfoxide. The mixture was slowly stirred in air at 37 °C for 2 hours. Subsequently, the peptide solution (pH = 6.0) was mixed with 100 μL of a solution of Compound 13 (0.2 M in DMSO, 10.0 equivalents) and 5 μL of a POL-PPh3 solution (20 mM in DMSO, 0.05 equivalent). The reaction equation is:

[0093]

[0094] (2) The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the product was purified by preparative high-performance liquid chromatography (HPLC) to obtain the modified product, and its regioselectivity was analyzed by LC-MS / MS, as Figures 11 - 13 .

[0095] Example 7

[0096] (1) 5.0 equivalents of solid TCEP-HCl were added to 1 mL of a 2 mM octreotide solution (2.0 mg, 2.0 μmol, 1.0 equivalent) in PBS (phosphate buffered saline) buffer containing 10% dimethyl sulfoxide. The mixture was slowly stirred in air at 37 °C for 2 hours. Subsequently, the peptide solution (pH 6.0) was mixed with 100 μL of a solution of Compound 13 (0.2 M in DMSO, 10.0 equivalents) and 5 μL of a POL-PPh3 solution (20 mM in DMSO, 0.05 equivalent), and the reaction equation is:

[0097]

[0098] (2) The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the product was purified by preparative high-performance liquid chromatography (HPLC) to obtain the modified product, and its regioselectivity was analyzed by LC-MS / MS, as Figures 14 - 16 .

[0099] Example 8

[0100] 5 μL of 13 (0.2 M, 10.0 equivalents) and 5 μL of POL-PPh3 solution (1.0 mM in DMSO, 0.05 equivalents) were added to 0.1 mM albumin (6.7 mg, 0.1 μmol) in PBS buffer at pH 6.0 (1.0 mL, 0.02 M). After slow stirring at 37 °C for 30 min, a 20 μL aliquot of the reaction mixture was evaluated by performing ESI-MS to obtain the modified product of BSA. Only the thiol groups participated in the reaction, and CD confirmed that the protein structure was well maintained (see the attachment for details Figures 17 - 20 ).

[0101] Examples 9 - 13

[0102] Examples 9 - 13 are basically the same as Example 1, and the differences are shown in Table 1 below:

[0103] Table 1 Difference Comparison

[0104]

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for bio-conjugation of thiol compounds catalyzed by phosphine, characterized in that, The method comprises the following steps: (1) Add an allylic compound, a mercapto compound and a phosphine catalyst into a reaction solvent, and stir and react at room temperature for 10 min to 4 h; Among them, the chemical structural formula of the allylic compound is: Among them, LG is a leaving group selected from any one of OAc, OBoc, Br, Cl, OH, OTs, OP(O)Et3, and OP(O)Me3; EWG is COR 3 , CN or COOR 4 ; R 3 is selected from any one of hydrogen, alkyl, and amino; R 4 is selected from alkyl, aryl, heteroaryl, 4-methyl-7-propoxy-2H-chromen-2-one, 1-((2R,4S,5S)-5-(hydroxymethyl)-4-(4-propyl-1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione, 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-N-(2-(4-propyl-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)pentaamide, 1-isopropyl-2,4-dimethylcyclohexyl, 5-ethyl-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyranyl, (R)-2,5,6,7,8-pentamethyl-2-((4S,8S)-4,8,11-trimethyldodecyl)chromanyl, and (3S,8S,9S,10R,13R,14S,17R)-3,10,13-trimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrenyl), any electron-withdrawing group; The chemical structural formula of the mercapto compound is: R 2 -SH; (2) After monitoring the reaction to completion by TLC, filter the reaction solution obtained in step (1), remove the solvent, and purify it to obtain a cysteine conjugate compound with the following chemical structural formula: wherein, R 1 is selected from any one of hydrogen, phenyl, aryl, polycyclic aryl, and heteroaryl; R 2 selected from alkyl, heteroaryl, fused polycyclic aryl, ethyl N-acetyl-L-cysteine, methyl N-acetyl-L-cysteine, methimazole, captopril, tiopronin, D-penicillamine, D-glucose, glutathione, maytansine, reduced lanreotide, reduced octreotide, reduced oxytocin, thiol-containing cysteine, dipeptide, polypeptide, protein, and any one of methyl, methoxy, chloro, fluoro, and bromo groups attached to the benzene ring.

2. The method according to claim 1, characterized in that, In step (1), the reaction solvent is a PBS phosphate buffer solution with a pH of 4 to 10, or a mixed solution of the PBS phosphate buffer solution and an organic solvent, and the organic solvent is selected from any one of dichloromethane, acetonitrile, methanol, ethanol, dimethyl sulfoxide, and N,N-dimethylformamide.

3. The method according to claim 1, characterized in that, In step (1), the phosphine catalyst is a homogeneous phosphine catalyst or a heterogeneous phosphine catalyst.

4. The method according to claim 3, wherein The homogeneous phosphine catalyst is selected from at least one of triphenylphosphine, tributylphosphine, trimethylphosphine, triethylphosphine, tricyclohexylphosphine, diphenylmethylphosphine, dimethylphenylphosphine, tris(o-methylphenyl)phosphine, 1,2-bis(diphenylphosphino)benzene, tris(2-furyl)phosphine, 1,2-bis(diphenylphosphino)ethane, 1,3-bis(diphenylphosphino)propane, ethyldiphenylphosphine, tris(p-tolyl)phosphine, bis(2-diphenylphosphinophenyl)ether, triisopropylideneacetonylphosphine, 2-bis(cyclohexylphosphino)-2',4',6'-triisopropylbiphenyl, 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene, 4,5-bis(diphenylphosphino)-9,9-dimethyloxanthrene, 2-(di-tert-butylphosphino)biphenyl, ethyldiphenylphosphine, (R)-(+)-2,2'-bis(diphenylphosphino)-1,1'-binaphthalene, 1,4-bis(diphenylphosphino)butane, 2-bis(cyclohexylphosphino)-2',6'-diisopropoxybiphenyl, and tris(4-methoxyphenyl)phosphine.

5. The method according to claim 3, wherein The heterogeneous phosphine catalyst is selected from at least one of NCPS-PPh3, EOF-17, POL-PPh3, PDVB-PPh3, and POPs-3.

6. The method according to claim 1, wherein In step (1), the molar volume ratio of the allylic compound, the mercapto compound, the phosphine catalyst, and the reaction solvent is 0.24 to 1.2 mmol: 0.2 to 1.0 mmol: 0.01 to 0.05 mmol: 2 to 10 mL.

7. The method according to claim 1, characterized in that, In step (1), the reaction system reacts under an air atmosphere or an inert gas atmosphere.

8. The method according to claim 1, wherein The alkyl group is selected from a linear alkyl group with 1 to 15 carbon atoms or a cycloalkyl group with 1 to 15 carbon atoms; The ortho-aryl group is selected from any one of a methyl group, a methoxy group, a nitro group, a tert-butyl group, a fluoro group, a chloro group, a bromo group, and a trimethylsilylethynyl group attached to the benzene ring; The fused-ring aryl group is selected from a naphthyl group or an anthracenyl group; The heteroaryl group is selected from any one of furan, indole, pyridine, and thiophene.

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