Method for reversible exchange of disulfide bond under mild biocompatible condition

Abstract

The invention provides a method for reversible disulfide bond exchange mediated by free radicals. According to the method, hydroxyl (p-toluenesulfonyloxy) iodobenzene (HTIB) is used as a free radical initiator, and controllable exchange of disulfide bonds is realized under the condition of biological compatibility. The technology has wide application potential, and is especially suitable for the field of biological medicines, such as chemical modification of amino acids, peptides, carbohydrates, proteins and drug molecules. Different from a traditional transition metal catalyzed disulfide bond exchange method, the method has the advantages that the reversible disulfide bond exchange reaction is efficiently realized under mild and biocompatible conditions, the method is applicable to the fields of protein engineering, drug development, dynamic covalent chemistry and the like, and a new way is provided for synthesis and modification of biomolecules. # imgabs0 #

Description

Technical Field

[0001] This invention relates to the field of organic chemistry, and more particularly to a method for disulfide bond exchange under mild, reversible, and biocompatible conditions, applicable to drug development, chemical biology, and dynamic covalent chemistry. Background Technology

[0002] In drug development, chemical biology, and dynamic combinatorial chemistry, mesothesis reactions, which can operate cleanly and reversibly under biocompatible conditions, offer significant advantages. Their reversibility is particularly valuable in systems requiring self-repair, recycling, or high adaptability, while biocompatibility enables their application in the biomedical field. Traditional olefin mesothesis and ring-opening mesothesis polymerization methods typically require harsh reaction conditions or use transition metal catalysts with potential toxicity in biological environments, limiting the versatility of these methods in practical applications. Furthermore, these methods are often accompanied by side reactions, making it difficult to achieve clean reaction processes.

[0003] Disulfide metathesis is a classic example of metathesis reactions in nature. Due to the fragility of disulfide bonds, they are easily broken, resulting in a mixture of various disulfides after the reaction reaches equilibrium. Disulfide metathesis has been extensively explored in medicinal chemistry and dynamic covalent chemistry (DCC) research, typically promoting disulfide exchange reactions by generating transient intermediates such as thiol radicals or thiolate anions. Existing techniques include ultraviolet irradiation, iodine, phosphine compounds, triethylphosphonamide, N-iodosuccinimide, transition metals, electrochemical methods, and ultrasound. However, these methods often suffer from various drawbacks, such as harsh reaction conditions, poor biocompatibility, easy oxidation of catalysts, difficulty in purifying asymmetric disulfides, and difficulty in achieving clean reversibility. Furthermore, for some applications, the reaction needs to be carried out in water at room temperature and neutral pH, which poses even greater challenges to process control. Summary of the Invention

[0004] This invention provides a method for reversible disulfide bond exchange under mild, biocompatible conditions. Utilizing a radical-mediated, bidirectionally controlled disulfide bond exchange method, hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB) is used as a radical initiator, achieving clean and reversible disulfide bond exchange under biocompatible conditions. By adjusting the amount of disulfide added and removing low-boiling-point dimethyl disulfide, the forward and reverse processes of the reaction can be effectively controlled. This invention is adaptable to a variety of substrates and possesses biocompatibility, making it suitable for applications in protein modification, glycosylation, and drug binding.

[0005] A key feature of this invention is its bidirectional reaction mechanism, particularly the design of the reverse reaction. This reverse reaction achieves a controllable and reversible reaction by removing the low-boiling-point dimethyl disulfide or other low-boiling-point disulfides generated in the reaction and reverting the asymmetric disulfide back to a symmetric disulfide. This characteristic has significant implications for applications in dynamic covalent chemistry and renewable materials.

[0006] To address the technical problem of this invention, the proposed technical solution is as follows: a method for reversible disulfide bond exchange under mild, biocompatible conditions, characterized by using hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB) as a free radical initiator, and achieving precise control of the disulfide bond exchange reaction by controlling the concentration of the disulfide feedstock and vacuum distilling of low-boiling-point disulfides, as shown in reaction formulas 1 and 2:

[0007] Reaction 1:

[0008] Reaction 2:

[0009] In reaction formula 1, R is defined as follows: R is any one of chain alkyl, cyclic alkyl, phenyl, substituted phenyl, heterocyclic aryl, or substituted heterocyclic aryl, or contains any one of cysteine ​​derivative, glutathione derivative, sugar derivative, captopril derivative, or thiamine derivative.

[0010] In reaction 1, R 1 Definition: R 1 It is any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, and diisopentyl;

[0011] In reaction formula 2, chemical formula IV is defined as: any one of aliphatic hydrocarbon cyclic disulfide ethers and their derivatives, or cyclic peptide compounds containing disulfide bonds and their derivatives.

[0012] In reaction 2, R 2 Definition: any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, diisopentyl or containing any one of cysteine ​​derivatives, glutathione derivatives, sugar derivatives, captopril derivatives, thiamine derivatives;

[0013] In reaction 2, R 3 Definition: any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, diisopentyl or containing any one of cysteine ​​derivatives, glutathione derivatives, sugar derivatives, captopril derivatives, thiamine derivatives;

[0014] The specific preparation steps are as follows:

[0015] (1) According to reaction formula 1, disulfide (Ⅰ), excess alkyl disulfide compound (Ⅱ), and catalytic amount of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB) were added to the reaction apparatus, along with an appropriate amount of aqueous solvent. The reaction temperature was maintained above 20°C, and the reaction was stirred. After the reaction was completed, the reaction mixture was separated and purified to obtain asymmetric disulfide compound (Ⅲ). According to reaction formula 2, cyclic disulfide (ⅠV), excess disulfide compound (V), and catalytic amount of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB) were added to the reaction apparatus, along with an appropriate amount of aqueous solvent. The reaction temperature was maintained above 20°C, and the reaction was stirred. After the reaction was completed, the reaction mixture was separated and purified to obtain asymmetric bis-disulfide compound (VI).

[0016] (2) According to reaction formula 1 and reaction formula 2, the reverse reaction is driven by removing the generated low-boiling disulfide compounds by vacuum distillation: In the reaction apparatus, disulfide compound (III) or (VI) is added; a catalytic amount of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB) is added; an aqueous solvent is added; the generated low-boiling disulfide compound (II) or (V) is removed by vacuum distillation while stirring; after the reaction is completed, the reaction mixture is separated and purified to obtain disulfide compound (I) or (IV).

[0017] Preferably, in formula (I) above, is any one of the following symmetrical disulfide compounds:

[0018]

[0019] Preferably, in formula (II) above, is a methyl disulfide compound;

[0020] Preferably, in formula (III) above, it is any one of the following disulfide compounds:

[0021]

[0022] The specific preparation steps are as follows:

[0023] (1) According to reaction formula 1, disulfide compound (Ⅰ), excess methyl disulfide compound (Ⅱ), and catalytic amount of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB) were added to the reaction apparatus, and an appropriate amount of aqueous solvent was added. The reaction temperature was maintained above 20°C, and the reaction was stirred. After the reaction was completed, the reaction mixture was separated and purified to obtain asymmetric disulfide compound (Ⅲ);

[0024] (2) According to reaction formula 1, the reverse reaction is driven by removing the generated low-boiling-point methyl disulfide compound through vacuum distillation: In the reaction apparatus, disulfide compound (III) is added; a catalytic amount of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB) is added; an aqueous solvent is added; the generated low-boiling-point disulfide compound (II) is removed by vacuum distillation while stirring; after the reaction is completed, the reaction mixture is separated and purified to obtain disulfide compound (I). Preferably, the method for bidirectional disulfide bond exchange under mild biocompatible conditions according to claim 1 is characterized in that the method includes forward and reverse control, and the reverse reaction, by removing the generated low-boiling-point disulfide compound (II), can convert the asymmetric disulfide back into a symmetric disulfide.

[0025] Preferably, the method for reversible disulfide bond exchange under mild biocompatible conditions according to claim 1 is characterized in that, but is not limited to, the reversible exchange of the following cyclic peptide compounds:

[0026]

[0027] The specific preparation steps are as follows:

[0028] In a reaction tube, hydroxy(p-toluenesulfonyl)iodobenzene (HTIB, 0.1-1.0 equiv.), a methyl disulfide compound (10-100 equiv.), and a peptide (1.0 equiv.) are sequentially added to a buffer solution at pH 6-8, and the reaction is stirred at room temperature. The reaction is monitored by LCMS, and after the reaction is complete, the resulting conjugated product is separated and purified. Preferably, the method for reversible disulfide bond exchange under mild biocompatible conditions according to claim 1 is characterized by, but not limited to, the chemical modification of insulin disulfide bonds.

[0029] In a reaction tube, hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 0.1-1.0 equiv.), methyl disulfide compound (10-100 equiv.), and human insulin (1.0 equiv.) were added sequentially to a buffer solution at pH 6-8, and the reaction was stirred at room temperature. The reaction was detected by LCMS. After the reaction was completed, the resulting conjugated product was separated and purified.

[0030] The present invention has the following beneficial effects:

[0031] (1) Strong biocompatibility: This method operates in a mild aqueous environment and is suitable for neutral pH and room temperature conditions, avoiding the harsh conditions of bioincompatibility in traditional metathesis reactions, making it widely used in the biomedical field.

[0032] (2) High reversibility and easy control: By generating and removing low-boiling-point disulfides, it can effectively achieve precise control of forward and reverse reactions, and is suitable for systems that require self-repair, recycling or reversible adjustment.

[0033] (3) Simple operation: Using high-valent iodine compound HTIB as a free radical initiator, no transition metal catalyst is required, which reduces the requirements for equipment and environmental conditions, and the reaction process is simple and easy to carry out.

[0034] (4) Clean reaction and reduced byproducts: This method reduces the generation of side reactions by rationally designing reaction conditions, and achieves high reaction selectivity and product purity.

[0035] (5) Wide range of applications: It is applicable to the coupling and decoupling processes of disulfide bonds in biomolecules and complex systems such as carbohydrates, drugs, and proteins. It can be used to prepare asymmetric disulfides and has strong versatility.

[0036] (6) Low cost: The HTIB used is an inexpensive and readily available high-valent iodine compound, which significantly reduces the reaction cost compared to transition metal catalysts and is suitable for large-scale industrial applications. Attached Figure Description

[0037] Figure 1 It is compound S1 1 H-NMR spectrum

[0038] Figure 2 It is compound S1 13 C-NMR spectrum

[0039] Figure 3 It is compound I1 1 H-NMR spectrum

[0040] Figure 4 It is compound I1 13 C-NMR spectrum

[0041] Figure 5 This is a chemical modification and mass spectrometry characterization of the cyclic peptide somatostatin.

[0042] Figure 6 This is a schematic diagram of the chemical modifications of insulin. Detailed Implementation

[0043] The present invention will be further described in detail and in complete detail below with reference to the implementation examples. The implementation examples listed will help to understand the present invention, but should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention are within the scope of the present invention.

[0044] Example 1:

[0045]

[0046] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of symmetrical disulfide and hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 0.04 mmol, 0.2 equivalents). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of diphenyl disulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0047]

[0048] 1-methyl-2-phenyldisulfane(I1)

[0049] The product was isolated by flash column chromatography (pure hexane) as a colorless oil. 28.3mg, 88% yield.

[0050] 1 H NMR (400MHz, CDCl3) δ7.56–7.49(m,2H),7.31–7.35(m,2H),7.20-7.25(m,1H),2.43(s,3H).

[0051] 13 C NMR (101MHz, CDCl3) δ136.98,129.06,127.68,126.91,22.94.

[0052] HRMS(ESI):Calcd for C7H8S2[M+H] + :157.2634; found:157.2622.

[0053] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of phenylmethyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0054]

[0055] 1,2-diphenyldisulfane (S1)

[0056] The product was isolated by flash chromatography(pure hexane)as awhite solid(mp60.9-62.1℃).19.8mg,91% yield.

[0057] 1 H NMR (400MHz, CDCl3) δ7.58–7.52(m,2H),7.38–7.31(m,2H),7.30–7.24(m,1H).

[0058] 13 C NMR (101MHz, CDCl3) δ137.07,129.11,127.55,127.20.

[0059] HRMS(ESI): Calcd for C 12 H 10 S2[M+H] + :219.0224; found:219.0224.

[0060] Example 2:

[0061]

[0062] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of symmetric dimethoxydisulfide and 0.04 mmol (0.2 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of dimethoxydisulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0063]

[0064] 1-(4-methoxyphenyl)-2-methyldisulfane(I2)

[0065] The product was isolated by flash column chromatography (hexane / ethylacetate 10:1) as a colorless oil. 28.8mg, 77% yield.

[0066] 1 H NMR (400MHz, CDCl3) δ7.53–7.45(m,2H),6.92–6.80(m,2H),3.81(s,3H),2.44(s,3H).

[0067] 13 C NMR (101MHz, CDCl3) δ159.76,132.12,127.87,114.73,55.42,22.89.

[0068] HRMS(ESI): Calcd for C8H 10 OS2[M+H] + :187.0173; found:187.0170.

[0069] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of methoxyphenyl methyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0070]

[0071] 1,2-bis(4-methoxyphenyl)disulfane(S2)

[0072] The product was isolated by flash chromatography(hexane / ethyl acetate10:1)as a white solid(mp41.2-44.6℃).24.3mg,97% yield.

[0073] 1 H NMR (400MHz, CDCl3) δ7.48–7.38(m,4H),6.91–6.82(m,4H),3.82(s,6H).

[0074] 13 C NMR (101MHz, CDCl3) δ159.94,132.68,128.45,114.65,55.39.

[0075] HRMS(ESI): Calcd for C 14 H 14 O2S2[M+H] + :279.0435; found:279.0437

[0076] Example 3:

[0077]

[0078] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of p-hydroxydiphenyl disulfide and 0.04 mmol (0.2 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of p-hydroxydiphenyl disulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0079]

[0080] 4-(methyldisulfaneyl)phenol(I3)

[0081] The product was isolated by flash column chromatography (hexane / ethylacetate 4:1) as a yellow oil. 28.7mg, 83% yield.

[0082] 1 H NMR (400MHz, CDCl3) δ7.48–7.40(m,2H),6.85–6.77(m,2H),4.96(s,1H),2.44(s,3H).

[0083] 13 C NMR (101MHz, CDCl3) δ155.69,132.25,128.15,116.17,22.88.

[0084] HRMS(ESI):Calcd for C7H8OS2[M+H] + :173.0017; found:173.0020.

[0085] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of p-hydroxyphenyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0086]

[0087] 4,4'-disulfanediyldiphenol(S3)

[0088] The product was isolated by flash chromatography(hexane / ethyl acetate2:1) as a light-yellow solid(mp149.5-150.6℃).24.3mg,97% yield.

[0089] 1 H NMR(400MHz,MeOD)δ7.39–7.22(m,4H),6.82–6.70(m,4H),5.11(s,2H).

[0090] 13 C NMR (101MHz, MeOD) δ157.98,133.28,126.86,115.68.

[0091] HRMS(ESI): Calcd for C 12 H 10 O2S2[M+H] + :251.0122; found:251.0120

[0092] Example 4:

[0093]

[0094] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of p-dichlorodisulfide and 0.04 mmol (0.2 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of p-dichlorodisulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0095]

[0096] 1-(4-chlorophenyl)-2-methyldisulfane(I4)

[0097] The product was isolated by flash column chromatography (pure hexane) as a colorless oil. 30.1mg, 79% yield.

[0098] 1 H NMR (400MHz, CDCl3) δ7.52–7.42(m,2H),7.35–7.27(m,2H),2.44(s,3H).

[0099] 13 C NMR (101MHz, CDCl3) δ135.56,132.93,129.16,129.02,22.85.

[0100] HRMS(ESI):Calcd for C7H7ClS2[M+H] + :190.9678; found:190.9679.

[0101] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of p-chlorophenylmethyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0102]

[0103] 1,2-bis(4-chlorophenyl)disulfane(S4)

[0104] The product was isolated by flash chromatography(pure hexane)as awhite solid(mp41.2-44.6℃).24.3mg,97% yield.

[0105] 1 H NMR (400MHz, CDCl3) δ7.47–7.39(m,4H),7.34–7.26(m,4H).

[0106] 13 C NMR (101MHz, CDCl3) δ135.16,133.67,129.35,129.32.

[0107] HRMS(ESI): Calcd for C 12 H8Cl2S2[M+H] + :286.9444; found:286.9441

[0108] Example 5:

[0109]

[0110] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of 2-pyridine disulfide and 0.04 mmol (0.2 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of 2-pyridine disulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0111]

[0112] 2-(methyldisulfaneyl)pyridine(I5)

[0113] The product was isolated by flash chromatography(hexane / ethyl acetate4:1)as a colorless oil.23.7mg,75% yield.

[0114] 1 H NMR (400MHz, CDCl3) δ8.47 (ddd, J=4.8, 1.8, 1.0Hz, 1H), 7.72–7.65 (m, 1H), 7.08 (ddd, J=7.0, 4.8, 1.4Hz, 1H). 13 C NMR (101MHz, CDCl3) δ159.95,149.78,137.03,120.58,119.48,22.97.

[0115] HRMS(ESI):Calcd for C6H7NS2[M+H] + :158.0020; found:158.0021.

[0116] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of 2-pyridinephenylmethyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0117]

[0118] 1,2-di(pyridin-2-yl)disulfane(S5)

[0119] The product was isolated by flash chromatography(hexane / ethyl acetate1:1)as a yellowish solid(mp56.1-57.9℃).20.4mg,97%yield.

[0120] 1 H NMR (400MHz, CDCl3) δ8.46 (dt, J=4.8, 1.4Hz, 1H), 7.72–7.55 (m, 2H), 7.11 (ddd, J=6.3, 4.8, 2.4Hz, 1H).

[0121] 13 C NMR (101MHz, CDCl3) δ158.91,149.56,137.40,121.12,119.67.

[0122] HRMS(ESI): Calcd for C 12 H8Cl2S2[M+H] + :221.0128; found:221.0127

[0123] Example 6:

[0124]

[0125] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of dibenzyl disulfide and 0.04 mmol (0.2 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of dibenzyl disulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0126]

[0127] 1-benzyl-2-methyldisulfane(I6)

[0128] The product was isolated by flash chromatography(pure hexane)as acolorless oil.54.4mg,80% yield.

[0129] 1 H NMR(400MHz, CDCl3)δ7.26–7.37(m,5H),3.91(s,2H),2.11(s,3H)

[0130] 13 C NMR (101MHz, CDCl3) δ137.57,129.33,128.54,127.40,43.05,23.01.

[0131] HRMS(ESI): Calcd for C8H 10 S2[M+H] + :171.0224; found:171.0223.

[0132] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of benzyl methyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0133]

[0134] 1,2-dibenzyldisulfane (S6)

[0135] The product was isolated by flash chromatography(pure hexane)as awhite solid(mp69.3-71.9℃).24.1mg,98% yield.

[0136] 1 H NMR (400MHz, CDCl3) δ7.51–7.29 (m, 10H), 3.70 (s, 4H).

[0137] 13 C NMR (101MHz, CDCl3) δ137.49,129.54,128.60,127.54,43.37.

[0138] HRMS(ESI): Calcd for C 14 H 14 S2[M+H] + :247.0537; found:247.0538

[0139] Example 7:

[0140]

[0141] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of dicyclohexyl disulfide and hydroxy(p-toluenesulfonyl)iodobenzene (HTIB, 0.04 mmol, 0.2 equivalents). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of dibenzyl disulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0142]

[0143] 1-cyclohexyl-2-methyldisulfane(I7)

[0144] The product was isolated by flash chromatography (pure hexane) as acolorless oil. 20.5mg, 63% yield.

[0145] 1 H NMR (400MHz, CDCl3) δ2.76(tt,J=10.7,3.7Hz,1H),2.40(s,3H),2.06–1.98(m,2H),1.87–1.75(m,2H),1.67–1.58(m,1H),1.45–1.15(m,5H).

[0146] 13 C NMR (101MHz, CDCl3) δ49.26, 32.88, 26.13, 25.64, 24.74.

[0147] HRMS(ESI): Calcd for C7H 14 S2[M+H] + :163.0537; found:163.0535.

[0148] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of cyclohexylmethyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0149]

[0150] 1,2-Dicyclohexyldisulfane (S7)

[0151] The product was isolated by flash chromatography(pure hexane)as acolorless oil.20.4mg,89% yield.

[0152] 1H NMR (400MHz, CDCl3) δ2.69 (tt, J=10.3, 3.9Hz, 2H), 2.05 (dt, J=8.5, 4.7Hz, 4H), 1 .79(dq,J=6.8,3.7,2.5Hz,4H),1.62(dt,J=9.0,4.1Hz,2H),1.41–1.12(m,10H).

[0153] 13 C NMR (101MHz, CDCl3) δ49.93, 32.86, 26.09, 25.72.

[0154] HRMS(ESI): Calcd for C 12 H 22 S2[M+H] + :231.1163; found:231.1165.

[0155] Example 8:

[0156]

[0157] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (5 equivalents) of dimethyl disulfide to a mixture of 0.1 mmol (1 equivalent) of di(2-hydroxyethyl) disulfide and 0.04 mmol (0.2 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of di(2-hydroxyethyl) disulfide, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum and purify the product by column chromatography.

[0158]

[0159] 2-(methyldisulfaneyl)ethan-1-ol(I8)

[0160] The product was isolated by flash chromatography(hexane / ethyl acetate4:1)as a colorless oil.22.3mg,90% yield.

[0161] 1H NMR (400MHz, CDCl3) δ3.88 (t, J = 5.9 Hz, 2H), 2.86 (t, J = 5.8 Hz, 2H), 2.42 (s, 3H), 2.24 (s, 1H).

[0162] 13 C NMR (101MHz, CDCl3) δ60.30, 40.49, 23.18.

[0163] HRMS(ESI):Calcd for C3H8S2[M+H] + :125.0017; found:125.0019.

[0164] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 0.2 mmol (1 equivalent) of (2-hydroxyethyl)methyl disulfide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly purify the reaction mixture by column chromatography.

[0165]

[0166] 2,2'-disulfanediylbis(ethan-1-ol)(S8)

[0167] The product was isolated by flash chromatography(hexane / ethyl acetate1:1)as a yellowish oil.25.2mg,82% yield.(0.2mmol scale instead)

[0168] 1 H NMR (400MHz, MeOD) δ4.86 (s, 2H), 3.81 (t, J = 6.5Hz, 4H), 2.86 (t, J = 6.5Hz, 4H).

[0169] 13 C NMR (101MHz, MeOD) δ59.93,48.31,48.10,47.88,47.67,47.46,47.25,47.03,40.76.

[0170] HRMS(ESI): Calcd for C4H 10 O2S2[M+H] + :155.0122; found:155.0121.

[0171] Example 9:

[0172]

[0173] Forward reaction procedure: In a 4 mL reaction vial, add 0.5 mmol (50 equivalents) of dimethyl disulfide to a mixture of 10 μmol (1 equivalent) of oxytocin and 0.1 mmol (10 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). Stir vigorously at 22 °C for 2 min. Add KPi buffer (pH = 7, 5 mL per mmol of oxytocin, source: Alfa, catalog number #38712), and continue stirring at 22 °C for 30 min. After the reaction is complete, extract the emulsion three times with ethyl acetate, combine the organic layers, and dry with MgSO4. Remove the solvent under vacuum, and purify the product by preparative HPLC.

[0174] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 10 μmol (1 equivalent) of dimethyl disulfide-modified oxytocin; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly analyze the reaction mixture by HPLC.

[0175] Example 10:

[0176]

[0177] Forward reaction procedure: In a 4 mL reaction vial, 0.5 mmol (50 equivalents) of dimethyl disulfide was added to a mixture of 10 μmol (1 equivalent) of lanreotide and 0.1 mmol (10 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). The mixture was stirred vigorously at 22 °C for 2 min. KPi buffer (pH = 7, 5 mL per mmol of lanreotide, source: Alfa, catalog number #38712) was added, and the mixture was stirred at 22 °C for another 30 min. After the reaction was complete, the emulsion was extracted three times with ethyl acetate, the organic layers were combined, and dried over MgSO4. The solvent was removed under vacuum, and the product was purified by preparative HPLC.

[0178] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 10 μmol (1 equivalent) of dimethyl disulfide-modified lanreotide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly analyze the reaction mixture by HPLC.

[0179] Example 11:

[0180]

[0181] Forward reaction procedure: In a 4 mL reaction vial, 0.5 mmol (50 equivalents) of dimethyl disulfide was added to a mixture of 10 μmol (1 equivalent) of auxin and 0.1 mmol (10 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). The mixture was stirred vigorously at 22 °C for 2 min. KPi buffer (pH = 7, 5 mL per mmol of auxin, source: Alfa, catalog number #38712) was added, and the mixture was stirred at 22 °C for another 30 min. After the reaction was complete, the emulsion was extracted three times with ethyl acetate, the organic layers were combined, and dried over MgSO4. The solvent was removed under vacuum, and the product was purified by preparative HPLC.

[0182] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 10 μmol (1 equivalent) of dimethyl disulfide-modified auxin; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly perform HPLC analysis on the reaction mixture.

[0183] Example 12:

[0184]

[0185] Forward reaction procedure: In a 4 mL reaction vial, 0.5 mmol (50 equivalents) of dimethyl disulfide was added to a mixture of 10 μmol (1 equivalent) of octreotide and 0.1 mmol (10 equivalents) of hydroxy(p-toluenesulfonyl)iodobenzene (HTIB). The mixture was stirred vigorously at 22 °C for 2 min. KPi buffer (pH = 7, 5 mL per mmol of octreotide, source: Alfa, catalog number #38712) was added, and the mixture was stirred at 22 °C for another 30 min. After the reaction was complete, the emulsion was extracted three times with ethyl acetate, the organic layers were combined, and dried over MgSO4. The solvent was removed under vacuum, and the product was purified by preparative HPLC.

[0186] Reverse reaction procedure: In a dry 100 mL round-bottom flask, add 10 μmol (1 equivalent) of dimethyl disulfide-modified octreotide; add 0.04 mmol (0.2 equivalent) of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 15.7 mg); add 2 mL of acetonitrile and 8 mL of KPi buffer at pH 7; place the mixture on a rotary evaporator and treat at 50 mbar pressure and 37 °C water bath temperature for 0.5 h to remove the generated dimethyl disulfide; monitor the reaction progress by thin-layer chromatography (TLC), and after the reaction is complete, directly analyze the reaction mixture by HPLC.

[0187] Example 13:

[0188] Forward reaction procedure: In a 4 mL reaction vial, 0.3 mmol (30 equivalents) of dimethyl disulfide was added to a mixture of 10 μmol (1 equivalent) of human insulin and hydroxy(p-toluenesulfonyl)iodobenzene (HTIB, 0.1 mmol, 10 equivalents). The mixture was stirred vigorously at 22 °C for 2 min. KPi buffer (pH = 7, 0.6 mL, source: Alfa, catalog number #38712) was added, and the mixture was stirred at 22 °C for another 30 min. After the reaction was complete, the emulsion was extracted three times with ethyl acetate, the organic layers were combined, and dried over MgSO4. The solvent was removed under vacuum, and the product was purified by preparative HPLC.

[0189] Comparative Example 1: Comparison of Catalysts

[0190]

[0191] Table 1

[0192] Serial Number catalyst solvent Yield 1 Dess-Martin periodinane KPi buffer (pH=7) 56 2 <![CDATA[I(OCOCF3)2Ph]]> KPi buffer (pH=7) 41 3 HTIB(0.2eq) KPi buffer (pH=7) 89 4 NCS KPi buffer (pH=7) 0 5 <![CDATA[I2]]> KPi buffer (pH=7) 49 6 NIS KPi buffer (pH=7) 49 7 <![CDATA[I(OAc)2Ph]]> KPi buffer (pH=7) 55 8 Select-fluor KPi buffer (pH=7) 0 9 <![CDATA[KIO4]]> KPi buffer (pH=7) 0 10 <![CDATA[NaIO4]]> KPi buffer (pH=7) 0 11 <![CDATA[[IPh2][Cl]]]> KPi buffer (pH=7) 0 12 <![CDATA[[IPh2][SO3CFi]]]> KPi buffer (pH=7) 0 13 <![CDATA[[IPh2][BF4]]]> KPi buffer (pH=7) 0 14 Togni reagent II KPi buffer (pH=7) 0 15 <![CDATA[PPh3]]> KPi buffer (pH=7) 51 16 HTIB(0.05eq) KPi buffer (pH=7) 55 17 HTIB(0.1eq) KPi buffer (pH=7) 70

[0193] After screening, hydroxy(p-toluenesulfonyloxy)iodobenzene was significantly superior to other catalysts. Comparative Example 2: Comparison of reaction solvents.

[0194]

[0195] Table 2

[0196] Serial Number catalyst solvent Yield 1 HTIB(0.2eq) KPi buffer (pH=7) 89 2 HTIB(0.2eq) <![CDATA[H2O]]> 87 3 HTIB(0.2eq) <![CDATA[CH3CN]]> 20 4 HTIB(0.2eq) KPi buffer (pH=6) 88 5 HTIB(0.2eq) PBS buffer (pH=8) 84

[0197] After screening, KPi buffer was found to be superior to other solvents.

[0198] Comparative Example 3: Reaction Time Comparison

[0199]

[0200] Table 3

[0201] Serial Number catalyst solvent Yield Serial Number 1 HTIB(0.2eq) KPibuffer (pH=7) 10min 68 2 HTIB(0.2eq) KPi buffer (pH=7) 30min 89 3 HTIB(0.2eq) KPibuffer (pH=7) 3h 89 4 HTIB(0.2eq) KPibuffer (pH=7) 12h 89

[0202] After screening, the reaction can be completed in 30 minutes.

Claims

1. A method for reversible exchange of disulfide bonds under mild biocompatible conditions, characterized in that: Using hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB) as a free radical initiator, the disulfide exchange reaction can be precisely controlled by controlling the concentration of the disulfide raw material in the reaction and distilling the low-boiling disulfide under reduced pressure, as shown in Reaction Formula 1 and Reaction Formula 2: Reaction 1: Reaction 2: In reaction formula 1, the definition of R is: R is any one of a chain alkyl group, a cyclic alkyl group, a phenyl group, a substituted phenyl group, a heterocyclic aromatic group, a substituted heterocyclic aromatic group, or contains any one of a cysteine ​​derivative, a glutathione derivative, a sugar derivative, a captopril derivative, and a thiamine derivative; In reaction formula 1, R 1 Definition of: R 1 Any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl and diisopentyl; In Reaction Formula 2, the definition of Chemical Formula IV is: any one of aliphatic hydrocarbon cyclic disulfide and its derivatives, cyclic peptide compounds containing disulfide bonds and their derivatives; In reaction formula 2, R 2 Definition: any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, diisopentyl or any one of cysteine ​​derivatives, glutathione derivatives, sugar derivatives, captopril derivatives, thiamine derivatives; In reaction formula 2, R 3 Definition: any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, diisopentyl or any one of cysteine ​​derivatives, glutathione derivatives, sugar derivatives, captopril derivatives, thiamine derivatives; The specific preparation steps are as follows: (1) According to reaction formula 1, a disulfide (I), an excess of an alkyl disulfide compound (II) and a catalytic amount of hydroxy (p-toluenesulfonyloxy) iodobenzene (HTIB) are added to a reaction device, an appropriate amount of an aqueous solvent is added, the reaction temperature is maintained at above 20°C, and the reaction is stirred; after the reaction is completed, the reaction mixture is separated and purified to obtain an asymmetric disulfide compound (III); According to reaction formula 2, a cyclic disulfide (IV), an excess of a disulfide compound (V) and a catalytic amount of hydroxy (p-toluenesulfonyloxy) iodobenzene (HTIB) are added to a reaction device, an appropriate amount of an aqueous solvent is added, the reaction temperature is maintained at above 20°C, and the reaction is stirred; after the reaction is completed, the reaction mixture is separated and purified to obtain an asymmetric bis disulfide compound (VI); (2) According to Reaction Formula 1 and Reaction Formula 2, the reverse reaction is driven by removing the generated low-boiling disulfide compound by vacuum distillation: in a reaction apparatus, add disulfide compound (III) or (VI); add a catalytic amount of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB); add an aqueous solvent; remove the generated low-boiling disulfide compound (II) or (V) by vacuum distillation while stirring; after the reaction is completed, separate and purify the reaction mixture to obtain disulfide compound (I) or (IV).

2. The method for reversible exchange of disulfide bonds under mild biocompatible conditions according to claim 1, characterized in that: In the above formula (I), is any one of the following symmetrical disulfide compounds: In the above formula (II), is a methyl disulfide compound; In the above formula (III), is any one of the following disulfide compounds: The specific preparation steps are as follows: (1) According to reaction formula 1, a disulfide compound (I), an excess of methyl disulfide compound (II) and a catalytic amount of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB) are added to a reaction device, and an appropriate amount of an aqueous solvent is added. The reaction temperature is maintained above 20° C. and the reaction is stirred. After the reaction is completed, the reaction mixture is separated and purified to obtain an asymmetric disulfide compound (III); (2) According to reaction formula 1, the reverse reaction is driven by removing the generated low-boiling methyl disulfide compound by vacuum distillation: in a reaction apparatus, add disulfide compound (III); add a catalytic amount of hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB); add an aqueous solvent; remove the generated low-boiling disulfide compound (II) by vacuum distillation while stirring; after the reaction is completed, separate and purify the reaction mixture to obtain disulfide compound (I).

3. The method for bidirectional disulfide bond exchange under mild biocompatible conditions according to claim 1, characterized in that: The method includes forward and reverse control, and the reverse reaction can convert the asymmetric disulfide back to the symmetric disulfide by removing the generated low-boiling disulfide compound (II).

4. The method for reversible exchange of disulfide bonds under mild biocompatible conditions according to claim 1, characterized in that: Reversible exchange of the following cyclic peptide compounds: The specific preparation steps are as follows: In a reaction tube, hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 0.1-1.0 equiv.), methyl disulfide compound (10-100 equiv.) and peptide (1.0 equiv.) are sequentially added to a pH 6-8 buffer and stirred at room temperature for reaction; the reaction is detected by LCMS, and after the reaction is completed, the obtained coupling product is separated and purified.

5. The method for reversible exchange of disulfide bonds under mild biocompatible conditions according to claim 1, characterized in that: Chemical modification of insulin disulfide bonds; In a reaction tube, hydroxy(p-toluenesulfonyloxy)iodobenzene (HTIB, 0.1-1.0 equiv.), methyl disulfide compound (10-100 equiv.) and human insulin (1.0 equiv.) are added to a pH 6-8 buffer in sequence, and the reaction is stirred at room temperature; the reaction is detected by LCMS, and after the reaction is completed, the obtained coupling product is separated and purified.

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