糖基化干扰素-γ的半合成

doi:10.6023/cjoc202402006

有机化学 ›› 2024, Vol. 44 ›› Issue (7): 2296-2304.DOI: 10.6023/cjoc202402006 上一篇下一篇

研究论文

糖基化干扰素-γ的半合成

周敏园^a, 赵洁^b, 叶发荣^b, 黄平^b, 邓明刚^a^,^*(), 王平^b^,^*()

a 华中农业大学生命科学与技术学院武汉 430070
b 上海交通大学张江高等研究院上海手性药物分子工程重点实验室化学糖生物学中心上海 200240

收稿日期:2024-02-05 修回日期:2024-03-17 发布日期:2024-04-10
作者简介:
† 共同第一作者.
基金资助:
国家自然科学基金(21672146); 国家自然科学基金(22077080); 国家自然科学基金(92253302); 上海交通大学医工交叉基金(21TQ1400210)

Semi-synthesis of Glycosylated Interferon-γ

Minyuan Zhou^a, Jie Zhao^b, Farong Ye^b, Ping Huang^b, Minggang Deng^a(), Ping Wang^b()

a College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070
b Center for Chemical Glycobiology, Shanghai Key Laboratory for Molecular Engineering of Chiral Drugs, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240

Received:2024-02-05 Revised:2024-03-17 Published:2024-04-10
Contact: * E-mail: wangp1@sjtu.edu.cn;dengmg@mail.hzau.edu.cn
About author:
† These authors contributed equally to this work.
Supported by:
National Natural Science Fund(21672146); National Natural Science Fund(22077080); National Natural Science Fund(92253302); Cross Fund of Shanghai Jiao Tong University Medical Engineering(21TQ1400210)

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摘要/Abstract

干扰素-γ (IFN-γ)是一种重要的细胞因子, 具有增强免疫活性、抗肿瘤和抗病毒的作用, 在医学研究和临床应用中具有广泛的潜力. 本研究报道了一种均一N-GlcNAc修饰的IFN-γ的化学半合成方法. 利用化学方法合成制备糖肽片段(Pyr1-Leu33)和多肽片段(Lys34-Ser39), 并通过大肠杆菌表达得到多肽片段(Ser40-Gly138). 采用“表达丝氨酸连接”和“自然化学连接-脱硫”相结合的策略, 成功完成了自C端到N端的N-GlcNAc修饰的IFN-γ蛋白的合成, 并成功地对目标糖蛋白进行了复性.

关键词: 自然化学连接, 表达丝氨酸连接, β-硫代氨基酸, 糖基化修饰

Interferon-gamma (IFN-γ) is an important cytokine with enhanced immune activity, anti-tumor and antiviral effects, and holds significant potential in medical research and clinical applications. In this study, we report a highly efficient semi-synthesis strategy of homogeneous N-GlcNAc modified IFN-γ. The glycopeptide fragment (Pyr1-Leu33) and peptide fragment (Lys34-Ser39) were prepared through chemical methods. And the peptide fragment (Ser40-Gly138) was obtained through Escherichia coli (E. coli) expression. Subsequently, using a combination of “expressed serine ligation” and “native chemical ligation-desulfurization”, we ligated these fragments from the C-terminal to the N-terminal, resulting in a full-length glycoprotein, which was successfully refolded to obtain the desired product.

Key words: native chemical ligation, expressed serine ligation, β-thioamino acid, glycosylation

引用此文

周敏园, 赵洁, 叶发荣, 黄平, 邓明刚, 王平. 糖基化干扰素-γ的半合成[J]. 有机化学, 2024, 44(7): 2296-2304.

Minyuan Zhou, Jie Zhao, Farong Ye, Ping Huang, Minggang Deng, Ping Wang. Semi-synthesis of Glycosylated Interferon-γ[J]. Chinese Journal of Organic Chemistry, 2024, 44(7): 2296-2304.

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图/表 5

图1. 糖基化IFN-γ的结构及逆合成分析和IFN-γ分解为合成片段1、2、重组片段3

Figure 1. Structure of glycosylated IFN-γ and its retrosynthetic analysis, and IFN-γ disconnected into synthetic glycopeptides fragment 1, 2 and recombinant peptide 3

图2. 多肽3的表达及纯化后的HPLC分析

Figure 2. Expression of peptide 3 and analytical HPLC

图3. Asn砌块8 (A)和多肽1 (B)的合成

Figure 3. Synthesis of Asn building block 8 (A) and peptide 1 (B) Reagents and conditions: a) DMC (5.0 equiv.), NaN3 (10.0 equiv.), 2,6-lutidine (5.0 equiv.), H2O, 0 ℃, 3 d; b) Ac2O (10.0 equiv.), pyridine (10.0 equiv.), DMAP (0.1 equiv.), 25 ℃, 4 h; c) H2, Pd/C (5%), MeOH, 25 ℃, 2 h; d) Fmoc-Asp-OtBu (1.0 equiv.), DIC (3.0 equiv.), DMAP (0.1 equiv.), dry DCM, 25 ℃, 12 h; e) TFA/DCM (V/V=1/1), 25 ℃, 2 h; f) Fmoc-AA-OH (4.0 equiv.), DIC (5.0 equiv.), Oxyma (4.0 equiv.), 45 ℃, 1 h; g) 10% N2H4?H2O/DMF, 25 ℃, 5 h; h) TFA/H2O/Phenol/TIPS (V/V/V/V=88/5/5/2), 25 ℃, 3 h; i) MPAA (0.2 mol/L), acac (1.2 equiv.), N2, pH 2.7~2.8, 37 ℃, 2 h. DMC, 2-chloro-1,3-dimethyli-midazolidinium chloride; DIC, diipropylcabodiimide; DMAP, 4-dimethylaminopyridine; DCM, dichlomthane; DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid; TIPS, triisopropylsilane; MPAA, 4-mercaptophenylacetic acid.

图4. β-硫代赖氨酸14 (A)和多肽2 (B)的合成

Figure 4. Synthesis of β-S-Lys 14 (A) and peptide 2 (B) Reagents and conditions: a) redox-active ester (2.0 equiv.), Eosin Y (0.1 equiv.), DIPEA (2.5 equiv.), white led light, DCM, N2, 25 ℃, 5 h; b) MeOH/H2O (V/V=3/1), LiOH (10 equiv.), 25 ℃, 1 h; c) 6 mol/L HCl in dioxane, 50 ℃, 8 h; d) 12 (2.0 equiv.), DIPEA (3.0 equiv.), EtOH, 25 ℃, overnight; e) phenylthiohypochlorite (1.0 equiv.), TFA, 25 ℃, 0.5 h; f) Boc2O (1.0 equiv.), DIPEA (2.0 equiv.), THF, 25 ℃, 1 h; g) Fmoc-AA-OH (4.0 equiv.), DIC (5.0 equiv.), Oxyma (4.0 equiv.), 45 ℃, 1 h; h) TFE/AcOH/DCM (V/V/V=1/1/3), 25 ℃, 3×1 h; i) 16 (1.0 equiv.), EDC (3.0 equiv.), HOOBt (3.0 equiv.), DMF, 25 ℃, 3 h; j) TFA/H2O/Phenol/TIPS (V/V/V/V=85/5/5/5), pyruvic acid (100 equiv.), 25 ℃, 3 h. DIPEA, N,N-diisopropylethylamine; THF, tetrahydrofuran; TFE, Tetrafluoroethene; acac, acetlacetone; EDC, 1-(3-dimethylaminoprpyl)-3-ethylcarbodi- imide hydrochloride; HOOBt, 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine.

图5. IFN-γ 19的合成(A)、HPLC分析和IFN-γ 20的HPLC分析(B)

Figure 5. Synthesis of IFN-γ 19 (A), and the analytical HPLC of IFN-γ 19 and IFN-γ 20 (B) Reagents and conditions: a) 2 (5.0 equiv.), 3 (3 mmol/L), pyridine-acetic acid (molar ratio 1/6), 25 ℃, 3 h; b) 0.1 mol/L HCl in HFIP, 1% TIPS, 0.5 h. c) 5% N2H4 in 6 mol/L GND?HCl, 0.2 mol/L Na2HPO4, 25 ℃, 0.5 h; d) 1 (1.0 equiv.), 17 (1.0 equiv.), TCEP (50 mmol/L), MPAA (100 mmol/L), pH 6.8, 25 ℃, 3 h; e) TCEP (300 mmol/L), GSH (100 mmol/L), VA-044 (50 mmol/L), pH 7.0, 40 ℃, 3 h; f) Buffer A, 50 mmol/L Tris?HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mol/L urea, pH 8.0, 4 ℃, 24 h. Buffer B, 50 mmol/L Tris?HCl, pH 8.0, 4 ℃, 24 h. HFIP, hexafluoroisopropanol; TCEP, tris(2-carboxyethyl)phosphine; GSH, reduced glutathione; VA-044, 2,2'-azobis(2-(2-imidazolin-2-yl)propane) dihydrochloride; EDTA, ethylene diamine tetraacetic acid.

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糖基化干扰素-γ的半合成

Semi-synthesis of Glycosylated Interferon-γ

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