Semi-synthesis of Glycosylated Interferon-γ

doi:10.6023/cjoc202402006

Chinese Journal of Organic Chemistry ›› 2024, Vol. 44 ›› Issue (7): 2296-2304.DOI: 10.6023/cjoc202402006 Previous Articles Next Articles

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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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Abstract

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

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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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Fig. & Tab. 5

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

Figure 2. Expression of peptide 3 and analytical HPLC

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.

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.

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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