蛋白质/多肽的精准合成: 单一位点选择性化学修饰与自动化合成技术★

doi:10.6023/cjoc202505023

摘要/Abstract

蛋白质/多肽的精准合成是开发创新药物、构建高性能分子探针及先进功能材料的关键基础. 近年来, 单一位点选择性化学修饰与自动化合成技术的突破性进展, 显著提升了序列控制的精确度, 实现了功能基团的定点引入及多样化修饰, 有效克服了传统非选择性修饰导致的产物异质性难题, 同时大幅提高了复杂多肽合成的效率与精度. 此综述总结了近五年(2020~2025)蛋白质/多肽单一位点修饰策略的研究进展, 重点探讨了基于位点特性的直接修饰、配体导向修饰以及关键点定向修饰等策略, 同时系统评述了自动化精准合成技术的最新发展. 这些技术的创新不仅大幅提升了蛋白质/多肽结构的可控合成与精准功能化水平, 更为药物开发、分子探针工程及生物偶联等应用领域提供了新途径. 展望未来, 新型生物正交试剂的开发应用、动态修饰技术的创新融合, 以及人工智能辅助的合成路线优化将成为该领域的重要研究方向, 有望推动蛋白质精准合成技术实现新的突破.

关键词: 蛋白质, 多肽, 精准化学合成, 单一位点修饰, 自动化合成

The precise synthesis of proteins and peptides is fundamental for developing innovative therapeutics, high-per- formance molecular probes, and advanced functional materials. Recent advances in single-site selective chemical modification and automated synthesis technologies have significantly improved precision in sequence control, enabled site-specific introduction of functional groups and diversified modification. These developments have effectively addressed the issue of product heterogeneity from traditional non-selective modifications while substantially improving the efficiency and precision of complex sequence synthesis. This review summarizes the research progress in single-site modification strategies for proteins/peptides over the past five years (2020~2025), focusing on site-specific direct modification, ligand-directed modification, and linchpin-directed modification. It also provides a systematic evaluation of recent developments in automated precision synthesis technologies. These technological innovations have not only enhanced the capability for controllable synthesis and precise functionalization of protein/peptide, but also created new opportunities in drug development, molecular probe engineering, and bioconjugation applications. Looking ahead, the development and application of novel bioorthogonal reagents, the integration of innovative dynamic modification technologies, and AI-assisted synthesis route optimization will emerge as key research directions, promising to drive breakthroughs in protein precision synthesis.

Key words: protein, peptide, precise chemical synthesis, single-site modification, automated synthesis

引用此文

任程, 李承喜. 蛋白质/多肽的精准合成: 单一位点选择性化学修饰与自动化合成技术^★[J]. 有机化学, 2025, 45(9): 3128-3147.

Cheng Ren, Chengxi Li. Precise Synthesis of Proteins/Peptides: Advances in Single-Site Selective Chemical Modification and Automated Synthesis Technologies^★[J]. Chinese Journal of Organic Chemistry, 2025, 45(9): 3128-3147.

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图/表 19

图1. 蛋白质/多肽单一位点直接修饰策略

Figure 1. Single-site direct chemical modification of proteins/peptides

图2. 蛋白质/多肽N端氨基单一位点选择性修饰

Figure 2. Single-site selective modification of the N-terminal amino group in proteins/peptides

图3. 蛋白质/多肽N端主链酰胺键单一位点修饰

Figure 3. Single-site selective modification of N-terminal backbone amide bonds in proteins/peptides

图4. 蛋白质/多肽N端脯氨酸单一位点选择性修饰

Figure 4. Single-site selective modification of N-terminal proline in proteins/peptides

图5. 蛋白质/多肽N端甘氨酸单一位点选择性修饰

Figure 5. Single-site selective modification of N-terminal glycine in proteins/peptides

图6. 蛋白质/多肽N端半胱氨酸单一位点选择性修饰

Figure 6. Single-site selective modification of N-terminal cysteine in proteins/peptides

图7. 蛋白质/多肽C端单一位点选择性修饰

Figure 7. Protein/peptide C-terminal single-site selective modification

图8. 配体导向单一位点修饰技术

Figure 8. Ligand-directed single-site modification

图9. 配体导向的赖氨酸单一位点选择性修饰

Figure 9. Ligand-directed lysine single-site selective modification

图10. 基于共价配体定向释放策略的半胱氨酸单一位点选择性修饰

Figure 10. CoLDR strategy for cysteine single-site selective modification

图11. 基于亲和力引导催化剂策略的单一位点选择性修饰

Figure 11. Single-site selective modification based on affinity-guided catalytic strategy

图12. 关键点定向修饰技术

Figure 12. Linchpin-directed modification

图13. FK1-spacer-FK2在赖氨酸残基上单一位点选择性修饰应用

Figure 13. Single-site selective modification of lysine residue by FK1-spacer-FK2

图14. FC1-spacer-FK2在赖氨酸残基上单一位点选择性修饰应用

Figure 14. Single-site selective modification of lysine residue by FC1-spacer-FK2

图15. 基于关键点定向修饰技术的天冬氨酸/组氨酸位点选择性修饰

Figure 15. Linchpin-directed single-site selective modification of aspartate or histidine residue

图16. 多肽自动化流动合成技术

Figure 16. Automated flow synthesis technology for peptides

图17. 多肽自动化平行自动合成技术

Figure 17. Automated parallel synthesis technology for peptides

图18. 蛋白质自动化流动合成技术

Figure 18. Automated flow synthesis technology for proteins

图19. 机器学习指导的自动化多肽精准合成

Figure 19. Automated precise synthesis of peptides guided by machine learning

参考文献 62

[1]	(a) Fu, H.; Mo, X.; Ivanov, A. A. Nat. Rev. Cancer 2025, 25, 189.
	(b) Roake, C. M.; Artandi, S. E. Nat. Rev. Mol. Cell Biol. 2020, 21, 384.
	(c) Wang, R.; Chen, B.; Elghobashi-Meinhardt, N.; Tie, J.-K.; Ayala, A.; Zhou, N.; Qi, X. Nature 2025, 639, 808.
[2]	(a) Dumontet, C.; Reichert, J. M.; Senter, P. D.; Lambert, J. M.; Beck, A. Nat. Rev. Drug Discov. 2023, 22, 641. doi: 10.1038/s41573-023-00709-2 pmid: 37308581
	(b) Cooper, B. M.; Iegre, J.; O' Donovan, D. H.; ÖlwegṴṴård Halvarsson, M.; Spring, D. R. Chem. Soc. Rev. 2021, 50, 1480. pmid: 37308581
[3]	(a) Hoyt, E. A.; Cal, P. M. S. D.; Oliveira, B. L.; Bernardes, G. J. L. Nat. Rev. Chem. 2019, 3, 147. pmid: 38095227
	(b) Chauhan, P.; V, R.; Kumar, M.; Molla, R.; Mishra, S. D.; Basa, S.; Rai, V. Chem. Soc. Rev. 2024, 53, 380. doi: 10.1039/d3cs00715d pmid: 38095227
	(c) Tamura, T.; Kawano, M.; Hamachi, I. Chem. Rev. 2025, 125, 1191. pmid: 38095227
	(d) Ishizawa, S.; Fujimura, K.; Oisaki, K.; Sato, S.; Ohata, J. ChemCatChem 2025, 17, e202402125. pmid: 38095227
	(e) Ma, Y.; Wang, Y.; Wang, F.; Lu, S.; Chen, X. Chin. Chem. Lett. 2025, 36, 110546. pmid: 38095227
	(f) Tantipanjaporn, A.; Wong, M.-K. Molecules 2023, 28, 1083. pmid: 38095227
	(g) Zielke, F. M.; Rutjes, F. P. J. T. Top. Curr. Chem. 2023, 381, 35. pmid: 38095227
	(h) Kim, Y.; Yi, H. B.; Seo, K.; Lee, H. S.; Shin, I. Trends Chem. 2025, 7, 240. pmid: 38095227
[4]	(a) Hartrampf, N.; Saebi, A.; Poskus, M.; Gates, Z. P.; Callahan, A. J.; Cowfer, A. E.; Hanna, S.; Antilla, S.; Schissel, C. K.; Quartararo, A. J.; Ye, X.; Mijalis, A. J.; Simon, M. D.; Loas, A.; Liu, S.; Jessen, C.; Nielsen, T. E.; Pentelute, B. L. Science 2020, 368, 980. doi: 10.1126/science.abb2491 pmid: 24616230
	(b) Simon, M. D.; Heider, P. L.; Adamo, A.; Vinogradov, A. A.; Mong, S. K.; Li, X.; Berger, T.; Policarpo, R. L.; Zhang, C.; Zou, Y.; Liao, X.; Spokoyny, A. M.; Jensen, K. F.; Pentelute, B. L. ChemBioChem 2014, 15, 713. doi: 10.1002/cbic.201300796 pmid: 24616230
	(c) Mijalis, A. J.; Thomas, D. A.; Simon, M. D.; Adamo, A.; Beaumont, R.; Jensen, K. F.; Pentelute, B. L. Nat. Chem. Biol. 2017, 13, 464. doi: 10.1038/NCHEMBIO.2318 pmid: 24616230
	(d) Li, C.; Callahan, A. J.; Phadke, K. S.; Bellaire, B.; Farquhar, C. E.; Zhang, G.; Schissel, C. K.; Mijalis, A. J.; Hartrampf, N.; Loas, A.; Verhoeven, D. E.; Pentelute, B. L. ACS Cent. Sci. 2022, 8, 205. pmid: 24616230
	(e) Wu, J.; Yang, X.; Pan, Y.; Zuo, T.; Ning, Z.; Li, C.; Zhang, Z. J. Flow Chem. 2023, 13, 385. pmid: 24616230
[5]	Matos, M. J.; Oliveira, B. L.; Martínez-Sáez, N.; Guerreiro, A.; Cal, P. M. S. D.; Bertoldo, J.; Maneiro, M.; Perkins, E.; Howard, J.; Deery, M. J.; Chalker, J. M.; Corzana, F.; Jiménez-Osés, G.; Bernardes, G. J. L. J. Am. Chem. Soc. 2018, 140, 4004.
[6]	(a) Sornay, C.; Vaur, V.; Wagner, A.; Chaubet, G. R. Soc. Open Sci. 2022, 9, 211563.
	(b) Spears, R. J.; Chudasama, V. Curr. Opin. Chem. Biol. 2023, 75, 102306.
[7]	Deng, J.-R.; Lai, N. C.-H.; Kung, K. K.-Y.; Yang, B.; Chung, S.-F.; Leung, A. S.-L.; Choi, M.-C.; Leung, Y.-C.; Wong, M.-K. Commun. Chem. 2020, 3, 67.
[8]	Chen, D.; Disotuar, M. M.; Xiong, X.; Wang, Y.; Chou, D. H.-C. Chem. Sci. 2017, 8, 2717.
[9]	Meudom, R.; Zheng, N.; Zhu, S.; Jacobsen, M. T.; Cao, L.; Chou, D. H.-C. Curr. Res. Chem. Biol. 2022, 2, 100013.
[10]	Tang, K. C.; Raj, M. Angew. Chem., Int. Ed. 2021, 60, 1797.
[11]	Mikkelsen, J. H.; Gustafsson, M. B. F.; Skrydstrup, T.; Jensen, K. B. Bioconjugate Chem. 2022, 33, 625. doi: 10.1021/acs.bioconjchem.2c00045 pmid: 35320668
[12]	Wang, S.; Zhou, Q.; Chen, X.; Luo, R.-H.; Li, Y.; Liu, X.; Yang, L.-M.; Zheng, Y.-T.; Wang, P. Nat. Commun. 2021, 12, 2257.
[13]	Miller, M. K.; Wang, H.; Hanaya, K.; Zhang, O.; Berlaga, A.; Ball, Z. T. Chem. Sci. 2020, 11, 10501. doi: 10.1039/d0sc02933e pmid: 34094308
[14]	Lin, Z.; Liu, B.; Lu, M.; Wang, Y.; Ren, X.; Liu, Z.; Luo, C.; Shi, W.; Zou, X.; Song, X.; Tang, F.; Huang, H.; Huang, W. J. Am. Chem. Soc. 2024, 146, 23752.
[15]	Liew, S. S.; Zhang, C.; Zhang, J.; Sun, H.; Li, L.; Yao, S. Q. Chem. Commun. 2020, 56, 11473.
[16]	Onoda, A.; Inoue, N.; Sumiyoshi, E.; Hayashi, T. ChemBioChem 2020, 21, 1274.
[17]	Barber, L. J.; Yates, N. D. J.; Fascione, M. A.; Parkin, A.; Hemsworth, G. R.; Genever, P. G.; Spicer, C. D. RSC Chem. Biol. 2023, 4, 56.
[18]	Barber, L. J.; Stankevich, K. S.; Spicer, C. D. JACS Au 2025, 5, 1983.
[19]	Hanaya, K.; Yamoto, K.; Taguchi, K.; Matsumoto, K.; Higashibayashi, S.; Sugai, T. Chem. Eur. J. 2022, 28, e202201677.
[20]	Hanaya, K.; Taguchi, K.; Wada, Y.; Kawano, M. Angew. Chem., Int. Ed. 2025, 64, e202417134.
[21]	Sim, Y. E.; Nwajiobi, O.; Mahesh, S.; Cohen, R. D.; Reibarkh, M. Y.; Raj, M. Chem. Sci. 2020, 11, 53.
[22]	Maza, J. C.; Ramsey, A. V.; Mehare, M.; Krska, S. W.; Parish, C. A.; Francis, M. B. Org. Biomol. Chem. 2020, 18, 1881.
[23]	Sahu, T.; Kumar, M.; T. K, S.; Joshi, M.; Mishra, R. K.; Rai, V. Chem. Commun. 2022, 58, 12451.
[24]	Machida, H.; Kanemoto, K. Angew. Chem., Int. Ed. 2024, 63, e202320012.
[25]	(a) Bandyopadhyay, A.; Cambray, S.; Gao, J. Chem. Sci. 2016, 7, 4589. doi: 10.1039/C6SC00172F pmid: 28044097
	(b) Huang, L.; Lee, L. C.-C.; Shum, J.; Xu, G.-X.; Lo, K. K.-W. Chem. Commun. 2024, 60, 6186. pmid: 28044097
[26]	Li, K.; Wang, W.; Gao, J. Angew. Chem., Int. Ed. 2020, 59, 14246.
[27]	Silva, M. J. S. A.; Cavadas, R. A. N.; Faustino, H.; Veiros, L. F.; Gois, P. M. P. Chem.-Eur. J. 2022, 28, e202202377.
[28]	Zheng, X.; Li, Z.; Gao, W.; Meng, X.; Li, X.; Luk, L. Y. P.; Zhao, Y.; Tsai, Y.-H.; Wu, C. J. Am. Chem. Soc. 2020, 142, 5097.
[29]	Wu, Y.; Li, C.; Fan, S.; Zhao, Y.; Wu, C. Bioconjugate Chem. 2021, 32, 2065.
[30]	Istrate, A.; Geeson, M. B.; Navo, C. D.; Sousa, B. B.; Marques, M. C.; Taylor, R. J.; Journeaux, T.; Oehler, S. R.; Mortensen, M. R.; Deery, M. J.; Bond, A. D.; Corzana, F.; Jiménez-Osés, G.; Bernardes, G. J. L. J. Am. Chem. Soc. 2022, 144, 10396. doi: 10.1021/jacs.2c02185 pmid: 35658467
[31]	Mir, M. H.; Parmar, S.; Singh, C.; Kalia, D. Nat. Commun. 2024, 15, 859.
[32]	Bloom, S.; Liu, C.; Kölmel, D. K.; Qiao, J. X.; Zhang, Y.; Poss, M. A.; Ewing, W. R.; MacMillan, D. W. C. Nat. Chem. 2018, 10, 205.
[33]	Zhang, L.; Floyd, B. M.; Chilamari, M.; Mapes, J.; Swaminathan, J.; Bloom, S.; Marcotte, E. M.; Anslyn, E. V. ACS Chem. Biol. 2021, 16, 2595. doi: 10.1021/acschembio.1c00631 pmid: 34734691
[34]	Le Du, E.; Garreau, M.; Waser, J. Chem. Sci. 2021, 12, 2467.
[35]	(a) Amaike, K.; Tamura, T.; Hamachi, I. Chem. Commun. 2017, 53, 11972. pmid: 38693710
	(b) Mortensen, M. R.; Skovsgaard, M. B.; Gothelf, K. V. ChemBioChem 2019, 20, 2711. doi: 10.1002/cbic.201900157 pmid: 38693710
	(c) Sheryl Sharma, S.; Naldrett, M. J.; Gill, M. J.; Checco, J. W. J. Am. Chem. Soc. 2024, 146, 13676. doi: 10.1021/jacs.4c04672 pmid: 38693710
[36]	Shiraiwa, K.; Cheng, R.; Nonaka, H.; Tamura, T.; Hamachi, I. Cell Chem. Biol. 2020, 27, 970. doi: S2451-9456(20)30239-7 pmid: 32679042
[37]	Zhang, X.; Jiang, L.; Huang, K.; Fang, C.; Li, J.; Yang, J.; Li, H.; Ruan, X.; Wang, P.; Mo, M.; Wu, P.; Xu, Y.; Peng, C.; Uesugi, M.; Ye, D.; Yu, F.-X.; Zhou, L. ACS Chem. Biol. 2020, 15, 632.
[38]	Xin, X.; Zhang, Y.; Gaetani, M.; Lundström, S. L.; Zubarev, R. A.; Zhou, Y.; Corkery, D. P.; Wu, Y.-W. Chem. Sci. 2022, 13, 7240.
[39]	Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.; Nishikawa, Y.; Fujisawa, A.; Hamachi, I. Nat. Commun. 2018, 9, 1870.
[40]	Kawano, M.; Murakawa, S.; Higashiguchi, K.; Matsuda, K.; Tamura, T.; Hamachi, I. J. Am. Chem. Soc. 2023, 145, 26202.
[41]	Kosar, M.; Sykes, D. A.; Viray, A. E. G.; Vitale, R. M.; Sarott, R. C.; Ganzoni, R. L.; Onion, D.; Tobias, J. M.; Leippe, P.; Ullmer, C.; Zirwes, E. A.; Guba, W.; Grether, U.; Frank, J. A.; Veprintsev, D. B.; Carreira, E. M. J. Am. Chem. Soc. 2023, 145, 15094.
[42]	Reddi, R. N.; Rogel, A.; Resnick, E.; Gabizon, R.; Prasad, P. K.; Gurwicz, N.; Barr, H.; Shulman, Z.; London, N. J. Am. Chem. Soc. 2021, 143, 20095.
[43]	Reddi, R. N.; Rogel, A.; Gabizon, R.; Rawale, D. G.; Harish, B.; Marom, S.; Tivon, B.; Arbel, Y. S.; Gurwicz, N.; Oren, R.; David, K.; Liu, J.; Duberstein, S.; Itkin, M.; Malitsky, S.; Barr, H.; Katz, B.-Z.; Herishanu, Y.; Shachar, I.; Shulman, Z.; London, N. J. Am. Chem. Soc. 2023, 145, 3346.
[44]	Raynal, L.; Nabarro, J.; Miller, L.; Dowle, A.; Moul, S.; Johnson, S.; Fascione, M.; Spicer, C. ChemRxiv. 2024, doi: 10.26434/chem-rxiv-2024-qskp0.
[45]	(a) Kumar, M.; Reddy, N. C.; Rai, V. Chem. Commun. 2021, 57, 7083.
	(b) Chauhan, P.; V, R.; Kumar, M.; Molla, R.; V. B, U.; Rai, V.; ACS Cent. Sci. 2023, 9, 137.
[46]	Adusumalli, S. R.; Rawale, D. G.; Singh, U.; Tripathi, P.; Paul, R.; Kalra, N.; Mishra, R. K.; Shukla, S.; Rai, V. J. Am. Chem. Soc. 2018, 140, 15114. doi: 10.1021/jacs.8b10490 pmid: 30336012
[47]	Adusumalli, S. R.; Rawale, D. G.; Thakur, K.; Purushottam, L.; Reddy, N. C.; Kalra, N.; Shukla, S.; Rai, V. Angew. Chem., Int. Ed. 2020, 59, 10332.
[48]	Rawale, D. G.; Gupta, M.; Thakur, K.; V, R.; Rai, V. Chem. Commun. 2024, 60, 7168.
[49]	Sahu, T.; Chilamari, M.; Rai, V. Chem. Commun. 2022, 58, 1768.
[50]	Reddy, N. C.; Molla, R.; Joshi, P. N.; T. K, S.; Basu, I.; Kawadkar, J.; Kalra, N.; Mishra, R. K.; Chakrabarty, S.; Shukla, S.; Rai, V. Nat. Commun. 2022, 13, 6038.
[51]	Li, J.; Hu, Q.-L.; Song, Z.; Chan, A. S. C.; Xiong, X.-F. Sci. China Chem. 2022, 65, 1356.
[52]	Nielsen, T.; Märcher, A.; Drobňáková, Z.; Hučko, M.; Štengl, M.; Balšánek, V.; Wiberg, C.; Nielsen, P. F.; Nielsen, T. E.; Gothelf, K. V.; Cló, E. Org. Biomol. Chem. 2020, 18, 4717.
[53]	Rawale, D. G.; Thakur, K.; Sreekumar, P.; T. K, S.; A, R.; Adusumalli, S. R.; Mishra, R. K.; Rai, V. Chem. Sci. 2021, 12, 6732.
[54]	Tian, D.; Tan, T. W.; Kuan Hai, R. T.; Wang, G.; Mohamed, F. P.; Yu, Z.; Ang, H. T.; Xu, W.; Tan, Q. W.; Ng, P. S.; Low, C. H.; Liu, B.; Quek Zekui, P.; Joy, J. K.; Cherian, J.; Mak, F. S.; Wu, J. J. Am. Chem. Soc. 2024, 146, 31321.
[55]	Saebi, A.; Brown, J. S.; Marando, V. M.; Hartrampf, N.; Chumbler, N. M.; Hanna, S.; Poskus, M.; Loas, A.; Kiessling, L. L.; Hung, D. T.; Pentelute, B. L. ACS Chem. Biol. 2023, 18, 518. doi: 10.1021/acschembio.2c00862 pmid: 36821521
[56]	Fittolani, G.; Callahan, A. J.; Loas, A.; Pentelute, B. L. Chem. Commun. 2025, 61, 5471.
[57]	Charalampidou, A.; Nehls, T.; Meyners, C.; Gandhesiri, S.; Pomplun, S.; Pentelute, B. L.; Lermyte, F.; Hausch, F. ACS Cent. Sci. 2024, 10, 649.
[58]	Williams, E. T.; Schiefelbein, K.; Schuster, M.; Ahmed, I. M. M.; De Vries, M.; Beveridge, R.; Zerbe, O.; Hartrampf, N. Chem. Sci. 2024, 15, 8756. doi: 10.1039/d4sc00481g pmid: 38873065
[59]	Ferentzi, K.; Nagy-Fazekas, D.; Farkas, V.; Perczel, A. React. Chem. Eng. 2024, 9, 58.
[60]	(a) Coley, C. W.; Green, W. H.; Jensen, K. F. Acc. Chem. Res. 2018, 51, 1281.
	(b) Zhou, Z.; Li, X.; Zare, R. N. ACS Cent. Sci. 2017, 3, 1337.
	(c) Gao, H.; Struble, T. J.; Coley, C. W.; Wang, Y.; Green, W. H.; Jensen, K. F. ACS Cent. Sci. 2018, 4, 1465.
[61]	Mohapatra, S.; Hartrampf, N.; Poskus, M.; Loas, A.; Gómez- Bombarelli, R.; Pentelute, B. L. ACS Cent. Sci. 2020, 6, 2277.
[62]	Li, C.; Zhang, G.; Mohapatra, S.; Callahan, A. J.; Loas, A.; Gómez-Bombarelli, R.; Pentelute, B. L. Adv. Sci. 2022, 9, 2201988.