Advances in Chemical Conjugation of Natural Cysteine: Techniques and Applications

Abstract

This review examines recent progress in chemical modification of natural cysteine residues, emphasizing biomedical and biotechnological applications. Capitalizing on the distinctive thiol group of cysteine (-SH) and natural scarcity in proteins, researchers have developed selective modification strategies operating under physiological conditions. The analysis systematically compares single thiol functionalization techniques (maleimide conjugation, cyclooctyne reactions) and disulfide rebridging approaches (phosphonamidate/allenamide linkers), evaluating their respective merits and limitations. Current applications span targeted drug delivery, biosensor development, and protein engineering innovations. The discussion highlights increasing attention toward disulfide bond manipulation for enhanced bioconjugation stability and diversity. While acknowledging the transformative potential of the field, the review identifies key challenges in the optimization of reaction specificity, product stability, and biological compatibility that will guide future research directions.

1 Introduction

2 Single Thiol Functionalization of Cysteine

3 Disulfide Functionalization of Cysteine

4 Conclusion

Key words

cysteine modification - bioconjugation - single thiol functionalization - disulfide rebridging - biomedical applications

Publication History

Received: 07 April 2025

Accepted after revision: 25 April 2025

Accepted Manuscript online:
25 April 2025

Article published online:
17 June 2025

• References

• 1 Wang K, Zhang S, Zhou X, Yang X, Li X, Wang Y, Fan P, Xiao Y, Sun W, Zhang P, Li W, Huang S. Nat. Methods 2024; 21: 92
• 2 Galli F. Amino Acids 2012; 42: 1
• 3 Yang M, Liu Y, Luo S, Liu C, Jiang N, Li C, Zhao H, Han Y, Chen W, Li L, Sun L. Acta Pharmacol. Sin. 2024; 45: 777
• 4 Blodgett JA, Zhang JK, Yu X, Metcalf WW. J. Antibiot. 2016; 69: 15
• 5 Kang MS, Kong TW. S, Khoo JY. X, Loh T.-P. Chem. Sci. 2021; 12: 13613
• 6 Tsuchikama K, An Z. Protein Cell 2018; 9: 33
• 7 Kim S, Ko W, Sung BH, Kim SC, Lee HS. Bioorg. Med. Chem. 2016; 24: 5816
• 8 Hudak JE, Barfield RM, de Hart GW, Grob P, Nogales E, Bertozzi CR, Rabuka D. Angew. Chem. Int. Ed. 2012; 51: 4161
• 9 Saadatmand SB, Ahmadi V, Hamidi SM. Sci. Rep. 2023; 13: 20625
• 10 Szymanska B, Lukaszewski Z, Zelazowska-Rutkowska B, Hermanowicz-Szamatowicz K, Gorodkiewicz E. Sensors 2021; 21: 3567
• 11 Pasquardini L, Cennamo N, Malleo G, Vanzetti L, Zeni L, Bonamini D, Salvia R, Bassi C, Bossi AM. Sensors 2021; 21: 3443
• 12 Drozd M, Karoń S, Malinowska E. Sensors 2021; 21: 3781
• 13 Zhang Z, Li L, Xu H, Lee C.-LK, Jia Z, Loh T.-P. J. Am. Chem. Soc. 2024; 146: 1776
• 14 Yates ND. J, Hatton NE, Fascione MA, Parkin A. ChemBioChem 2023; 24: e202300313
• 15 Bahou C, Szijj PA, Spears RJ, Wall A, Javaid F, Sattikar A, Love EA, Baker JR, Chudasama V. Bioconjugate Chem. 2021; 32: 672
• 16 De Geyter E, Antonatou E, Kalaitzakis D, Smolen S, Iyer A, Tack L, Ongenae E, Vassilikogiannakis G, Madder A. Chem. Sci. 2021; 12: 5246
• 17 Jbara M, Rodriguez J, Dhanjee HH, Loas A, Buchwald SL, Pentelute BL. Angew. Chem. Int. Ed. 2021; 60: 12109
• 18 Liu S, Ye H, Yi L, Xi Z. Chem. Commun. 2023; 59: 1497
• 19 Teng S, Zhang Z, Li B, Li L, Tan MC. L, Jia Z, Loh T.-P. Angew. Chem. Int. Ed. 2023; 62: e202311906
• 20 Chen F, Zheng M, Nobile V, Gao J. Chem. Eur. J. 2022; 28: e202200058
• 21 Ziegler K, Frimmer M, Möller W, Fasold H. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1982; 319: 254
• 22 Gui W, Davidson GA, Zhuang Z. RSC Chem. Biol. 2021; 2: 450
• 23 Makhnyr VM. Chem. Nat. Compd. 1985; 21: 553
• 24 Wang Q, Guo S, Gao Y. Adv. Biosci. Biotechnol. 2016; 7: 454
• 25 Bhat KP, Ümit Kaniskan H, Jin J, Gozani O. Nat. Rev. Drug Discovery 2021; 20: 265
• 26 Xiao C.-W, Hendry A, Kenney L, Bertinato J. Sci. Rep. 2023; 13: 19943
• 27 Levine PM, Craven TW, Bonneau R, Kirshenbaum K. Org. Lett. 2014; 16: 512
• 28 Bhattacharyya J, Bellucci JJ, Chilkoti A. Biomaterials from Nature for Advanced Devices and Therapies . Neves NM, Reis RL. Wiley; Hoboken: 2016: 106-126
• 29 Yang X, Chen C, Hu Q, Yan J, Zhou C. Neurosci. Lett. 2010; 469: 385
• 30 We Gao J, Yang GL, Chen C, Chin J. Chin Mater Med 2013; 38: 748
• 31 Hoyle CE, Bowman CN. Angew. Chem. Int. Ed. 2010; 49: 1540
• 32 Zhang Y, Zang C, An G, Shang M, Cui Z, Chen G, Xi Z, Zhou C. Nat. Commun. 2020; 11: 1015
• 33 Zhang C, Dai P, Vinogradov AA, Gates ZP, Pentelute BL. Angew. Chem. Int. Ed. 2018; 57: 6459
• 34 Paulsen CE, Carroll KS. Chem. Rev. 2013; 113: 4633
• 35 Giles NM, Watts AB, Giles GI, Fry FH, Littlechild JA, Jacob C. Chem. Biol. 2003; 10: 677
• 36 Kuninori T, Nishiyama J. Agric. Biol. Chem. 1985; 49: 2453
• 37 Alley SC, Benjamin DR, Jeffrey SC, Okeley NM, Meyer DL, Sanderson RJ, Senter PD. Bioconjugate Chem. 2008; 19: 759
• 38 Lewis MR, Shively JE. Bioconjugate Chem. 1998; 9: 72
• 39 Shen B.-Q, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, Parsons-Reponte KL, Tien J, Yu S.-F, Mai E, Li D, Tibbitts J, Baudys J, Saad OM, Scales SJ, McDonald PJ, Hass PE, Eigenbrot C, Nguyen T, Solis WA, Fuji RN, Flagella KM, Patel D, Spencer SD, Khawli LA, Ebens A, Wong WL, Vandlen R, Kaur S, Sliwkowski MX, Scheller RH, Polakis P, Junutula JR. Nat. Biotechnol. 2012; 30: 184
• 40 Wei C, Zhang G, Clark T, Barletta F, Tumey LN, Rago B, Hansel S, Han X. Anal. Chem. 2016; 88: 4979
• 41 Baldwin AD, Kiick KL. Bioconjugate Chem. 2011; 22: 1946
• 42 Lyon RP. Nat. Biotechnol. 2014; 32: 1059
• 43 Kantner T, Watts AG. Bioconjugate Chem. 2016; 27: 2400
• 44 Zhang Y, Zhou X, Xie Y, Greenberg MM, Xi Z, Zhou C. J. Am. Chem. Soc. 2017; 139: 6146
• 45 van Geel R, Prujin GJ. M, van Delft FL, Boelens WC. Org. Process Res. Dev. 2018; 22: 286
• 46 Shiu H, Chan T, Ho C, Liu Y, Wong M, Che C. Chem. Eur. J. 2009; 15: 3839
• 47 Ariyasu S, Hayashi H, Xing B, Chiba S. Bioconjugate Chem. 2017; 28: 897
• 48a Koniev O, Leriche G, Nothisen M, Remy J.-S, Strub J.-M, Schaeffer-Reiss C, Van Dorsselaer A, Baati R, Wagner A. Bioconjugate Chem. 2014; 25: 202
• 48b Kolodych S, Koniev O, Baatarkhuu Z, Bonnefoy J.-Y, Debaene F, Cianférani S, Van Dorsselaer A, Wagner A. Bioconjugate Chem. 2015; 26: 197
• 48c Ariyasu S, Hayashi H, Xing B, Chiba S. Bioconjugate Chem. 2017; 28: 897
• 49 Tessier R, Nandi RK, Dwyer BG, Abegg D, Sornay C, Ceballos J, Erb S, Cianférani S, Wagner A, Chaubet G, Adibekian A, Waser J. Angew. Chem. Int. Ed. 2020; 59: 10961
• 50 Bernardim B, Dunsmore L, Li H, Hocking B, Nuñez-Franco R, Navo CD, Jiménez-Osés G, Burtoloso AC. B, Bernardes GJ. L. Bioconjugate Chem. 2020; 31: 1604
• 51 Kasper M, Glanz M, Stengl A, Penkert M, Klenk S, Sauer T, Schumacher D, Helma J, Krause E, Cardoso MC, Leonhardt H, Hackenberger CP. R. Angew. Chem. Int. Ed. 2019; 58: 11625
• 52 Kasper M, Stengl A, Ochtrop P, Gerlach M, Stoschek T, Schumacher D, Helma J, Penkert M, Krause E, Leonhardt H, Hackenberger CP. R. Angew. Chem. Int. Ed. 2019; 58: 11631
• 53 Park Y, Baumann AL, Moon H, Byrne S, Kasper M.-A, Hwang S, Sun H, Baik M.-H, Hackenberger CP. R. Chem. Sci. 2021; 12: 8141
• 54 Abbas A, Xing B, Loh T. Angew. Chem. Int. Ed. 2014; 53: 7491
• 55 Cameron AJ, Harris PW. R, Brimble MA. Angew. Chem. Int. Ed. 2020; 59: 18054
• 56 Tiambeng TN, Roberts DS, Brown KA, Zhu Y, Chen B, Wu Z, Mitchell SD, Guardado-Alvarez TM, Jin S, Ge Y. Nat. Commun. 2020; 11: 3903
• 57 Jafari MR, Lakusta J, Lundgren RJ, Derda R. Bioconjugate Chem. 2016; 27: 509
• 58 Pedzisa L, Li X, Rader C, Roush WR. Org. Biomol. Chem. 2016; 14: 5141
• 59 Petit E, Bosch L, Costa AM, Vilarrasa J. J. Org. Chem. 2019; 84: 11170
• 60 Wang B, Li C, He D, Ding K, Tian Q, Feng G, Qin A, Tang BZ. Small 2023; 2307309
• 61 Smith NJ, Rohlfing K, Sawicki LA, Kharkar PM, Boyd SJ, Kloxin AM, Fox JM. Org. Biomol. Chem. 2018; 16: 2164
• 62 Yu J, Yang X, Sun Y, Yin Z. Angew. Chem. Int. Ed. 2018; 57: 11598
• 63a Bernardim B, Cal PM. S. D, Matos MJ, Oliveira BL, Martínez-Sáez N, Albuquerque IS, Perkins E, Corzana F, Burtoloso AC. B, Jiménez-Osés G, Bernardes GJ. L. Nat. Commun. 2016; 7: 13128
• 63b Bernardim B, Matos MJ, Ferhati X, Compañón I, Guerreiro A, Akkapeddi P, Burtoloso AC. B, Jiménez-Osés G, Corzana F, Bernardes GJ. L. Nat. Protoc. 2019; 14: 86
• 64 Evans P, Taylor RJ. K. J. Sulfur Chem. 2005; 26: 481
• 65 Masri MS, Friedman M. J. Protein Chem. 1988; 7: 49
• 66 Zhang D, Devarie-Baez NO, Li Q, Lancaster JR, Xian M. Org. Lett. 2012; 14: 3396
• 67 Toda N, Asano S, Barbas CF. III. Angew. Chem. Int. Ed. 2013; 52: 12592
• 68 Davydova M, Le Roi GD, Adumeau P, Zeglis BM. J. Vis. Exp. 2019; e59063
• 69 Bernardes GJ. L, Casi G, Trüssel S, Hartmann I, Schwager K, Scheuermann J, Neri D. Angew. Chem. Int. Ed. 2012; 51: 941
• 70 List T, Casi G, Neri D. Mol. Cancer Ther. 2014; 13: 2641
• 71 Tokunaga K, Sato M, Kuwata K, Miura C, Fuchida H, Matsunaga N, Koyanagi S, Ohdo S, Shindo N, Ojida A. J. Am. Chem. Soc. 2020; 142: 18522
• 72 Khoury PR, Goddard JD, Tam W. Tetrahedron 2004; 60: 8103
• 73 Schneider C, Niisuke K, Boeglin WE, Voehler M, Stec DF, Porter NA, Brash AR. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 18941
• 74 Spokoyny AM, Zou Y, Ling JJ, Yu H, Lin Y.-S, Pentelute BL. J. Am. Chem. Soc. 2013; 135: 5946
• 75 Zhang C, Spokoyny AM, Zou Y, Simon MD, Pentelute BL. Angew. Chem. Int. Ed. 2013; 52: 14001
• 76 Zhang C, Welborn M, Zhu T, Yang NJ, Santos MS, Van Voorhis T, Pentelute BL. Nat. Chem. 2016; 8: 120
• 77 Li J, Deng J.-J, Yin Z, Hu Q.-L, Ge Y, Song Z, Zhang Y, Chan AS. C, Li H, Xiong X.-F. Chem. Sci. 2021; 12: 5209
• 78 Matos MJ, Navo CD, Hakala T, Ferhati X, Guerreiro A, Hartmann D, Bernardim B, Saar KL, Compañón I, Corzana F, Knowles TP. J, Jiménez-Osés G, Bernardes GJ. L. Angew. Chem. Int. Ed. 2019; 58: 6640
• 79 Wan C, Hou Z, Yang D, Zhou Z, Xu H, Wang Y, Dai C, Liang M, Meng J, Chen J, Yin F, Wang R, Li Z. Chem. Sci. 2023; 14: 604
• 80 Bacauanu V, Merz ZN, Hua ZL, Lang SB. J. Am. Chem. Soc. 2023; 145: 25842
• 81 Peltek OO, Muslimov AR, Zyuzin MV, Timin AS. J. Nanobiotechnol. 2019; 17: 90
• 82 Wang W, Yang C, Wang T, Deng H. Cell Death Dis. 2022; 13: 267
• 83 Wiedemann C, Kumar A, Lang A, Ohlenschläger O. Front. Chem. 2020; 8: 280
• 84 Yang J, Petitjean SJ. L, Koehler M, Zhang Q, Dumitru AC, Chen W, Derclaye S, Vincent SP, Soumillion P, Alsteens D. Nat. Commun. 2020; 11: 4541
• 85 Walsh SJ, Omarjee S, Galloway WR. J. D, Kwan TT.-L, Sore HF, Parker JS, Hyvönen M, Carroll JS, Spring DR. Chem. Sci. 2019; 10: 694
• 86 Hoogenboom R. Angew. Chem. Int. Ed. 2010; 49: 3415
• 87 Balan S, Choi J, Godwin A, Teo I, Laborde CM, Heidelberger S, Zloh M, Shaunak S, Brocchini S. Bioconjugate Chem. 2007; 18: 61
• 88 Moore JE, Ward WH. J. Am. Chem. Soc. 1956; 78: 2414
• 89 Chen Y, Yang W, Wu J, Sun W, Loh T.-P, Jiang Y. Org. Lett. 2020; 22: 2038
• 90 Koniev O, Dovgan I, Renoux B, Ehkirch A, Eberova J, Cianférani S, Kolodych S, Papot S, Wagner A. Med. Chem. Commun. 2018; 9: 827
• 91 Patel M, Forte N, Bishop CR, Porter MJ, Dagwell M, Karu K, Chudasama V, Baker JR. J. Am. Chem. Soc. 2024; 146: 274
• 92 Ahangarpour M, Kavianinia I, Hume PA, Harris PW. R, Brimble MA. J. Am. Chem. Soc. 2022; 144: 13652
• 93 Hong V, Presolski SI, Ma C, Finn MG. Angew. Chem. Int. Ed. 2009; 48: 9879
• 94 Laserna V, Istrate A, Kafuta K, Hakala TA, Knowles TP. J, Alcarazo M, Bernardes GJ. L. Bioconjugate Chem. 2021; 32: 1570
• 95 Griebenow N, Bräse S, Dilmac AM. RSC Adv. 2015; 5: 54301
• 96 Stieger CE, Franz L, Körlin F, Hackenberger CP. R. Angew. Chem. Int. Ed. 2021; 60: 15359
• 97 Guermazi R, Royer L, Galmiche L, Clavier G, Audebert P, Hedhli A. J. Fluoresc. 2016; 26: 1349
• 98 Canovas C, Moreau M, Bernhard C, Oudot A, Guillemin M, Denat F, Goncalves V. Angew. Chem. Int. Ed. 2018; 57: 10646
• 99 Rochet LN. C, Bahou C, Wojciechowski JP, Koutsopetras I, Britton P, Spears RJ, Thanasi IA, Shao B, Zhong L, Bučar D.-K, Aliev AE, Porter MJ, Stevens MM, Baker JR, Chudasama V. Chem. Sci. 2023; 14: 13743
• 100 Badescu G, Bryant P, Bird M, Henseleit K, Swierkosz J, Parekh V, Tommasi R, Pawlisz E, Jurlewicz K, Farys M, Camper N, Sheng X, Fisher M, Grygorash R, Kyle A, Abhilash A, Frigerio M, Edwards J, Godwin A. Bioconjugate Chem. 2014; 25: 1124
• 101 Wu L.-H, Zhou S, Luo Q.-F, Tian J.-S, Loh T.-P. Org. Lett. 2020; 22: 8193
• 102 Martínez-Sáez N, Sun S, Oldrini D, Carboni F, Compañón I, Deery MJ, Vendruscolo M, Corzana F, Adamo R, Bernardes GJ. L. Angew. Chem. Int. Ed. 2017; 56: 14963
• 103 Burkhard JA, Wuitschik G, Rogers-Evans M, Müller K, Carreira EM. Angew. Chem. Int. Ed. 2010; 49: 9052
• 104 Hennrich U, Kopka K. Pharmaceuticals 2019; 12: 114
• 105 Grass P, Marbach P, Bruns C, Lancranjan I. Metabolism 1996; 45: 27
• 106 Vingtdeux V, Zhao H, Chandakkar P, Acker CM, Davies P, Marambaud P. Mol. Med. 2016; 22: 841