While internal cysteines have a long history as easy target for protein derivatization and for attaching payloads, e.g., by reacting them with maleimides, their modification frequently interferes with the formation of disulfide bridges (which however sometimes are essential for correct protein function) or the role of cysteines as essential parts of the catalytic core of enzymes. 1,2-Aminothiols in contrast allow specific modifications without affecting structurally relevant amino acids.

In solid phase peptide synthesis (SPPS), 1,2-aminothiols may be incorporated as non-canonical amino acids at any position. N-terminal cysteines (NCys) are special cases of 1,2-aminothiols. In recombinant proteins, providing an NCys is an easy task utilizing TEV protease; as a third option, 1,2-aminothiols may be introduced by SPPS in a protected form as thiazolidine and later deprotected with methoxyamine (O-methylhydroxylamine). This can be done in neutral aqueous solutions, usually in situ, right before the actual click-like reaction is initiated.

Exemplary peptide sequence with an N-terminal cysteine (NCys), an internal 1,2-aminothiol and a thiazolidine, the protected version of an 1,2-aminothiol.

1,2-Aminothiols are an excellent match for mono- and dicyanopyridines to generate cyclic peptides, but they offer even more possibilities: 1,2-aminothiols also undergo click-like reactions with cyclopropenones (CPO, forming 1,4-thiazepan-5-one rings) and with cyanobenzothiazoles (CBT), they may be used as non-canonical amino acids and for branching peptide chains. Furthermore, 1,2-aminothiols also can serve as template for native chemical ligation (NCL), which also may be utilized for ubiquitinoylation with ubiquitin thioesters.

1,2-Aminothiols offer several possibilities for bioorthogonal click-like derivatization at neutral aqueous conditions, e.g., with 2-cyanopyridines, cyanobenzothiazoles, or cylopropenones.

Iris Biotech provides a selection of non-canonical amino acid building blocks which allow the direct introduction of 1,2 aminothiols and thiazolidines at any position during SPPS.

→  You want to learn more about Click Chemistry or click-like reactions? Download our brochure or read our blog about the CBT click reaction! 

References:

The Cyanopyridine-Aminothiol Click Reaction: Expanding Horizons in Chemical Biology; C. Nitsche; SynLett. 2024; 35: A-E. http://dx.doi.org/10.1055/a-2214-7612

Biocompatible and Selective Generation of Bicyclic Peptides; S. Ullrich, J. George, A. Coram, R. Morewood, C. Nitsche; Angew Chem Int Ed. 2022; 61(43): e20228400. https://doi.org/10.1002/anie.202208400

Platform for Orthogonal N‑Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents; A. Istrate, M. Geeson, C. Navo, B. Sousa, M. Marques, R. Taylor, T. Journeaux, S. Oehler, M. Mortensen, M. Deery, A. Bond, F. Corzana, G. Jiménez-Osés, G. Bernardes; J Am Chem Soc. 2022; 144(23): 10396-10406. https://pubs.acs.org/doi/10.1021/jacs.2c02185

Chemical Synthesis of Ubiquitin, Ubiquitin-Based Probes, and Diubiquitin; F. El Oualid, R. Merkx, R. Ekkebus, D. Hameed, J. Smit, A. de Jong, H. Hilkmann, T. Sixma, H. Ovaa; Angew Chem Int Ed. 2010; 49(52): 10149-10153. https://doi.org/10.1002/anie.201005995

Highly Efficient and Chemoselective Peptide Ubiquitylation; K. Ajish Kumar, M. Haj-Yahya, D. Olschewski, H. Lashuel, A. Brik; Angew Chem Int Ed. 2009; 48(43): 8090-8094. https://doi.org/10.1002/anie.200902936

Traceless and Site-Specific Ubiquitination of Recombinant Proteins; S. Virdee, P. Kapadnis, T. Elliott, K. Lang, J. Madrzak, D. Nguyen, L. Riechmann, J. Chin; J Am Chem Soc. 2011; 133(28): 10708–10711. https://doi.org/10.1021/ja202799r

Tobacco Etch Virus protease: A shortcut across biotechnologies; F. Cesaratto, O. Burrone, G. Petris; J Biotechnol. 2016; 231: 239-249. https://doi.org/10.1016/j.jbiotec.2016.06.012

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations; V. Agouridas, O. El Mahdi, V. Diemer, M. Cargoët, J. Monbaliu, O. Melnyk; Chem. Rev. 2019; 119(12): 7328-7443. https://doi.org/10.1021/acs.chemrev.8b00712