While nitriles in general may react with sulfhydryl groups (e.g., from cysteines) in a rather unspecific way, the advantage of alpha-cyanopyridines (2-picolinonitrils) is their strong selectivity for 1,2-aminothiols (e.g., provided as N-terminal cysteine or internally as non-canonical amino acid). Thus, this reaction may be used for the synthesis of cyclic peptides or to fuse ligands to peptide chains.

The reaction between a 2-cyanopyridine and a 1,2-aminothiol forms a 2-thiazoline.

This click-like reaction is biorthogonal, biocompatible, catalyst-free, and selective; it proceeds readily in aqueous solutions at physiological pH and ambient temperature. The formation of the resulting 2-thiazoline (alternative name: 2,5-dihydrothiazole) is driven by the release of ammonia. The reaction product is stable at physiological conditions. Cyanopyridine click-like reactions may be used for macrocyclization, stapling, bi- and even tricyclization, or to attach payloads to peptides produced by SPPS. The required 1,2-aminothiols can be provided in three ways: a) as N-terminal cysteine (NCys), b) as intra-chain 1,2 aminothiol or c) as 1,3-thiazolidine which can be deprotected later in situ. In vitro, the click reaction is optimally carried out at a pH between 7 and 8. In recombinant proteins, N-terminal cysteines may be provided by the action of TEV protease.

With a 2,6 dicyanopyridine (2,6-DCP) side chain as bidentate anchor, the formation of bicyclic peptides is rather simple, just two 1,2-aminothiols need to be provided. The cyclization itself will take place within a few minutes in the presence of TCEP (tris-carboxyethylphosphine; LS-3405) as reductant, in a neutral buffer at pH 7.5 and ambient temperature.

Example for the synthesis of a bicyclic peptide: When a peptide with an N-terminal cysteine, a C-terminal 1,2-aminothiol and an intra-chain dicyanopyridine (2,6-DCP) is incubated for a few minutes at aqueous neutral conditions, the reaction between the 2,6-DCP and the 1,2 aminothiols will take place quickly. TCEP serves as reduction agent and ensures that the thiols remain in their reduced form.

2,6-Dicyanopyridine may also be used in a non-bound form to make cyclic peptides, when the two 1,2-aminothiols are provided as side chains, introduced as non-canonical amino acids. For the controlled synthesis of tricyclic peptides, this method even may be combined with the previously presented synthesis of bicyclic peptides, when two more 1,2-aminothiols initially are introduced in a protected fashion as 1,3 thiazoles, are deprotected with methoxyamine after the first cyclization and the 2,6-DCP is used to form the thiazoline bridge.

Of course, the 1,2-aminothiol does not need to be provided as amino acid within a peptide backbone; it also may be used to attach other molecules to peptides, e.g., payloads or reporters, where the cyanopyridine has been incorporated by SPPS.

Iris Biotech provides Fmoc protected building blocks which may be used to conveniently introduce 2-cyanopyridyl residues into a peptide, as well as building blocks for 2,6-dicyano-functionalization (see related products below). Furthermore, we also offer one biotinylated cyanopyridine and three molecules which enable CuAAC Click chemistry with azido- and propargyl groups, respectively, and with DBCO for strain-promoted Click reactions. The cyanopyridines have been introduced as substituted alanines or via 2-cyano-nicotinamides (CINA), which have been attached as amides to side chain amino groups of the building blocks.

→ You want to learn more about 1,2-aminothiols? Read our blog!

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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

Tobacco Etch Virus protease: A shortcut across biotechnologies; F. Cesaratto, O. Burrone, G. Petris; J Biotechnol. 2016; 231(10): 239-249. https://doi.org/10.1016/j.jbiotec.2016.06.012