Regio- and chemoselective conjugations to peptides and proteins frequently are challenging, as the reagents must be compatible with functional groups, the coupling conditions should be mild and physiological, and the reaction ideally may be performed in aqueous media. Furthermore, yields should be high. Selective modifications are important, because unselective labeling creates heterogenous products, complicates cleanup, reduces yields and thus increases waste and costs.

The selective modification of the N-terminus (i.e., an alpha-amino group) is frequently required when, e.g., payloads are attached to antibodies to generate ADCs, or proteins need to be precisely labeled, e.g., with fluorescent dyes or biotin. Besides, the N-terminus is an attractive site for modification as it represents a unique position in proteins and is typically not involved in folding. Classical reagents for the conjugation of molecules to amines are NHS and TFP esters, iso(thio)cyanates and the reductive amination with aldehydes. However, all of them react rather unspecific.

Herein, we introduce a novel reagent for the specific N-terminal labeling of proteins and peptides: 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde (1-NP-triazole-4-CHO) (RL-8650).

This nitrophenol-functionalized triazole aldehyde reacts with primary amines under mild conditions and high conversion rates with reported yields of 90% and more. This allows to introduce labels, e.g., for imaging. The coupling reaction may be performed in aqueous solution buffered with 10 mM potassium phosphate or MOPS at pH 7.5, by adding the aldehyde dissolved in DMF and incubating for 4-16 hours. Work-up may be performed by chromatography.

Activation of a primary amine (e.g., carrying a label = green star) with 1-NP-triazole-4-carbaldehyde. The amine binds to the aldehyde, forming an imine, which undergoes a rearrangement thus integrating the amine nitrogen as part of the triazole. Then, p-nitrophenylaniline is eliminated and an activated aldehyde triazole is formed which then can be coupled to the N-terminus of a peptide.

With the success of Click Chemistry the triazole moiety gained more and more presence in drug design and discovery. Several triazole derivatives with interesting biological activities are already reported in literature making it an attractive structural motive for peptide modification.

The activated triazole aldehyde – bearing the desired label (illustrated as green star in the scheme below) – can be specifically conjugated to the N-terminus of a peptide forming a N-terminal 4-imidazolinone. The reaction may be performed overnight, in an aqueous buffer at physiological pH, e.g., 10 mM phosphate buffer, at 37 °C.

Coupling of the aldehyde-functionalized triazole reagent to the N-terminus of a peptide forming a imidazolinone.

In addition, our portfolio comprises 4-(diethoxymethyl)-1-nitrophenyl-triazole (RL-8655), a derivative of 1-NP-triazole-4-CHO (RL-8650) in which the aldehyde function is protected as acetal. This building block may serve as negative control or as starting material for derivatives where the aldehyde needs to be protected. The acetal can be deprotected under mild acidic conditions.

Chemical structure of 4-(diethoxymethyl)-1-nitrophenyl-triazole (RL-8655).

References:

Tandem synthesis of 1-formyl-1,2,3-triazoles; J. T. Fletcher, J. A. Christensen, E. M. Villa; Tetrahedron Lett. 2017; 58(47): 4450-4454. https://doi.org/10.1016/j.tetlet.2017.10.023

Synthesis and antibiotic activity of a small molecules library of 1,2,3-triazole derivatives; M. Aufort, J. Herscovici, P. Bouhours, N. Moreau, C. Girard; Bioorg Med Chem Lett. 2008; 18(3): 1195-1198. https://doi.org/10.1016/j.bmcl.2007.11.111

Triazolecarbaldehyde reagents for one-step N-terminal protein modification, A. Onoda, N. Inouse, E. Sumiyoshi, T. Hayashi; ChemBioChem. 2019; 21(9): 1274-1278. https://doi.org/10.1002/cbic.201900692