Iminoboronates are efficient intermediates for selective, rapid and reversible N-terminal cysteine functionalisation

Area of Science:

• Chemical Biology
• Organic Chemistry
• Bioconjugation Chemistry

Background:

• Selective functionalization of proteins is crucial for developing targeted therapeutics and diagnostics.
• Existing methods often lack the required chemoselectivity or operate under harsh conditions.
• N-terminal cysteines present a unique and accessible handle for specific protein modification.

Purpose of the Study:

• To develop a novel chemoselective ligation strategy for N-terminal cysteines.
• To investigate the mechanism and scope of the reaction between formyl benzeno boronic acids and N-terminal cysteines.
• To demonstrate the utility of this method for bioconjugation and peptide modification.

Main Methods:

• Reaction of formyl benzeno boronic acids (2FBBA) with N-terminal cysteines under mild aqueous conditions (pH 7.4, 23 °C).
• Density Functional Theory (DFT) calculations to elucidate the reaction mechanism and stereoselectivity.
• Application of the 2FBBA ligation to model peptides (C-ovalbumin, laminin fragment) and a biologically relevant peptide (calcitonin).
• Orthogonal functionalization strategies combining 2FBBA with maleimide chemistry.

Main Results:

• 2FBBA selectively reacts with N-terminal cysteines to form boronated thiazolidines with a B-N bond.
• The reaction proceeds rapidly (2.38 ± 0.23 × 10^2 M^-1 s^-1) and tolerates adjacent amino acid variations.
• DFT calculations confirm diastereoselectivity and highlight the role of the boronic acid in activation and stabilization.
• Stable functionalization of model peptides and calcitonin was achieved, even in the presence of competing thiols.
• Dual and orthogonal modification of calcitonin was demonstrated.

Conclusions:

• Formyl benzeno boronic acids offer a highly chemoselective and efficient method for N-terminal cysteine functionalization.
• The developed ligation strategy is robust, proceeds under mild conditions, and is suitable for complex bioconjugate synthesis.
• This methodology provides a powerful tool for creating well-defined bioconjugates with potential applications in drug delivery and diagnostics.