Light-Mediated Desulfurization of Peptides in Multi-Well Format for Combinatorial Library Synthesis

Native chemical ligation (NCL) combined with post-ligation desulfurization is a powerful strategy for the chemical joining of unprotected peptide segments to form larger polypeptides or to synthesize head-to-tail cyclized peptides. However, a simple, robust, and safe desulfurization protocol suitable for high-throughput synthesis workflows has yet to be developed. Here, we present a photochemical strategy in which desulfurization is achieved using 270 nm ultraviolet (UV) irradiation in the presence of tris(2-carboxyethyl)phosphine (TCEP). Optimization of reaction conditions resulted in reaction efficiency comparable with current chemical desulfurization methods. Desulfurization of a combinatorial peptide library confirmed that the photochemical desulfurization strategy is compatible with all canonical amino acids as well as thiol-protected cysteines. Implementation of the optimized conditions in a 96-well plate format using a custom-built light-emitting diode (LED) array confirmed that product formation is dependent on local light exposure. Increasing the distance between the plate and the light source enabled more uniform irradiation, giving homogenous product formation. Altogether, this study establishes UV-mediated desulfurization as a safe and scalable alternative for high-throughput peptide synthesis workflows.