Initially, we evaluated the utility of using metal chlorides for forming thioether linkages between Trp and Cys using the model peptides (Ac-L-Cys(Acm)(O)-R-NH2 (1) and Ac-GALFR-Trp-FG-NH2 (2a)). We employed AcOH as solvent because this solubilizes a variety of peptides (Table , Figures and). The requisite substrate peptide 1 was easily obtainable by oxidation of the precursor peptide (Ac-L-Cys(Acm)-R-NH2) using NaIO4 in MeCN/H2O (Figure ). Incubation of a mixture of 1 (1.5 mM) and 2a (1.0 mM) in AcOH at 25 °C for 15 h in the absence of chloride anion failed to give Cys-Trp-linked peptide 3a with both substrates remaining intact (Table , entry 1, Figure ). Addition of tetra-n-butylammonium chloride ((nBu)4NCl) resulted in the slight formation of the desired 3a in 6% conversion yield (entry 2, Figure ). The use of lithium chloride (LiCl) or calcium chloride (CaCl2) improved the reaction outcome, such that the desired 3a was obtained over in 40% conversion yield (entries 3 and 4, Figures and)). Further improvement resulted from the use of magnesium chloride (MgCl2). The reaction of 1 (1.5 mM) with 2a (1.0 mM) in AcOH in the presence of 30 mM MgCl2 at 25 °C for 15 h proceeded in over 95% conversion (entry 5, Figure ). The use of other solvent systems (AcOH/H2O, hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE)) failed to give 3a (entries 6-8, Figures and)). The use of HFIP led to the formation of MgCl2-precipitated mixtures. The presence of a large volume of water resulted in decomposition of the transiently formed S-chlorocysteine to yield cysteine sulfenic acid (Cys-S-OH), which self-dimerized to give peptides S2 (Figure ). Addition of a small amount of TFA (0.1% TFA in AcOH) accelerated the linking reaction (entry 9, Figures ) and S5(H)) to afford 3a in 60% isolated yield (Figure ). Here, MgCl2 serves as an indispensable Lewis acid, no desired reaction being observed in the absence of MgCl2 (entry 10, Figure ). Replacement of TFA with sodium trifluoroacetate significantly decreased the reaction efficacy (entries 9 vs. 11, Figure ). This indicates that TFA functions as a crucial acidic accelerator for the reaction, although its precise role remains unclear. Further application of the MgCl2mediated Cys-Trp linking reaction to a range of proteins requires widening the available solvent combinations that are appropriate to protein modifications. Although the most ideal solvent for proteins is typically aqueous, the system that we developed doesn't work well under aqueous condi- tions (entry 6). Recently, ionic liquids have become a potential choice as an alternate to water in protein chemistry, and bioconjugation using an ionic liquid (1-butyl-1-methylpyrolidinium trifluoromethanesulfonate (BMPy•OTf)) has been reported. In light of this, we next examined the applicability of MgCl2-mediated Trp sulfenylation under ionic acid conditions. We initially attempted reacting 1 (1.5 mM) with 2a (1.0 mM) in BMPy•OTf in the presence of 30 mM MgCl2 and 0.1% TFA at 50 °C. However, the reaction mixture became high viscous and achieving complete dissolution of the materials was highly time-consuming. None-the-less, we were able to obtain the desired product 3a in moderate yield after reacting for 12 h (entry 12, Figure ).