To further explore the optimised reaction, we considered buffer composition, the phosphine additive, and alternative desulfurisation conditions. Cyclisation of model 7a in PBS did not proceed cleanly (Figure ); while employing HEPES buffer did lead to clean conversion of the starting peptide, the undesired desulfurised linear by-product was the dominant product (Figure ). Thus, cyclisation in conjugation buffer containing a high concentration of chaotropic salt is more effective. Replacing TCEP with the water soluble phosphine, 3,3',3''-phosphanetriyltris(benzenesulfonic acid) trisodium salt (TPPTS) did not lead to effective conversion of the starting peptide 7a (Figure ). Employing 1,3,5-triaza-7phosphaadamantane (PTA) appeared to eliminate the production of the undesired linear desulfurised by-product (Figure ). However, repetition of the reaction on an isolable scale and purification by preparative HPLC revealed the byproduct and other impurities eluting under the broader product peak (Figure ) which ultimately limited the isolated yield. Finally, the peptide H-(alG)AFAC-OH (7b) was synthesised on 2-CTC resin to probe the cyclisation on peptides bearing a Cterminal carboxylic acid. While conversion to the desired product was observed (Figure ), the ratio of by-product to cyclised was less favourable when compared to C-terminal amide peptides. In addition, for this simple model, the retention times for the desired product and by-product were very similar making separation a significant challenge.