Methionine-Containing Peptides: Avoiding Secondary Reactions in the Final Global Deprotection

Abstract

The solid-phase synthesis of Met-containing peptides using a fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) protection scheme is inevitably accompanied by two stubborn side reactions, namely, oxidation and S-alkylation (tert-butylation), which result in the formation of Met(O) and sulfonium salt impurities of the target peptide, respectively. These two reactions are acid-catalyzed, and they occur during the final trifluoroacetic (TFA)-based acidolytic cleavage step. Herein, we developed two new cleavage solutions that eradicate the oxidation and reduce S-alkylation. TFA-anisole-trimethylsilyl chloride (TMSCl)-Me 2 Striisopropylsilane (TIS) containing 1 mg of triphenyl phosphine per mL of solution was the optimal mixture for Cys-containing peptides, while for the remaining peptides, TIS was not required. Both cleavage solutions proved to be excellent when sensitive amino acids such as Cys and Trp were involved. TMSCl did not affect either of these sensitive amino acids. Reversing the sulfonium salt to free Met-containing peptide was achieved by heating the peptide at 40 °C for 24 h using 5% acetic acid.

Advancing Sustainable Peptide Synthesis by Solvent Quality Control: Understanding and Mitigating Hydroperoxide in 1-Butyl-2-pyrrolidinone (NBP)

“…On the other hand, solutions of Met in DMF, NFM, and DMSO gave minimal Met oxidation (entries 5–7), and the fact that these solvents were opened for >2 months suggested that they were not as prone to R-OOH formation on storage as NBP and NMP. Finally, as Met oxidation during TFA cleavage is a relatively facile process which has to be suppressed by suitable scavengers, ,, we assessed Met oxidation in TFA (entry 8), and as anticipated, an appreciable amount of Met(O) was formed. Nevertheless, the extent of the oxidation was far less than for the R-OOH-contaminated NBP and NMP (entries 1 and 4), suggesting that oxidation during SPPS, rather than during cleavage, might be a bigger concern when pyrrolidinone solvents such as NMP or NBP are used in peptide synthesis.…”

“…While we previously reported that oxidation-sensitive amino acids such as Cys and Trp undergo N -oxyl radical-caused degradation in DMF, ,, we did not observe appreciable Met oxidation under these conditions. In fact, while preventing Met oxidation during peptide resin cleavages has been thoroughly investigated, Met oxidation during peptide synthesis has been less studied . In fact, in some cases Met oxidation during synthesis can be so cumbersome, that a less oxidation-prone AA instead of Met is used, and we thus set out to investigate the impact of hydroperoxides in NBP, as well as other SPPS solvents, on the oxidation of Met and other oxidizable AAs and peptides.…”

Modulators of the Hop-HSP90 Protein-Protein Interaction Disrupt Early-Stage of KSHV Lytic Replication

“…Finally, it was interesting to note that replacement of the V-4 residue of peptide 1 with proline (17) had a significantly detrimental effect on PPI disruption activity, while the proline analogue of 4 (18) 52 was also inactive. Peptides 1 -3, all of which were tetrazole-containing analogues of MEEVD, which displayed the most promising Hop TPR2A -HSP90 CTD PPI disruption, were then assessed for their ability to disrupt the fulllength Hop-HSP90 PPI, again using an ELISA based solid phase assay (Figure 4A).…”

Incorporation of a Highly Reactive Oxalyl Thioester-Based Interacting Handle into Proteins

“…The desired product was accompanied by numerous byproducts, including substantial amounts of free-thiol intermediates. We then came across a recent report by Yang et al, who established that TIS can reduce Cys disulfide bonds formed on the solid phase during the cleavage and deprotection step in TFA. , We therefore substituted hydrosilane with thioanisole. Interestingly, doing so resulted in the clean formation of the target oxo SEA peptide 6a , albeit with slow kinetics (Table , entry 2).…”