Peptide Synthesis - Protecting Groups - N-terminal Protecting Groups - t-Boc Protecting Group

t-Boc Protecting Group

The original method for the synthesis of proteins relied on (tert-butyloxycarbonyl or more simply "Boc") to temporarily protect the α-amino group. In this method, the Boc group is covalently bound to the amino group to suppress its nucleophilicity. The C-terminal amino acid is covalently linked to the resin through a linker. Next, the Boc group is removed with acid, such as trifluoroacetic acid (TFA). This forms a positively-charged amino group (in the presence of excess TFA; note image on the right illustrates neutral amino group), which is neutralized (via in-situ or non in-situ methods) and coupled to the incoming activated amino acid. Reactions are driven to completion by the use of excess (two- to four-fold) activated amino acid. After each deprotection and coupling step, a wash with N,N-dimethylformamide (DMF) is performed to remove excess reagents, allowing for high yields (~99%) during each cycle.

t-Boc protecting strategies retain usefulness in reducing peptide aggregation during synthesis. t-Boc groups can be added to amino acids with t-Boc anhydride and a suitable base. Some researchers prefer Boc SPPS for complex syntheses . In addition, when synthesizing nonnatural peptide analogs, which are base-sensitive (such as depsipeptides), the t-Boc protecting group is necessary, because Fmoc SPPS uses a base to deprotect the α-amino group.

Permanent side chain protecting groups are typically benzyl or benzyl-based groups. Final removal of the peptide from the linkage occurs simultaneously with side-chain deprotection with anhydrous hydrogen fluoride via hydrolytic cleavage. The final product is a fluoride salt which is relatively easy to solubilize. Importantly, scavengers such as cresol are added to the HF in order to prevent reactive t-butyl cations from generating undesired products. In fact, the use of harsh hydrogen fluoride may degrade some peptides, which was the premise for the development of a milder, base-labile method of SPPS—namely, the Fmoc method.