An Iodine-Free and Directed-Disulfide-Bond-Forming Route to Insulin Analogues

An iodine-free synthetic route to insulin analogues has been established via a directed disulfide bond formation strategy. This method is completely compatible with oxidation-sensitive residues. The key step is constructing the third disulfide bond via a novel procedure involving phenyl­acetyl­amino­methyl group (Phacm), immobilized Penicillin G Acylase, and Ellman’s reagent. We expect that this method could be broadly utilized for synthesizing insulin-like and other cysteine-rich peptides, in particular, where oxidation-sensitive residues are present in the sequence

Irreversible Sortase A‑Mediated Ligation Driven by Diketopiperazine Formation

Sortase A (SrtA)-mediated ligation has emerged as an attractive tool in bioorganic chemistry attributing to the remarkable specificity of the ligation reaction and the physiological reaction conditions. However, the reversible nature of this reaction limits the efficiency of the ligation reaction and has become a significant constraint to its more widespread use. We report herein a novel set of SrtA substrates (LPETGG-isoacyl-Ser and LPETGG-isoacyl-Hse) that can be irreversibly ligated to N-terminal Gly-containing moieties via the deactivation of the SrtA-excised peptide fragment through diketopiperazine (DKP) formation. The convenience of the synthetic procedure and the stability of the substrates in the ligation buffer suggest that both LPETGG-isoacyl-Ser and LPETGG-isoacyl-Hse are valuable alternatives to existing irreversible SrtA substrate sequences

Atrial Natriuretic Peptide-Fc, ANP-Fc, Fusion Proteins: Semisynthesis, In Vitro Activity and Pharmacokinetics in Rats

Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short <i>in vivo</i> half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semisynthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied among 2, 11, or 16 amino acids. In addition, either one (“monomeric”) or two (“dimeric”) ANP peptides were linked to Fc to study whether this modification had an effect on <i>in vitro</i> activity and/or <i>in vivo</i> half-life. The various constructs were studied for <i>in vitro</i> activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16- and ∼375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc’s were found to be approximately 2 orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8–5.5 h), but a trend was observed where the <i>C</i>_max of the monomeric constructs was approximately 3-fold higher than that of the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases