3 research outputs found
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><sub>max</sub> 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