Accelerated chemical synthesis of peptides and small proteins.
The chemical synthesis of peptides and small proteins is a powerful complementary strategy to recombinant protein overexpression and is widely used in structural biology, immunology, protein engineering, and biomedical research. Despite considerable improvements in the fidelity of peptide chain assembly, side-chain protection, and postsynthesis analysis, a limiting factor in accessing polypeptides containing greater than 50 residues remains the time taken for chain assembly. The ultimate goal of this work is to establish highly efficient chemical procedures that achieve chain-assembly rates of approximately 10-15 residues per hour, thus underpinning the rapid chemical synthesis of long polypeptides and proteins, including cytokines, growth factors, protein domains, and small enzymes. Here we report Boc chemistry that employs O-(7-azabenzotriazol-1-yl)-N,N, N',N'-tetramethyluronium hexafluorophosphate (HATU)/dimethyl sulfoxide in situ neutralization as the coupling agent and incorporates a protected amino acid residue every 5 min to produce peptides of good quality. This rapid coupling chemistry was successfully demonstrated by synthesizing several small to medium peptides, including the "difficult" C-terminal sequence of HIV-1 proteinase (residues 81-99); fragment 65-74 of the acyl carrier protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor antagonist; and the pro-inflammatory chemotactic protein CP10, an 88-residue protein, by means of native chemical ligation. The benefits of this approach include enhanced ability to identify and characterize "difficult couplings," rapid access to peptides for biological and structure-activity studies, and accelerated synthesis of tailored large peptide segments (<50 residues) for use in chemoselective ligation methods.
Miranda
L P
LP
Centre for Drug Design & Development, The University of Queensland, Brisbane, Queensland 4072, Australia.
Alewood
P F
PF
D000595:Amino Acid Sequence; D020916:Conotoxins; D004121:Dimethyl Sulfoxide; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000138:chemical synthesis; D009842:Oligopeptides / Q000138:chemical synthesis; D010455:Peptides / Q000138:chemical synthesis*; D011506:Proteins / Q000138:chemical synthesis*; D014230:Triazoles; D014508:Urea / Q000031:analogs & derivatives