Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary.

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet(7)]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the alpha-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximately 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Engineering of a synthetic receptor to alter peptide binding selectivity.

BACKGROUND: Molecular recognition processes are ubiquitous in nature: substrate binding by enzymes, antigen recognition by antibodies, and hormone activation of receptors provide three classic examples. To better understand these large systems it is valuable to study smaller, well defined host molecules. Previously we found sequence-selective peptide binding with a class of C3 symmetric synthetic receptors. In this work we rationally altered that host structure in order to produce a corresponding change in binding selectivity. RESULTS: A novel C3 symmetric receptor was designed and synthesized such that, unlike previous host molecules, it contained hydrogen-bond accepting functionality within the binding cavity. Screening of this host against a combinatorial tripeptide library revealed an exquisite selectivity for sequences with D-Pro-D-Asn carboxyl termini. Computer simulations and NMR studies indicate that hydrogen bonding of the D-Asn side-chain amide to the amine functionality within the cavity is responsible for this selectivity. CONCLUSIONS: Computer-aided design and combinatorial library screening methods combine to provide a powerful approach to induce and evaluate the results of rational changes in the molecular recognition properties of molecules. Using this approach, we modified the binding properties of a class of molecules to select for hydrogen-bonding residues instead of hydrophobic residues and concomitantly increased the overall sequences selectivity. Structural studies indicate that these changes indeed result from the type of binding mode proposed as part of the initial design. This approach can increase our understanding of molecular recognition processes, and should allow the rational design of larger, more selective systems.