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

Abstract

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet 7 ]cyt b562, by thioestermediated 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 ␣-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 fulllength 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 Ϸ45 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. Because of its unmatched flexibility, total chemical synthesis is emerging as a powerful tool for protein engineering (1, 2). Chemical access to proteins inherently provides the ability to incorporate unnatural amino acids into proteins in a completely general fashion and has opened many new avenues for the study of protein function. The large number and variety of unnatural amino acids available enables researchers to systematically probe size, shape, acidity, hydrogen bonding, and electronic structure effects on the reactivity properties of the protein molecule. Studies of bioinorganic systems in particular can benefit greatly from the ability to place unnatural metal-chelating residues into metalloprotein structures in a site-specific manner.Chemical ligation reactions (3) have been used in the total synthesis of myriad engineered protein targets with unusual backbone and side chain groups. Several ligation chemistries have been used (4-7). In particular, the native chemical ligation (NCL) reaction (8) has enabled the synthesis of many wild-type and engineered protein targets by allowing full-length polypeptide sequences to be built by linking readily obtained smaller unprotected peptide segments with native amide bonds. In the original NCL scheme (9), a thioester-containing peptide reacts in a chemoselective manner with a second peptide that has a cysteine as its N-terminal residue. The side chain thiol of that cysteine undergoes thiol exchange with the thioester moiety of the first peptide. A thioester-linked intermediate is formed, whic...

DiGeorge Critical Region 8 (DGCR8) Is a Double-cysteine-ligated Heme Protein

“…7B). In contrast, previous studies showed that substitution of a methionine ligand with SeMet caused the Soret ␣ and ␤ peaks to red-shift on the range of 3-7 nm (35,36). The signals in the MCD and EPR spectra of SeMetlabeled HBD-His 6 were also unchanged from those of the native Met-containing protein (Fig.…”

Der gewundene Pfad zum Erythropoietin entlang der Grenze von Chemie und Biologie

“…Die Kentsche NCL-Strategie zur Verknüpfung einzelner Glycopeptidfragmente bençtigt einen Cysteinrest an jeder Ligationsstelle; dort werden die Peptide für die Kupplung aktiviert. Wie viele natürlich vorkommende Proteine und Glycoproteine verfügt EPO über [31] und Dawson [32] bahnbrechende Beiträge zur Entwicklung abspaltbarer Thiolbasierter Auxiliare für die NCL an cysteinfreien Stellen geleistet hatten.…”

Chemoselektive Ligations‐ und Modifikationsstrategien für Peptide und Proteine

“…[140] Kürzlich haben Danishefsky et al das Tmb-Auxiliar in Verbindung mit einem C-terminalen Peptidphenolester 93 mit einem ortho-Disulfid (siehe Abschnitt 2.3.3) in sterisch anspruchsvollen Kupplungen zweier Glycopeptide 93 und 94, die jeweils eine N-verbrückte Chitobiose-Einheit trugen, an einer Gly-Gln-Ligationsstelle eingesetzt. [141] Hierbei wurde beobachtet, dass saure Bedingungen für die Auxiliarabspaltung zu einer Anreicherung des Thioester-Intermediats 95 durch Protonierung des sekundären Amins führen, was auf eine N!S-Verschiebung schließen lässt.…”