Extending the Applicability of Native Chemical Ligation

Canne, Lynne E.; Bark, Steven J.; Kent, Stephen B. H.

doi:10.1021/ja960398s

Cited by 275 publications

(199 citation statements)

References 28 publications

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“…The auxiliary must include a thiol group suitably disposed to mimic the side chain of the Cys residue that is crucial to thioester-mediated NCL reactions (10), hence the use of the N ␣ -(2-mercaptoethyl) moiety as the core building block in the auxiliary. Further, the auxiliary must be stable to conditions of peptide synthesis but removable after the ligation under conditions that do not damage the newly formed polypeptide product.…”

Section: Methodsmentioning

confidence: 99%

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

Low

Hill

Carrasco

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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157

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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...

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Section: Methodsmentioning

confidence: 99%

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

Low

Hill

Carrasco

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

157

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“…A prototype procedure for the use of an auxiliary-functional-group approach to native amide-forming, thioester-mediated chemical ligation has been reported (121). This work defined the principles of an effective approach to ligation at non-Cys residues, but the chemistry used had to be refined and extended because severe shortcomings were observed in the original investigation, as revealed by studies in model systems (121). In this respect, recently reported work from the Dawson laboratory at The Scripps Research Institute may represent a more effective chemistry for ligation at residues other than Cys (121a), using the same auxiliary-functional-group approach.…”

Section: Future Developments Ligation Sitesmentioning

confidence: 99%

Synthesis of Native Proteins by Chemical Ligation

Dawson

Kent²

2000

Annu. Rev. Biochem.

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1,587

1,526

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Key Words chemical protein synthesis, thioester, protein, peptide, solid phase synthesis, polymer-supported synthesis, protein engineering s Abstract In just a few short years, the chemical ligation of unprotected peptide segments in aqueous solution has established itself as the most practical method for the total synthesis of native proteins. A wide range of proteins has been prepared. These synthetic molecules have led to the elucidation of gene function, to the discovery of novel biology, and to the determination of new three-dimensional protein structures by both NMR and X-ray crystallography. The facile access to novel analogs provided by chemical protein synthesis has led to original insights into the molecular basis of protein function in a number of systems. Chemical protein synthesis has also enabled the systematic development of proteins with enhanced potency and specificity as candidate therapeutic agents.

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“…82 Ligations were carried out in either 8 M urea/0.1 M Na 2 HPO 4 at pH 7.0 or in 6 M Gnd$HCl/0.1 M Na 2 HPO 4 at pH 7.5. Appreciable yields were obtained in each study.…”

Section: 81mentioning

confidence: 99%