Alternative chemistries for the synthesis of thrombospondin‐1 type 1 repeats

Abstract

Synthetic protein engineering has benefited from the development of diverse methods for the synthesis of functionalized peptide fragments and approaches for their subsequent assembly into full length polypeptides. Here we describe a series of synthetic approaches for the total chemical synthesis of the second type 1 repeat of thrombospondin‐1 (TSR2) that vary in both the location of the ligation site and α‐amine protecting group strategy (Boc/Fmoc) used for the synthesis of the associated peptide fragments. These syntheses illustrate that challenging peptide sequences can result from the protecting group strategy as well as from sequence‐dependent factors. Importantly, we find that such challenges can be overcome by altering the chemistry used for solid phase peptide synthesis, the choice of ligation site, and the resin used as a solid support. From these studies, we have developed a robust synthetic route to the TSR2 polypeptide consisting of native chemical ligation between an N‐terminal fragment synthesized by Boc‐SPPS and a C‐terminal fragment synthesized by Fmoc‐SPPS. Finally, the folded TSR2 domain is obtained following an optimized oxidative folding protocol using an excess of oxidized glutathione. This optimized synthesis will enable the use of unnatural amino acids to probe the unusual structure and anti‐angiogenic activity of this protein domain. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 405–413, 2010.

9‐Fluorenylmethoxycarbonyl‐Based Solid‐Phase Synthesis of Peptide α‐Thioesters

“…The synthesis of a 29-residue peptide-benzimidazolone succeeded in 36 % yield. The usefulness of the approach has been demonstrated in the total chemical synthesis of HIV-1 Tat protein, [52] the second type 1 repeat of thrombospondin-1 (TSR2), [53] and in the semisynthesis of the N-terminal domain of the anthrax lethal factor. [54]…”

A Reversible Protection Strategy To Improve Fmoc‐SPPS of Peptide Thioesters by the N‐Acylurea Approach

“…In order to eliminate the possibility that resin effects alone might modulate reactivity to account for the discrepancy between our results and other reports, [11] syntheses of the H4C and H4N peptides were carried out on Rink substituted ChemMatrix and PAL-PEG-PS resins respectively; branched products were still generated (Supplemental Figure S2). These results suggest that resin effects can modulate but not eliminate reactivity of the second Dbz amine under peptide synthesis conditions.…”

“…[5] This deactivation has been reported to be sufficient to minimize acylation at the second amine during chain extension, although extraneous acetylation during capping steps has been reported by multiple researchers and some over-acylation has been observed during coupling cycles. [10–11] Peptides H4C and H4N are extremely glycine-rich, and each has a C-terminal Gly-Gly sequence. These sequences present a particular challenge to the combination of steric and electronic factors that modulate reactivity at the second amine of Dbz during chain extension.…”

“…2): the desired species, addition of a second chain by acylation during Gly coupling (A 1 : 9%; F 1 : 6% by integration of RP-HPLC chromatogram) and the acetylated species (A A : 12%; F A 7%). It should be noted that optimized capping conditions have been reported to minimize acetylation at the unprotected Dbz amine [11] in comparison to standard capping cocktails. However, even minimal non-productive acetylation product would accumulate at each step of a long peptide synthesis.…”

9‐Fluorenylmethyloxycarbonyl‐basierte Festphasensynthese von α‐Peptidthioestern

“…Die Synthese eines 29-meren Peptidbenzimidazolons gelang in 36 % Ausbeute. Der Nutzen dieser Methode wurde bei der chemischen Totalsynthese des HIV-1-Tat-Proteins [52] und der zweiten Typ-1-Wiederholung von Thrombospondin-1 (TSR2) [53] sowie bei der Semisynthese der N-terminalen Domäne des Anthrax-Lethalfaktors [54]…”