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Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

Biopolymers (2015-02-07)
Paul W R Harris, Margaret A Brimble
ABSTRACT

The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanidine hydrochloride solution
Sigma-Aldrich
HATU, ≥98.0% (CHN)
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Fmoc-Arg(Pbf)-OH, ≥98.0% (HPLC)
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Fmoc-His(Trt)-OH, ≥98.0% (sum of enantiomers, HPLC)
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1-Hydroxybenzotriazole hydrate, ≥97.0% (T)
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Fmoc-Asn(Trt)-OH, ≥97.0%
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Tris(2-carboxyethyl)phosphine hydrochloride, powder
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N,N-Dimethylformamide, for molecular biology, ≥99%
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HATU, 97%
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N,N-Dimethylformamide, anhydrous, 99.8%
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Tris(2-carboxyethyl)phosphine hydrochloride, BioUltra, ≥98% (NMR)
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4-Mercaptophenylacetic acid, 97%
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Tris(2-carboxyethyl)phosphine hydrochloride, BioUltra, suitable for electrophoresis, SDS-PAGE tested
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1-Hydroxybenzotriazole hydrate, wetted with not less than 20 wt. % water, 97%
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1-Hydroxybenzotriazole hydrate, wetted with not less than 14 wt. % water, 98%
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Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
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Guanidine hydrochloride, for molecular biology, ≥99%
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Guanidine hydrochloride, ≥99% (titration), organic base and chaeotropic agent
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HBTU, ≥98.0% (T)
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Guanidine hydrochloride, BioUltra, for molecular biology, ≥99.5% (AT)
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Fmoc-Gln(Trt)-OH, ≥98.0% (HPLC)
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Fmoc-Lys(Boc)-OH, ≥98.0% (HPLC)
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Guanidine hydrochloride, ≥99.0% (AT)
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Fmoc-Glu(OtBu)-OH, ≥98.0% (HPLC)
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Guanidine hydrochloride, ≥98%
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Fmoc-Trp(Boc)-OH, ≥97.0% (HPLC)
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Acetonitrile, for DNA synthesis
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Ultrapure Acetonitrile, for DNA synthesis
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Triisopropylsilane, 98%