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Amino Acids. 2014 Feb;46(2):395-400. doi: 10.1007/s00726-013-1629-3. Epub 2013 Dec 6.

An optimized Fmoc synthesis of human defensin 5.

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  • 1Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Dr. Aiguader 88, 08003, Barcelona, Spain.

Abstract

Human α-defensin 5 (DEF5), expressed by the Paneth cells of human small intestine, plays an important role in host defense against microbial infections. DEF5, a 32-residue peptide adopting a three-stranded β-sheet fold stabilized by three internal disulfide bonds, is not efficiently produced by recombinant expression techniques and is, therefore, an interesting goal for chemical synthesis. While DEF5 production by Boc-based solid-phase synthesis has been described, to date no synthetic account by the more convenient Fmoc method has been published. Herein, we report an optimized solid-phase synthesis of DEF5 using the Fmoc strategy. Starting from a rather problematic initial synthesis using standard Wang resin and coupling protocols, the sequence elongation process has been monitored by mini-cleavage and MS analysis at strategic points, to identify problematic spots and act accordingly. For expediency, some of the optimization rounds have been run on defensin 5 amide. Main modifications have included the ChemMatrix(®) resin, known to decrease chain aggregation, and the use of pseudoproline dipeptide units at selected positions. Combination of some of these improvements results in a significantly purer product, to the extent that it can undergo in situ anaerobic oxidative folding to the native form without the need of an intermediate purification step. A typical synthesis run yielded about 15 mg of >95 % pure material. This approach should facilitate production of DEF5 and of selected analogs for structure-activity studies and other applications.

PMID:
24311274
[PubMed - indexed for MEDLINE]
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