title: An optimized Fmoc synthesis of human defensin 5.
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.
authors: Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Dr. Aiguader 88, 08003, Barcelona, Spain.
authors: Ermelinda
authors: E
authors: Vernieri
authors: Javier
authors: J
authors: Valle
authors: David
authors: D
authors: Andreu
authors: Beatriz G
authors: BG
authors: de la Torre
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D005449:Fluorenes / Q000737:chemistry*; D006801:Humans; D017510:Protein Folding; D060327:Solid-Phase Synthesis Techniques; D023084:alpha-Defensins / Q000138:chemical synthesis* / Q000302:isolation & purification
chemical_list: D000596:Amino Acids; C500225:DEFA5 protein, human; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D023084:alpha-Defensins