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.

Perspectives for clinical use of engineered human host defense antimicrobial peptides

“…described a new methodology based on the use of the fluoromethyloxycarbonyl chloride (Fmoc) strategy for an optimized solid-phase synthesis of HD5 (Vernieri et al. 2014). As a result, they obtained a correctly folded HD5, highly pure (> 95%) and with an overall yield of 15 mg per run.…”

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

“…These include the elegant work reported from the laboratories of Craik and Andreu. 103,[190][191][192][193][194][195][196][197] For example, the formation of disulfide bonds by employing thioester-mediated strategy or oester-mediated strategy has been reported. [191][192][193][194]198 Similarly the syntheses of knottin peptide thionin using stepwise chemoselective disulfide bond formation and synthesis of human defensins 5 with the use of pseudoproline dipeptide units at selected points have been reported by Andreu and colleagues.…”

“…[191][192][193][194]198 Similarly the syntheses of knottin peptide thionin using stepwise chemoselective disulfide bond formation and synthesis of human defensins 5 with the use of pseudoproline dipeptide units at selected points have been reported by Andreu and colleagues. 197,199 Also, formation of multiple intra-molecular disulfide bonds by using cysteine residues with different side chain-protecting groups opens the opportunity to overcome the formation of undesired bridges and polymerised side products as a result of simultaneous oxidation of all thiol groups. [200][201][202] On the other hand, switching to other forms of cyclic bonds such as lactamisation, olefin metathesis, ruthenium-catalysed ring closure metathesis, Cu(I)-catalysed azide-alkyne cycloaddition, and thioether formation would also be beneficial for the therapeutic use of these peptides.…”

Oligomerization of neutral peptides derived from the JC virus agnoprotein through a cysteine residue

“…We also synthesized an Ala mutant at Cys40 of peptide 2 to investigate the role of the thiol group. We used ChemMatrix ® resin (Vernieri et al 2014) for obtaining the crude products with improved purities. All peptides were purified using preparative HPLC and identified by MS analysis (Supporting Information, Figure S1).…”