Functional Diversification of Oyster Big Defensins Generates Antimicrobial Specificity and Synergy against Members of the Microbiota.

Big defensins are two-domain antimicrobial peptides (AMPs) that have highly diversified in mollusks. Cg-BigDefs are expressed by immune cells in the oyster Crassostrea gigas, and their expression is dampened during the Pacific Oyster Mortality Syndrome (POMS), which evolves toward fatal bacteremia. We evaluated whether Cg-BigDefs contribute to the control of oyster-associated microbial communities. Two Cg-BigDefs that are representative of molecular diversity within the peptide family, namely Cg-BigDef1 and Cg-BigDef5, were characterized by gene cloning and synthesized by solid-phase peptide synthesis and native chemical ligation. Synthetic peptides were tested for antibacterial activity against a collection of culturable bacteria belonging to the oyster microbiota, characterized by 16S sequencing and MALDI Biotyping. We first tested the potential of Cg-BigDefs to control the oyster microbiota by injecting synthetic Cg-BigDef1 into oyster tissues and analyzing microbiota dynamics over 24 h by 16S metabarcoding. Cg-BigDef1 induced a significant shift in oyster microbiota ß-diversity after 6 h and 24 h, prompting us to investigate antimicrobial activities in vitro against members of the oyster microbiota. Both Cg-BigDef1 and Cg-BigDef5 were active at a high salt concentration (400 mM NaCl) and showed broad spectra of activity against bacteria associated with C. gigas pathologies. Antimicrobial specificity was observed for both molecules at an intra- and inter-genera level. Remarkably, antimicrobial spectra of Cg-BigDef1 and Cg-BigDef5 were complementary, and peptides acted synergistically. Overall, we found that primary sequence diversification of Cg-BigDefs has generated specificity and synergy and extended the spectrum of activity of this peptide family.

Crystal-to-Crystal Synthesis of Triazole-Linked Pseudo-proteins via Topochemical Azide-Alkyne Cycloaddition Reaction.

Isosteric replacement of amide bond(s) of peptides with surrogate groups is an important strategy for the synthesis of peptidomimetics (pseudo-peptides). Triazole is a well-recognized bio-isostere for peptide bonds, and peptides with one or more triazole units are of great interest for different applications. We have used a catalyst-free and solvent-free method, viz., topochemical azide-alkyne cycloaddition (TAAC) reaction, to synthesize pseudo-proteins with repeating sequences. A designed ß-sheet-forming l-Ala-l-Val dipeptide containing azide and alkyne at its termini (N3-Ala-Val-NHCH2C≡CH, 1) was synthesized. Single-crystal XRD analysis of the dipeptide 1 showed parallel ß-sheet arrangement along the b-direction and head-to-tail arrangement of such ß-sheets along the c-direction. This head-to-tail arrangement along the c-direction places the complementary reacting motifs, viz., azide and alkyne, of adjacent molecules in proximity. The crystals of dipeptide 1, upon heating at 85 °C, underwent crystal-to-crystal polymerization, giving 1,4-triazole-linked pseudo-proteins. This TAAC polymerization was investigated by various time-dependent techniques, such as NMR, IR, DSC, and PXRD. The crystal-to-crystal nature of this transformation was revealed from polarizing microscopy and PXRD experiments, and the regiospecificity of triazole formation was evidenced from various NMR techniques. The MALDI-TOF spectrum showed the presence of pseudo-proteins >7 kDa.

Bio-inspired synthesis of rare and unnatural carbohydrates and cyclitols through strain driven epimerization.

We report a bio-inspired, strain driven epimerization of trans-ketals to cis-ketals through an enolate intermediate. Swern oxidation of a hydroxyl group adjacent to a trans-ketal effects both oxidation and its epimerization to cis-ketal. This novel and general strategy allows inversion of up to three contiguous stereocenters and has been illustrated by the synthesis of several unnatural/rare isomers of carbohydrates/cyclitols from their naturally abundant isomers.