Characterisation of the structure and self-assembly of a small cyclic peptide: an analysis using NMR spectroscopy, diffusion and heteronuclear relaxation measurements

Pseudodesmin A, a member of the viscosin group, is a cyclic lipodepsipeptide (CLP) consisting out of an oligopeptide that is cyclised through a lactone bond between its C-terminal carboxyl group and the hydroxyl group of a threonine side chain and a 3-hydroxydecanoic acid moiety bonded to the N-terminal end of the peptide. It is found to self-assemble in apolar organic solvents, which is reminiscent of its expected ability of forming ion pores in cellular membranes. The goal of this dissertation is to investigate the self-assembly in organic solvents and the structure of the assemblies formed mainly by translational diffusion and heteronuclear relaxation NMR measurements. After covering some theory and background concerning diffusion, translational diffusion NMR, NMR relaxation, and CLPs, the elucidation of the pseudodesmin A conformation – by both X-ray diffraction and NMR – will be discussed. Next, the self-assembly is studied by translational diffusion measurements at different concentrations in chloroform and acetonitrile/chloroform mixtures. Using these results, a model is proposed for the supramolecular assembly that explains the selective self-assembly in non-polar environment, the limitless nature of the assembly and the biological function of the ion pore. This model, confirmed by the detection of intermolecular rOe contacts, encompasses a side by side aggregation of the amphipathic monomer alpha-helical units followed by a stacking of these aggregates – along a direction closely parallel to the alpha-helix – to cylindrically shaped structures. Heteronuclear 13C-alpha relaxation is then used to gain more insight in the organisation of the supramolecular structure and to confirm some aspects of the proposed model. The dependence of the 13C-alpha R1 and R2 relaxation rate constants on the 13C-1H bond vector orientation within a molecular structure is used to obtain information concerning the rotational diffusion properties of the assembly and the orientation of the monomer within the supramolecular assembly. This is used to acquire the shape and average dimensions of the supramolecular assembly, which confirm the model of cylindrical assemblies that grow in only one dimension, nearly parallel with the helix structure. Finally, a theoretical simulation is performed to assess the impact of the self-association equilibria on the relaxation rate constants

Synthesis of 5-substituted 2'-deoxyuridine-5'-phosphonate analogues and evaluation of their antiviral activity

A small series of 5-(hetero)aryl-modified nucleoside phosphonates was synthesized via an 8-step procedure including a Wittig reaction and Suzuki-Miyaura coupling. An unanticipated anomerization during phosphonate deprotection allowed us to isolate both anomers of the 5-substituted 2'-deoxy-uridine phosphonates and assess their antiviral activity against a broad panel of viruses

Aminophosphines : a double role in the synthesis of colloidal indium phosphide quantum dots

Aminophosphines have recently emerged as economical, easy-to-implement precursors for making InP nanocrystals, which stand out as alternative Cd-free quantum dots for optoelectronic applications. Here, we present a complete investigation of the chemical reactions leading to InP formation starting from InCl3 and tris(dialkylamino)phosphines. Using nuclear magnetic resonance (NMR) spectroscopy and single crystal X-ray diffraction, we demonstrate that injection of the aminophosphine in the reaction mixture is followed by a transamination with oleylamine, the solvent of the reaction. In addition, mass spectrometry and NMR indicate that the formation of InP concurs with that of tetra(oleylamino)phosphonium chloride. The chemical yield of the InP formation agrees with this 4 P(+III) -> P(-III) + 3 P(+V) disproportionation reaction occurring, since full conversion of the In precursor was only attained for a 4:1 P/In ratio. Hence it underlines the double role, of the aminophosphine as both precursor and reducing agent. These new insights will guide further optimization of high quality InP quantum dots and might lead to the extension of synthetic protocols toward other pnictide nanocrystals

Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species

Orfamide type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudornonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudornonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens

Molecular model for the self-assembly of the cyclic lipodepsipeptide pseudodesmin A

Self-assembly of peptides into supramolecular structures represents an active field of research with potential applications ranging from material science to medicine. Their study typically involves the application of a large toolbox of spectroscopic and imaging techniques. However, quite often, the structural aspects remain underexposed. Besides, molecular modeling of the self-assembly process is usually difficult to handle, since a vast conformational space has to be sampled. Here, we have used an approach that combines short molecular dynamics simulations for peptide dimerization and NMR restraints to build a model of the supramolecular structure from the dimeric units. Experimental NMR data notably provide crucial information about the conformation of the monomeric units, the supramolecular assembly dimensions, and the orientation of the individual peptides within the assembly. This in silico/in vitro mixed approach enables us to define accurate atomistic models of supramolecular structures of the bacterial cyclic lipodepsipeptide pseudodesmin A