Jean-Christophe Rossi

An Expeditious Multigram-Scale Synthesis of Lysine Dendrigraft (DGL) Polymers by Aqueous N-Carboxyanhydride Polycondensation

The synthesis and characterisation of new arborescent architectures of poly(L-lysine), called lysine dendrigraft (DGL) polymers, are described. DGL polymers were prepared through a multiple-generation scheme (up to generation 5) in a weakly acidic aqueous medium by polycondensing N(epsilon)-trifluoroacetyl-L-lysine-N-carboxyanhydride (Lys(Tfa)-NCA) onto the previous generation G(n-1) of DGL, which was used as a macroinitiator. The first generation employed spontaneous NCA polycondensation in water without a macroinitiator; this afforded low-molecular-weight, linear poly(L-lysine) G1 with a polymerisation degree of 8 and a polydispersity index of 1.2. The spontaneous precipitation of the growing N(epsilon)-Tfa-protected polymer (GnP) ensures moderate control of the molecular weight (with unimodal distribution) and easy work-up. The subsequent alkaline removal of Tfa protecting groups afforded generation Gn of DGL as a free form (with 35-60% overall yield from NCA precursor, depending on the DGL generation) that was either used directly in the synthesis of the next generation (G(n+1)) or collected for other uses. Unprotected forms of DGL G1-G5 were characterised by size-exclusion chromatography, capillary electrophoresis and (1)H NMR spectroscopy. The latter technique allowed us to assess the branching density of DGL, the degree of which (ca. 25%) turned out to be intermediate between previously described dendritic graft poly(L-lysines) and lysine dendrimers. An optimised monomer (NCA) versus macroinitiator (DGL G(n-1)) ratio allowed us to obtain unimodal molecular weight distributions with polydispersity indexes ranging from 1.3 to 1.5. Together with the possibility of reaching high molecular weights (with a polymerisation degree of ca. 1000 for G5) within a few synthetic steps, this synthetic route to DGL provides an easy, cost-efficient, multigram-scale access to dendritic polylysines with various potential applications in biology and in other domains.

DYNAMIC CO-EVOLUTION OF PEPTIDES AND CHEMICAL ENERGETICS, A GATEWAY TO THE EMERGENCE OF HOMOCHIRALITY AND THE CATALYTIC ACTIVITY OF PEPTIDES

We propose a scenario for the dynamic co-evolution of peptides and energy on the primitive Earth. From a multi component system consisting of hydrogen cyanide, several carbonyl compounds, ammonia, alkyl amine, carbonic anhydride, borate and isocyanic acid, we show that the reversibility of this system leads to several intermediate nitriles, that irreversibly evolve to α-amino acids and N-carbamoyl amino acids via selective catalytic processes. On the primitive Earth these N-carbamoyl amino acids combined with energetic molecules (NOx) may have been the core of a molecular engine producing peptides permanently and assuring their recycling and evolution. We present this molecular engine, a production example, and its various selectivities. The perspectives for such a dynamic approach to the emergence of peptides are evoked in the conclusion.

Diastereoselectivity in prebiotically relevant 5(4H)-oxazolone-mediated peptide couplings

A stereochemical study of a potentially prebiotic peptide-forming reaction was carried out as the first part of a systems chemistry investigation of potential paths for symmetry breaking. Substantial diastereomeric excesses result from a fast epimerization of the 5(4H)-oxazolone intermediate in aqueous solution.

Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO2-promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO2-promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide–phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.

Multi-Technique Characterization of Poly-L-lysine Dendrigrafts–Cu(II) Complexes for Biocatalysis

Poly-L-lysine is a biocompatible polymer used for drug or gene delivery, for transport through cellular membranes, and as nanosized magnetic resonance imaging contrast agents. Cu(II)-poly-L-lysine complexes are of particular interest for their role in biocatalysis. In this study, poly-L-lysine dendrigrafts (DGLs) at different generations (G2, G3, and G4) are synthesized and characterized in absence and presence of Cu(II) by means of electron paramagnetic resonance (EPR), UV-Vis, potentiometric titration and circular dichroism (CD). The analysis is performed as a function of the [Cu(II)]/[Lys] (=R) molar ratio, pH and generation by identifying differently flexible complexes in different dendrimer regions. The amine sites in the lateral chains become increasingly involved with the increase of pH. The good agreement and complementarity of the results from the different techniques provide an integrate view of the structural and dynamic properties of Cu(II)-DGL complexes implementing their use as biocatalysts.

Mixed Anhydride Intermediates in the Reaction of 5(4H)-Oxazolones with Phosphate Esters and Nucleotides.

Abstract 5(4H)‐Oxazolones can be formed through the activation of acylated α‐amino acids or of peptide C termini. They constitute potentially activated intermediates in the abiotic chemistry of peptides that preceded the origin of life or early stages of biology and are capable of yielding mixed carboxylic‐phosphoric anhydrides upon reaction with phosphate esters and nucleotides. Here, we present the results of a study aimed at investigating the chemistry that can be built through this interaction. As a matter of fact, the formation of mixed anhydrides with mononucleotides and nucleic acid models is shown to take place at positions involving a mono‐substituted phosphate group at the 3’‐ or 5’‐terminus but not at the internal phosphodiester linkages. In addition to the formation of mixed anhydrides, the subsequent intramolecular acyl or phosphoryl transfers taking place at the 3’‐terminus are considered to be particularly relevant to the common prebiotic chemistry of α‐amino acids and nucleotides.

Diastereoselective Synthesis of a Dyn[3]arene with Distinct Binding Behaviors toward Linear Biogenic Polyamines

The extension of the family of dyn[ n]arenes toward a three-membered macrocycle is reported. Through a templated approach, a single diastereoisomer of a dyn[3]arene that bears six carboxyl groups could be isolated by precipitation in 59-63% yield and excellent purity (≥95%). A combination of experimental and computational experiments in water at physiological pH revealed that the macrocycle could bind parent biogenic polyamines with a unique diversity of surface-binding modes. Whereas no binding event could be accurately measured with 1,3-diaminopropane, spermidine formed a classical stoichiometric complex with the dyn[3]arene in the millimolar concentration range. On the other hand, the data obtained for spermine could only be attributed to a more complex binding event with the formation of a 2:1 complex at high [host]/[guest] ratios and redistribution toward a 1:1 complex upon further addition of guest.

Size characterization of lipid-based self-emulsifying pharmaceutical excipients during lipolysis using Taylor dispersion analysis with fluorescence detection

Self-emulsifying drug delivery systems based on lipids have gained in interest in recent years due to their capacity to enhance the bioavailability of poorly water soluble drugs. Their oral intake suggests that they will be in contact with gastric and pancreatic enzymes during their passage through the gastrointestinal tract. The study of the evolution of such systems in the presence of enzymes is thus essential to develop better drug delivery vehicles. In this work, the lipolysis of two lipid based self-emulsifying drug delivery systems, Labrasol® and Gelucire® 44/14 by pancreatic enzymes and under conditions mimicking the gastrointestinal tract are presented. The following of the digestion is realized by Taylor dispersion analysis using fluorescent detection. A hydrophobic marker was used to tag the microdroplets. Results have shown that, Labrasol® droplets decrease exponentially in size with lipolysis time, from 11.8 nm to 3.5 nm in 20 min. On the contrary, Gelucire® 44/14 droplets increased sigmoïdally in size from 5.6 to 24.7 nm. Even after 120 min lipolysis, both systems maintained a solubilizing capacity of the hydrophobic marker.

The Prebiotic C-Terminal Elongation of Peptides can be Initiated by N-Carbamoyl Amino Acids

The formation of peptides upon 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-promoted activation of N-carbamoylamino acids (CAA), was considered in the scope of our recent works on carbodiimide promoted C-terminus elongation of peptides in a prebiotic context. Thus EDC promoted activation of CAA derivatives of Tyr(Me) or Ala in dilute aqueous medium pH 5.5-6.5 in the presence of excess of AA, resulted in peptide formation by C-terminus activation/elongation. Kinetic results similar to those of EDC-mediated activation of N-acyl-AA lead us to postulate the formation of a 2-amino-5(4H)-oxazolone intermediate by cyclization of the activated CAA, in spite of the absence of epimerization occurred at CAA residues. Thus, in a prebiotic context, CAA may have played a similar role as N-acyl-AA in the initiation of C-terminus peptide elongation.