Process improvement in amino acid N-carboxyanhydride synthesis by N-carbamoyl amino acid nitrosation.

Amino acid N-carboxyanhydrides (NCA), convenient monomer for polypeptide synthesis, are easily prepared in high purity as the result of N-carbamoyl amino acids (CAA) nitrosation by gaseous NOx (4:1 NO + O2 mixture, or NOCl) in toluene. Removal of polar side products is then efficiently carried out during subsequent work-up and crystallisation so that the resulting NCA obtained in good yield is suitable for controlled, primary amine-initiated polymerisation.

Hydrolysis of nitriles using an immobilized nitrilase: applications to the synthesis of methionine hydroxy analogue derivatives.

Mild and selective hydrolysis of a large range of nitriles leading to carboxylic acids was achieved under neutral conditions by an immobilized and genetically modified enzyme preparation from Alcaligenes faecalis ATCC8750. This immobilized nitrilase has been shown to be an effective catalyst for the stereoselective hydrolysis of mandelonitrile 1a to R-(-)-mandelic acid 1c. This method is particularly useful for the production of hydroxy analogues of methionine derivatives 2c-4c that could have an interest in cattle feeding and for the transformation of compounds containing other acid- or base-sensitive groups 3a-10a. A series of aliphatic dinitriles 11a-15a was hydrolyzed to the corresponding cyano acids. The suitability of the immobilized catalyst as a robust and versatile biocatalyst is discussed, and models to account for the stereoselectivity of the enzymic hydrolysis have been proposed.

Heart-cutting two-dimensional electrophoresis in a single capillary.

The possibility of performing two-dimensional capillary electrophoresis (2D-CE) separations in a single capillary was investigated. For this purpose, a fraction stemming from the first dimension of the separation was selected and isolated in the capillary by evacuating out of the capillary the other undesirable compounds. Next, the isolated fraction was submitted to a second separation medium that was introduced in the capillary by electroosmotic flow. The second separation medium was able to reach the isolated fraction since the solutes were migrating in counter-electroosmotic mode. Since only one fraction is submitted to the second dimension of the separation, this new methodology is closer to a heart-cutting approach than to a true comprehensive 2D separation. However, it has the advantage of not requiring any special coupling device between capillaries since the two dimensions of the separation are performed in the same capillary. In this work, a simple mixture of synthetic polymers taken as model compounds was separated according to: (i) the charge density by free solution CE in the first dimension, and (ii) the molar masses by CE in the presence of an entangled polymer solution in the second dimension. The different strategies that were investigated to isolate the fraction at the end of the first dimension are described in detail. The influence of important experimental parameters (capillary diameter, applied pressure for mobilizing the solutes, diffusion coefficients of the solutes) on the performances of the two-dimensional separations were studied. A careful attention was paid to the influence of these parameters on the efficiency of the separations. The experimental results demonstrate that heart-cutting 2D electrophoretic separations can be performed in a capillary format using a single capillary.

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 alpha-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.

Nonaqueous capillary zone electrophoresis of synthetic organic polypeptides.

Poly(Nepsilon-trifluoroacetyl-L-lysine) was used as a model solute to investigate the potential of nonaqueous capillary electrophoresis (NACE) for the characterization of synthetic organic polymers. The information obtained by NACE was compared to that derived from size exclusion chromatography (SEC) experiments, and the two techniques were found to be complimentary for polymer characterization. On one hand, NACE permitted (i) the separation of oligomers according to their molar mass and (ii) the separation of the polymers according to the nature of the end groups. On the other hand, SEC experiments were used for the characterization of the molar mass distribution for higher molar masses. Due to the tendency of the solutes (polypeptides) to adsorb onto the fused-silica capillary wall, careful attention was paid to the rinsing procedure of the capillary between runs in order to keep the capillary surface clean. For that purpose, the use of electrophoretic desorption under denaturating conditions was very effective. Optimization of the separation was performed by studying (i) the influence of the proportion of methanol in a methanoVacetonitrile mixture and (ii) the influence of acetic acid concentration in the background electrolyte. Highly resolved separation of the oligomers (up to a degree of polymerization n of approximately 50) was obtained by adding trifluoroacetic acid to the electrolyte. Important information concerning the polymer conformations could be obtained from the mobility data. Two different plots relating the effective mobility data to the degree of polymerization were proposed for monitoring the changes in polymer conformations as a function of the number of monomers.