Comparison of methods for measurement of organic compounds at ultra-trace level: analytical criteria and application to analysis of amino acids in extraterrestrial samples.

The need for criteria to compare different analytical methods for measuring extraterrestrial organic matter at ultra-trace levels in relatively small and unique samples (e.g., fragments of meteorites, micrometeorites, planetary samples) is discussed. We emphasize the need to standardize the description of future analyses, and take the first step toward a proposed international laboratory network for performance testing.

Synthesis of Nepsilon-protected-L-lysine and gamma-benzyl-L-glutamate N-carboxyanhydrides (NCA) by carbamoylation and nitrosation.

This paper reports on an original process to synthesize N-carboxyanhydrides, which consists of nitrosating N-carbamoylamino acids with a NO/O(2) gas mixture in acetonitrile. The synthesis of several N-carbamoylamino acids of L-lysine was described using potassium cyanate in water. The latter were then nitrosated to yield the corresponding NCA with more or less efficiency. Indeed, the NCA carrying an acid-sensitive protecting group led to a partial deprotection to give the L-lysine NCA salt. The NCA of N(epsilon)-trifluoroacetyl-L-lysine, N(epsilon)-benzyloxycarbonyl-L-lysine and gamma-benzyl-L-glutamate were successfully synthesized with satisfactory yields. Their polymerizability was compared to that of the N(epsilon)-trifluoroacetyl-L-lysine NCA initiated by n-hexylamine in N,N-dimethylformamide. It also showed that this new process of NCA synthesis could be applied to the synthesis of polypeptides and more generally to the protein chemistry.

Antibodies directed against L and D isovaline using a chemical derivatizing reagent for the measurement of their enantiomeric ratio in extraterrestrial samples: first step-production and characterization of antibodies.

Determining the enantiomeric ratio of amino acids in meteorites requires very sensitive and precise measurements. In this study, an immunochemical approach, combined with new chemical derivatizing agents, was investigated for the measurement of the enantiomeric ratio of isovaline. In the initial step, L and D isovaline were derivatized with epsilon-benzyloxycarbonyl-L-lysine-(t-butyl ester)-chloroethylnitrosourea (Z-L-Lys-(OtBu)-CENU). The Z group was hydrolyzed and the resulting isovaline derivatives (L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) were conjugated with protein using glutaraldehyde and reduced with sodium borohydride. Rabbits were immunized with the immunogenic conjugates thus obtained. Antibodies were characterized using many compounds, both derivatized and underivatized, in competitive ELISA tests. These competition experiments performed enabled us to establish the following results: 1) unconjugated L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline were poorly recognized; 2) all related L-Lys(OtBu)-alpha-hydrogenated amino acids (L and D) were not recognized at all, which eliminates the possibility of the measurements being distorted by contamination; 3) only conjugated L-Lys(OtBu)-alpha-amino-isobutyric acid (AIB) was recognized by the antibody, 4) the enantiomeric discrimination of L and D isovaline through their derivatives (diastereoisomeric L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) was in accordance with the measurement of their enantiomeric ratio. Immunopurification was shown to enhance antibody specificity. The strategy employed shows potential for the quantification of meteoritic amino acids.

Kinetic study of the polymerization of alpha-amino acid N-carboxyanhydrides in aqueous solution using capillary electrophoresis.

N-Carboxyanhydrides of amino acids (NCAs) are very reactive monomers able to polymerize into oligopeptides. They are assumed to be prebiotic precursors of the first polypeptides. Few reports have been published on the study of NCA polymerization in aqueous solution. In this work, a kinetic study focused on the hydrolysis of NCA and its coupling with amino acids and homopeptides (up to tripeptide) was carried out, taking L-valine derivatives as model compounds. For that purpose, capillary electrophoresis appeared to be an effective and reliable technique for the measurement of the kinetic constants. The electrophoretic separation conditions, the procedure for stopping NCA reactivity, as well as the conditions of reaction are discussed in detail. We report the variation of the kinetic constant of the coupling reaction of the NCA of valine with an oligovaline as a function of its degree of polymerization. Finally, a temperature study also allowed us to estimate the activation energies associated with the NCA of valine hydrolysis and its coupling reaction with valine.

Chiral determination of amino acids by capillary electrophoresis and laser-induced fluorescence at picomolar concentrations.

In this publication we present results on the determination of enantiomers of amino acids at very low concentrations. A fluoresceine-based chiral dye was synthesized to allow the separation of diastereoisomers of D- and L-amino acids. We used capillary electrophoresis with different non-ionic surfactants (Brij). The separation parameters were optimized and separations of D- and L-isovaline, an unusual terrestrial amino acid, were obtained. The sensitivity limits were also determined using a commercial laser-induced fluorescence detector. The quantitation of these amino acids is very important to understand the process of chiral selection on Earth.

N-carbamoyl amino acid solid-gas nitrosation by NO/NOx: A new route to oligopeptides via alpha-amino acid N-carboxyanhydride. Prebiotic implications.

Alpha-N-Carbamoyl amino acid (CAA), whose conditions of formation in a prebiotic hydrosphere have been described previously (Taillades et al. 1998), could have been an important intermediate in prebiotic peptide synthesis through reaction with atmospheric NOx. Nitrosation of solid CAA (glycine or valine derivative) by a 4/1 NO/O2 gaseous mixture (1 atm) yields N-carboxyanhydride (NCA) quantitatively in less than 1 h at room temperature. The crude solid NCA undergoes quantitative oligomerization (from trimer to nonamer under the conditions we used) when treated with a (bi)carbonate aqueous buffer at pH 9. We therefore suggest that part of the prebiotic amino acid activation/polymerization process may have taken place in a dry phase ("drying-lagoon" scenario).

N-carbamoyl-alpha-amino acids rather than free alpha-amino acids formation in the primitive hydrosphere: a novel proposal for the emergence of prebiotic peptides.

Our previous kinetic and thermodynamic studies upon the reactional system HCHO/HCN/NH3 in aqueous solutions are completed. In the assumed prebiotic conditions of the primitive earth ([HCHO] and [HCN] near 1 g L-1, T = 25 degrees C, pH = 8, [NH3] very low), this system leads to 99.9% of alpha-hydroxyacetonitrile and 0.1% of alpha-aminoacetonitrile (precursor of the alpha-amino acid). The classical base-catalyzed hydration of nitriles, slow and not selective, can not modify significantly this proportion. On the contrary, we found two specific and efficient reactions of alpha-aminonitriles which shift the initial equilibrium in favor of the alpha-aminonitrile pathway. The first reaction catalyzed by formaldehyde generates alpha-aminoamides, precursors of alpha-aminoacids. The second reaction catalyzed by carbon dioxide affords hydantoins, precursors of N-carbamoyl-alpha-aminoacids. In the primitive hydrosphere, where the concentration in carbon dioxide was estimated to be higher than that of formaldehyde, the formation of hydantoins was consequently more efficient. The rates of hydrolysis of the alpha-aminoacetamide and of the hydantoin at pH 8 being very similar, the synthesis of the N-carbamoyl-alpha-amino acid seems then to be the fatal issue of the HCHO/HCN/NH3 system that nature used to perform its evolution. These N-protected alpha-amino acids offer new perspectives in prebiotic chemistry, in particular for the emergence of peptides on the prebiotic earth.