Detection of potential TNA and RNA nucleoside precursors in a prebiotic mixture by pure shift diffusion-ordered NMR spectroscopy.

In the context of prebiotic chemistry, one of the characteristics of mixed nitrogenous-oxygenous chemistry is its propensity to give rise to highly complex reaction mixtures. There is therefore an urgent need to develop improved spectroscopic techniques if onerous chromatographic separations are to be avoided. One potential avenue is the combination of pure shift methodology, in which NMR spectra are measured with greatly improved resolution by suppressing multiplet structure, with diffusion-ordered spectroscopy, in which NMR signals from different species are distinguished through their different rates of diffusion. Such a combination has the added advantage of working with intact mixtures, allowing analyses to be carried out without perturbing mixtures in which chemical entities are part of a network of reactions in equilibrium. As part of a systems chemistry approach towards investigating the self-assembly of potentially prebiotic small molecules, we have analysed the complex mixture arising from mixing glycolaldehyde and cyanamide, in a first application of pure shift DOSY NMR to the characterisation of a partially unknown reaction composition. The work presented illustrates the potential of pure shift DOSY to be applied to chemistries that give rise to mixtures of compounds in which the NMR signal resolution is poor. The direct formation of potential RNA and TNA nucleoside precursors, amongst other adducts, was observed. These preliminary observations may have implications for the potentially prebiotic assembly chemistry of pyrimidine threonucleotides, and therefore of TNA, by using recently reported chemistries that yield the activated pyridimidine ribonucleotides.

Analysis of mono- and oligosaccharides in ionic liquid containing matrices.

Ionic liquids (ILs), that is, salts with melting points <100°C, have recently attracted a lot of attention in biomass processing due to their ability to dissolve lignocellulosics. In this work, we studied how two imidazolium-based, hydrophilic, cellulose dissolving ionic liquids 1,3-dimethylimidazolium dimethylphosphate [DMIM]DMP and 1-ethyl-3-methylimidazolium acetate [EMIM]AcO affect the usually employed analytical methods for mono- and oligosaccharides, typical products from hydrolytic treatments of biomass. HPLC methods were severely hampered by the presence of ILs with loss of separation power and severe baseline problems, making their use for saccharide quantification extremely challenging. Problems in DNS photometric assay and chromatography were also encountered at high ionic liquid concentrations and many capillary electrophoresis (CE) methods did not allow an efficient analysis of saccharides in these matrices. In this paper we describe an optimized CE method with pre-column derivatization for the qualitative and quantitative analysis of mono- and oligosaccharides in sample matrices containing moderate (20-40% (v/v)) concentrations of ILs. The IL content and type in the sample matrix was found to affect both peak shape and quantification parameters. Generally, the presence of high IL concentrations (≥20% (v/v)) had a dampening effect on the detection of the analytes. IL in lower concentrations of <20% (v/v) was, however, found to improve peak shape and/or separation in some cases. The optimized CE method has good sensitivity in moderate concentrations of the ionic liquids used, with limits of detection of 5mg/L for cellooligomers up to the size of cellotetraose and 5-20mg/L for cellopentaose and cellohexaose, depending on the matrix. The method was used for analysing the action of a commercial ß-glucosidase in ILs and for analysing saccharides in the IL containing hydrolysates from the hydrolysis of microcrystalline cellulose with Trichoderma reesei endoglucanase Cel5A. According to the results, [DMIM]DMP and [EMIM]AcO] showed clear differences in enzyme inactivation.

[Simultaneous determination of meso-erythritol and L-erythrulose in fermentation broth using high performance liquid chromatography].

A high performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of meso-erythritol and L-erythrulose in fermentation broth. The chromatographic conditions were as follows: Lichrospher 5-NH2 column (250 mm x 4.6 mm) with the temperature of 30 degrees C, acetonitrile-water (90: 10, v/v) as mobile phase with the flow rate of 1.0 mL/min. meso-Erythritol was detected by refractive index (RI) detector at 35 degrees C and L-erythrulose was detected by ultraviolet (UV) detector at 277 nm at room temperature. The linear range for meso-erythritol was 1.00 - 100.00 g/L with a correlation coefficient of 0.998 5. The limit of detection (LOD) and the limit of quantification (LOQ) for meso-erythritol were 0.10 g/L and 0.45 g/L, respectively. The linear range for L-erythrulose was 1.00 - 100.00 g/L with a correlation coefficient of 0.995 8. The LOD and LOQ for L-erythrulose were 0.50 g/L and 0.87 g/L, respectively. The relative standard deviations (RSDs) of intraday and interday for meso-erythritol were less than 3.28% and 5.30%, respectively. The intraday and interday RSDs for L-erythrulose were less than 2.16% and 2.25%, respectively. The recoveries of meso-erythritol and L-erythrulose in fermentation broth were greater than 99%. The samples from fermentation broth were detected at different time points. The measurement by the novel HPLC method was not affected by the other components in the fermentation broth. Furthermore, the HPLC method can be used for the determination of the substrate meso-erythritol and the product L-erythrulose simultaneously.

Methods for enzymatic and colorimetric determinations of D-erythrulose (D-tetrulose).

The specific determinations of D-erythrulose by enzymatic assay or colorimetric method, which permit the quantitative determination of between 20 and 400 nmol of the sugar, are described. Enzymatic determination of D-erythrulose made use of the D-erythrulose reductase purified from beef or chicken liver, which catalyzes specifically the reduction of D-erythrulose with concomitant conversion of NADH to NAD+. The colorimetric microdetermination of erythrulose could be carried out by utilizing the phenol-sulfuric acid reaction under low temperature. These methods are simple, rapid, and sensitive, and give reproducible results.

Mechanism of cellulose synthesis in Agrobacterium tumefaciens.

Extracts of Agrobacterium tumefaciens incorporated UDP-[14C]glucose into cellulose. When the extracts were fractionated into membrane and soluble components, neither fraction was able to synthesize cellulose. A combination of the membrane and soluble fractions restored the activity found in the original extracts. Extracts of cellulose-minus mutants showed no significant incorporation of UDP-glucose into cellulose. When mixtures of the extracts were made, the mutants were found to fall into two groups: extracts of mutants from the first group could be combined with extracts of the second group to obtain cellulose synthesis. No synthesis was observed when extracts of mutants from the same group were mixed. The groups of mutants corresponded to the two operons identified in sequencing the cel genes (A. G. Matthysse, S. White, and R. Lightfoot. J. Bacteriol. 177:1069-1075, 1995). Extracts of mutants were fractionated into membrane and soluble components, and the fractions were mixed and assayed for the ability to synthesize cellulose. When the membrane fraction from mutants in the celDE operon was combined with the soluble fraction from mutants in the celABC operon, incorporation of UDP-glucose into cellulose was observed. In order to determine whether lipid-linked intermediates were involved in cellulose synthesis, permeablized cells were examined for the incorporation of UDP-[14C]glucose into material extractable with organic solvents. No radioactivity was found in the chloroform-methanol extract of mutants in the celDE operon, but radioactive material was recovered in the chloroform-methanol extract of mutants in the celABC operon. The saccharide component of these compounds was released after mild acid hydrolysis and was found to be mainly glucose for the celA insertion mutant and a mixture of cellobiose, cellotriose, and cellotetrose for the celB and celC insertion mutants. The radioactive compound extracted with chloroform-methanol form the celC insertion mutant was incorporated into cellulose by membrane preparations from celE mutants, which suggests that this compound is a lipid-linked intermediate in cellulose synthesis.