Development of a rapid quantitative method of analysis of carbohydrate fatty acid ester reaction mixtures using 1H qNMR.

Two new analytical methods, applying absolute 1H qNMR, were developed to monitor product yield and quantify unreacted carbohydrate and fatty acid reactants, in the synthesis of carbohydrate fatty acid esters (CFAE). These methods provide a mass balance of the crude reaction mixtures and diversify the analytical screening and quantitation approaches available within the synthesis of these molecules. Both methods were validated for the model reaction of methyl α-d-glucopyranoside (MAG) and lauric acid (LA) to form the mono ester product, methyl 6-O-dodecanoyl-α-d-glucopyranoside. Analysis in CD3OD by 1H qNMR, with fumaric acid (FA) as an internal standard (IS), allowed monitoring of all reaction components. Alternatively, using CDCl3 and (E)-stilbene as IS enabled the analysis of CFAE and fatty acid. Parameters calculated for method validation included specificity and selectivity, linearity, accuracy, intermediate precision, limit of detection (LOD), limit of quantification (LOQ) and robustness. Both methods provided excellent linearity with R2 > 0.997. The accuracy, precision, and robustness of the method in CD3OD was <2 % uncertainty making it suitable for complete reaction analysis. The method completed in CDCl3 resulted in accuracy, intermediate precision, and robustness of <5 %, except for accuracy in the lowest levels of concentration (>5 %). For all related analytes in the CD3OD and CDCl3 methods, the LOD and LOQ were determined to ensure applicability for the intended use in the assessment of reaction crude composition. Finally, the system suitability was assessed in a scaled lipase catalysed CFAE synthetic reaction. The determined qNMR product yields were verified against isolated purified product yields with <5 % uncertainty.

Process Optimisation Studies and Aminonitrile Substrate Evaluation of Rhodococcus erythropolis SET1, A Nitrile Hydrolyzing Bacterium.

A comprehensive series of optimization studies including pH, solvent and temperature were completed on the nitrile hydrolyzing Rhodococcus erythropolis bacterium SET1 with the substrate 3-hydroxybutyronitrile. These identified temperature of 25 °C and pH of 7 as the best conditions to retain enantioselectivity and activity. The effect of the addition of organic solvents to the biotransformation mixture was also determined. The results of the study suggested that SET1 is suitable for use in selected organo-aqueous media at specific ratios only. The functional group tolerance of the isolate with unprotected and protected ß-aminonitriles, structural analogues of ß-hydroxynitriles was also investigated with disappointingly poor isolated yields and selectivity obtained. The isolate was further evaluated with the α- aminonitrile phenylglycinonitrile generating acid in excellent yield and ee (>99 % (S) - isomer and 50 % yield). A series of pH studies with this substrate indicated pH 7 to be the optimum pH to avoid product and substrate degradation.

A rotaxane mimic of the photoactive yellow protein chromophore environment: effects of hydrogen bonding and mechanical interlocking on a coumaric amide derivative.

Hydrogen bonding in a [2]rotaxane is shown to stabilise the phenolate anion of a coumaric amide chromophore by almost 3 pKa units; however, the effect on the UV spectral shift in the anion is small and, significantly given the photochemistry of PYP, despite the hydrogen bonding olefin photoisomerisation in the anionic rotaxane remains heavily suppressed.

Coumaric amide rotaxanes: effects of hydrogen bonding and mechanical interlocking on the photochemistry and photophysics.

Secondary amide derivatives of coumaric and ferulic acid are shown to undergo photoisomerization, forming a photostationary mixture of E- and Z-isomers. When the same chromophores are incorporated in rotaxanes, the extent of conversion to the Z-isomers is much smaller. Low temperature fluorescence experiments show that the energy barrier for non-radiative decay of the excited state is higher in the rotaxanes than in the corresponding threads, but the barriers are low in all cases.