Analysis and protease-catalysed synthesis of sucrose alkanoate regioisomers

Aleksander Lie

Research output: Book/ReportPh.D. thesis

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The aims of the presented research were to develop quantifiable methods for reversed-phase high-performance liquid chromatography analysis of sucrose alkanoate regioisomers and to investigate the activity and regioisomeric distribution in the biocatalytic esterification of sucrose with vinyl laurate in DMF using serine proteases and a metalloprotease.
A broad range of elution strategies for the chromatographic analysis of sucrose alkanoate regioisomers was systematically investigated using design of experiments strategies and statistical and multivariate analysis and modelling. Efficiency evaluation of the elution strategies, in terms of the resolution metric general resolution deviation for multiple peaks (RDm) and analysis time, showed that the best results were offered by step-down gradient elution for sucrose caprate and isocratic elution with increased flow for sucrose laurate. Step-down gradient elution of sucrose caprate offered improvements in separation by 13-38 % and reductions in analysis time by 3-9 % compared to isocratic elution. For sucrose laurate, isocratic elution with increased flow showed improvements in separation of up to 29 % and reductions of 6-34 % in analysis time compared to isocratic elution. The optimised elution strategies for the sucrose alkanoates resulted in the separation of regioisomers with resolutions adequate for quantification, and the regioisomers were successfully identified and assigned by nuclear magnetic resonance spectroscopy.
The acylation of sucrose with vinyl laurate acyl donor in the hydrophilic, aprotic solvent N,N-dimethylformamide with or without protease biocatalyst resulted in the formation of sucrose laurate, with 2-O-lauroyl sucrose the most abundant monoester regioisomer synthesised. The serine protease ALP-901 produced the highest yield after 48 hours reaction time (53 %), while the reaction with thermolysin achieved the overall highest yield (63 %) after 6 hours, with only monoesters synthesised. The highest concentration of 2-O-lauroyl sucrose observed was 23.7 mM after 24 hours in the thermolysin-catalysed reaction. The detected appearance of the sucrose laurate regioisomers largely corresponded to the apparent rates of formation, based on monoester concentration and regioisomeric distribution after 48 hours, and 2-O-lauroyl sucrose was among the first regioisomers to appear in all investigated reactions. The concentration of sucrose laurate was observed to decline with reaction times above 6 hours in the reaction with thermolysin due to oligoester formation, and declining concentration with increasing reaction time was also observed for some specific sucrose laurate regioisomers in the reactions with other enzyme formulations. Differences in regioisomeric distribution after 48 hours appeared partly to stem from differences in overall reaction rates, as the different reaction conditions resulted in similar distributions after different reaction times. The esterification of sucrose with vinyl laurate with no protein in the reaction mixture appeared to be catalysed by the presence of aluminosilicate molecular sieves in the reaction medium. Mass spectrometry analysis of sucrose laurate product confirmed the molecular mass.

Original languageEnglish
Number of pages96
ISBN (Print)978-87-93100-54-1
Publication statusPublished - 2014

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  • Ph.d.-grad

    Jérôme Le Dréau, Anne Randorff Højen, Aleksander Lie, Zakir Hussain Rather & Gry Kjærsdam Telléus


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