Dietary isothiocyanates (ITCs) obtained from Brassica vegetables demonstrate several modes of diverse activity, conferring antioxidant, anti-inflammatory, antibacterial, antifungal, and antitumor properties. ITCs are one of several hydrolysis products of glucosinolates, secondary metabolites which are substrates for the thioglucohydrolase myrosinase. Despite the extensive body of literature describing the properties of naturally-occurring ITCs and glucosinolates, recent pursuits toward the development of synthetic, non-natural ITCs have consequently led to an exploration of their non-natural glucosinolate precursors. Since evaluation of the myrosinase-dependent hydrolysis of non-natural glucosinolates can be problematic using established UV-Vis spectroscopic methods, an alternative HPLC-based analysis method was developed. In this account, we describe the 1) preparation of two synthetic glucosinolates and the 2) parallel kinetic analysis of three glucosinolates using traditional UV-Vis spectroscopy and a novel HPLC assay which determines initial velocities through analysis of reaction progress curves. The results of our study demonstrate that the kinetic data is consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis�Menten and specific activity analyses. Additionally, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports. Together, these studies validate a novel HPLC protocol for the kinetic evaluation of non-natural glucosinolates which may prove useful in the evaluation of other enzymatic systems.

Synthesis and parallel kinetic evaluation of <i>Sinapis alba</i> myrosinase-mediated glucosinolate hydrolysis using UV-visible spectroscopic and HPLC analytical methods

247th National Meeting (2014)

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Synthesis and parallel kinetic evaluation of Sinapis alba myrosinase-mediated glucosinolate hydrolysis using UV-visible spectroscopic and HPLC analytical methods