High-performance liquid chromatography-based method to evaluate kinetics of glucosinolate hydrolysis by Sinapis alba myrosinase

Abstract

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites which are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic, non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established UV-Vis spectroscopic methods. To overcome this limitation, an alternative HPLC-based analytical approach was developed where initial reaction velocities were generated from non-linear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV-Vis methodology. The results of this study demonstrate that kinetic data is consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports.

HPLC-based kinetics assay facilitates analysis of systems with multiple reaction products and thermal enzyme denaturation

“…Although natural glucosinolates and their ITCs have been relatively well-described, fewer accounts have been made concerning non-natural analogues, those compounds bearing sidechains structures not observed in natural sources [19]. Screening efforts have identified non-natural ITC leads with promising activity against human disease [20,21,22,23].…”

“…However, the broader structural diversity of non-natural analogues challenged the traditional ultraviolet-visible (UV–Vis) spectrophotometric methods of monitoring this enzymatic method of unmasking bioactive ITCs [16]. In 2014, a novel HPLC approach for analyzing reaction kinetics was described and validated against the UV–Vis method; key advantages of the HPLC approach were the ability to chromatographically separate and simultaneously track analytes with diverse polarity/solubility profiles, such as 1 and 2 [19]. This methodology has more recently been expanded to LC–MS systems to aid in the evaluation of samples from complex mixtures and has broader implications as an analytical approach [24].…”

“…Importantly, this study demonstrated that the synthetic non-natural glucosinolates 5 was a substrate for myrosinase and, upon exposure to the enzyme, was converted to its analogous ITC 8 [23,27]. More recent efforts further demonstrate that non-natural ITCs can be generated in situ via the reaction of non-natural glucosinolates and myrosinase [19]. Together, this combined work supports the premise that non-natural ITCs and their glucosinolate prodrugs are viable targets for the rational design of novel anticancer agents, a field that has not been extensively explored.…”

“…Although non-natural glucosinolates are known substrates for myrosinase, the kinetic studies conducted to date have been focused on the goals of proof-of-principle [23] and validation of the general HPLC methodology [19]. A comprehensive evaluation of myrosinase toward non-natural substrates has not been conducted to demonstrate that these analogues mirror the outcome(s), tolerance, and mechanism exhibited by natural glucosinolates.…”

“…The validated HPLC approach is ideally suited for these enzymological studies, as it offers the possibility of simultaneously and independently evaluating glucosinolate and any resultant organic product(s), a feature which has broader methodological considerations and applications [19]. Initial rates ( V 0 ) for glucosinolate hydrolysis and product formation would be obtained from nonlinear reaction progress curves fit with the Lambert W (x) [19,33]; these elucidated V 0 would provide explicit rate information, an advantage over many previous accounts which solely reported relative rates [31,32]. …”