Synthesis and Evaluation of Chromogenic and Fluorogenic Analogs of Glycerol for Enzyme Assays

Abstract

The branched glycerol analogs 1 and 2 were prepared. Mono-ester derivatives of these triols undergo a chromogenic or fluorogenic reaction in the presence of NaIO 4 . In contrast, both the diesters and the triols are themselves not chromogenic or fluorogenic. Diester derivatives of these triols can be used as probes for lipases. The tris-phosphate derivative of 1 is a fluorogenic substrate for various phosphatases.Introduction. ± Enzyme assays are indispensable tools in enzymology, where they are used to identify enzymes and to evaluate their purity and activity [1]. When an enzyme is discovered, one important question is always the identity of its natural substrate, which is presumably also its best substrate in terms of kinetics behavior. In the context of enzyme assays, the related problem is to find the optimal assay method for any given enzyme, in particular the most-sensitive method. Simple assays giving readily recordable spectroscopic signals are preferred in the context of high-throughput screening for new enzymes by biodiversity mining or directed evolution. Herein, we report the synthesis and evaluation of the branched triols 1 and 2 as building blocks for enzyme substrates. Triols 1 and 2 are close analogs of glycerol, and their ester derivatives can be used to prepare chromogenic and fluorogenic analogs of glycerides, the natural substrates of lipases. The synthesis of these triols and their derivatives and their applications in enzyme assays are presented.

A Sensitive and Selective High‐Throughput Screening Fluorescence Assay for Lipases and Esterases

“…These substrates release diol 6 upon reaction with the enzymes, and a fluorescence signal is produced in the presence of NaIO 4 and bovine serum albumin (BSA) by liberation of umbelliferon (8) through an oxidation/b-elimination sequence via aldehyde 7 [5]. A first attempt at optimizing the structures of these substrates towards lipases consisted in modifying diol 6 into a branched analog of glycerol to produce diglyceride-like aliphatic ester substrates [6]. However, this strategy did not lead to the expected improvement in sensitivity.…”

Enzyme assays for high-throughput screening

“…This concept is quite versatile [3] and was used recently for assaying CC-lyases such as transaldolases [8] and transketolase [9], Bayer-villigerases [10 ], HIV protease [11] and thermostable esterases [12 ,13] (Figure 1). Efficient substrates for lipases and esterases were also reported that operate by a similar indirect release of umbelliferone [14][15][16]. A recently reported b-lactamase substrate operates by a similar mechanism [17].…”

Fluorescence Assays for Biotransformations

“…The standard substrates for phosphatases are nitrophenyl phosphates and related monophosphates of aromatic phenols. Di -and triphosphates 45 and 46 related to the Clips -O principle discussed above have been investigated (Scheme 1.14 ) [58] . The substrates are obtained from the diols by phosphorylation with dibenzyl phosphoramidite, followed by oxidation to the phosphate and debenzylation by hydrogenation.…”