Trimethyl-Substituted Carbamate as a Versatile Self-Immolative Linker for Fluorescence Detection of Enzyme Reactions.

Noriaki Nakamura,Shohei Uchinomiya,Akio Ojida,Kazuya Inoue

doi:10.3390/molecules25092153

Abstract

Self-immolative linker is a useful building block of molecular probes, with broad applications in the fields of enzyme activity analysis, stimuli-responsive material science, and drug delivery. This manuscript presents N-methyl dimethyl methyl (i.e., trimethyl) carbamate as a new class of self-immolative linker for the fluorescence detection of enzyme reactions. The trimethyl carbamate was shown to spontaneously undergo intramolecular cyclization upon formation of a carboxylate group, to liberate a fluorophore with the second time rapid reaction kinetics. Interestingly, the auto-cleavage reaction of trimethyl carbamate was also induced by the formation of hydroxyl and amino groups. Fluorescent probes with a trimethyl carbamate could be applicable for fluorescence monitoring of the enzyme reactions catalyzed by esterase, ketoreductase, and aminotransferase, and for fluorescence imaging of intracellular esterase activity in living cells, hence demonstrating the utility of this new class of self-immolative linker.

Highlights

Fluorescent probes are invaluable chemical tools for the detection of enzyme activities in biological research
With the versatile trimethyl carbamate as self-immolative linker in hand, we evaluated its utility in fluorescence detection of enzyme reactions
We have reported the development of trimethyl carbamate as a new class of self-immolative linker

Summary

Introduction

Fluorescent probes are invaluable chemical tools for the detection of enzyme activities in biological research. A variety of small-molecule fluorescent probes have been developed to detect various enzyme reactions, such as esterase/protease-catalyzed hydrolysis and oxidoreductase-catalyzed redox reactions [1,2,3,4]. The probe should be sufficiently stable under biological conditions to avoid non-enzymatic degradation, such as hydrolysis, which would generate unwanted background fluorescence signal. To fulfill these requirements, several types of self-immolative linker have been devised and exploited as a building unit in probe design [5,6,7]. Self-immolative linker is inserted between fluorophore and reactive group as a functional adaptor

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Conclusion