Artificial Fluorescent Glucosinolates (F-GSLs) Are Transported by the Glucosinolate Transporters GTR1/2/3

Abstract

The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and distribution of the specialized plant metabolite glucosinolates. Due to representing both antinutritional and health-promoting compounds, there is increasing interest in characterizing GTRs from various plant species. We generated seven artificial glucosinolates (either aliphatic or benzenic) bearing different fluorophores (Fluorescein, BODIPY, Rhodamine, Dansylamide, and NBD) and investigated the ability of GTR1/2/3 from Arabidopsis thaliana to import the fluorescent glucosinolates (F-GSLs) into oocytes from Xenopus laevis. Five out of the seven F-GSLs synthesized were imported by at least one of the GTRs. GTR1 and GTR2 were able to import three F-GSLs actively above external concentration, while GTR3 imported only one actively. Competition assays indicate that the F-GSLs are transported by the same mechanism as non-tagged natural glucosinolates. The GTR-mediated F-GSL uptake is detected via a rapid and sensitive assay only requiring simple fluorescence measurements on a standard plate reader. This is highly useful in investigations of glucosinolate transport function and provides a critical prerequisite for elucidating the relationship between structure and function through high-throughput screening of GTR mutant libraries. The F-GSL themselves may also be suitable for future studies on glucosinolate transport in vivo.

Enzyme-Responsive Fluorescent Labeling Strategy for In Vivo Imaging of Gut Bacteria

“…Basically, myrosinase fluorescent substrates have been reported. Recently, Klahn and co-workers presented a kind of azide-containing artificial glucosinolate GSL-N3, which can be used to rapidly assemble fluorescent glucosinolates and subsequently release ITCs in the presence of myrosinase , . Similarly, Tatiboet's group also introduced fluorescent glucosinolates for plasmatic protein labeling in HEK293 cells .…”

Bioresponsive pseudoGlucosinolates (psGSLs) release Isothiocyanates (ITCs) in the Presence of Nitroreductases

“…[39] Recently, we also have reported the synthesis of GSL-DNSA (Scheme 1B), which was utilized to image uptake through GTR receptors of Arabidopsis thaliana. [40] The major limitation for application of these probes in broader chemical biology context is the dependence on myrosinase, a thioglycosidase limited to the producing plants. Beyond that ITCs are hydrolysis sensitive electrophiles showing bad pharmacokinetic properties leaving their interesting biological activities unexploited.…”

“…Beyond that ITCs are hydrolysis sensitive electrophiles showing bad pharmacokinetic properties leaving their interesting biological activities unexploited. [1,41,42] Within our attempts to design bio-responsive molecular entities [43][44][45][46][47] and explore the chemistry of artificial GSLs, [38,40,48] we aimed to translate the natural myrosinase-mediated release mechanism of ITCs from GSLs (Scheme 1A) towards artificial GSLs, which are bioresponsive towards enzymes other than the thioglucosidase myrosinase or even chemical triggers. Therefore, we envisaged the design of artificial GSLs, in which the thioglycosidic trigger is substituted by a chemically masked para-aminobenzylthiol unit (Scheme 1C), which we named pseudoglucosinolates (psGSLs).…”

“…B: Artificial, fluorescent and azide containing GSLs by Klahn and co-workers. [38,40] C: Design concept of novel pseudoglucosinolates (psGSLs). [48] PG = protective group.…”

Novel Derivatives of Nitrobenzofurazan with Chromogenic and Fluorogenic Properties

“…Furthermore, NBD-Cl serves as a reagent for the synthesis of other valuable benzo-oxadiazole-type compounds used in various physical, chemical, or biological processes [ 13 ]. For instance, medicines like cephalosporins [ 14 ], acetylcysteine, captopril [ 15 ], serotonin [ 16 ], glucosinolates [ 17 ], ciprofloxacin [ 18 ], or materials like mesoporous silica [ 19 ] or polymer microparticles [ 20 ] have been derivatized with NBD-chloride.…”