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

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.

Copper-catalyzed S-arylation of Furanose-Fused Oxazolidine-2-thiones.

The 1,3-oxazolidine-2-thiones (OZTs) are important chiral molecules, especially in asymmetric synthesis. These compounds serve as important active units in biologically active compounds. Herein, carbohydrate anchored OZTs were explored to develop a copper-catalyzed C-S bond formation with aryl iodides. Chemoselective S-arylation was observed, with copper iodide and dimethylethylenediamine (DMEDA) as the best ligand in dioxane at 60-90 °C. The corresponding chiral oxazolines were obtained in reasonable to good yields under relatively mild reaction conditions. This approach is cheap, as using one of the cheapest transition metals, a simple protocol and various functional group tolerance make it a valuable strategy for getting S-substituted furanose-fused OZT. The structures of the novel carbohydrates were confirmed by NMR spectroscopy and an HRMS analysis.

Diverted Natural Lossen-type Rearrangement for Bioconjugation through in Situ Myrosinase-Triggered Isothiocyanate Synthesis.

Fluorescein isothiocyanate (FITC) is one of the most extensively used fluorescent probes for the labeling of biomolecules. The isothiocyanate function reacts with lysine residues of proteins to provide a chemically stable thiourea linkage without releasing any byproduct. However, diversification of isothiocyanate-based reagents is still hampered by the lack of mild conditions to generate isothiocyanate chemical functions, as well as by their poor stability and limited solutions available to increase water solubility, restricting the use of isothiocyanate labeling to highly water-soluble fluorophores. Inspired by plant biological processes, we report a safe and biocompatible myrosinase-assisted in situ formation of isothiocyanate conjugates from a highly water-soluble and stable glucosinolate precursor. This method was applied for the fluorescence labeling of a plasmatic protein and fluorescence imaging of living cells.

UGT74B1 from Arabidopsis thaliana as a versatile biocatalyst for the synthesis of desulfoglycosinolates.

Thioglycosides, even if rare in Nature, have gained increased interest for their biological properties. Chemical syntheses of this class of compounds have been largely studied but little has been reported on their biosynthesis. Herein, combining experiments from the different fields of enzymology, bioorganic chemistry and molecular modeling, we wish to demonstrate the versatility of the glucosyltransferase UGT74B1 and its synthetic potency for the preparation of a variety of natural and unnatural desulfoglycosinolates.

Synthesis of 3-fluoropyrrolidines and 4-fluoropyrrolidin-2-ones from allylic fluorides.

Various 3-fluoropyrrolidines and 4-fluoropyrrolidin-2-ones were prepared by 5-exo-trig iodocyclisation from allylic fluorides bearing a pending nitrogen nucleophile. These bench-stable precursors were made accessible upon electrophilic fluorination of the corresponding allylsilanes. The presence of the allylic fluorine substituent induces syn-stereocontrol upon iodocyclisation with diastereomeric ratios ranging from 10:1 to > 20:1 for all N-tosyl-3-fluoropent-4-en-1-amines and amides. The sense and level of stereocontrol is strikingly similar to the corresponding iodocyclisation of structurally related allylic fluorides bearing pending oxygen nucleophiles. These results suggest that the syn selectivity observed upon ring closure involves I(2)-π complexes with the fluorine positioned inside.

Multigram scale preparation of a semi-synthetic N-trifluoroacetyl protected chondroitin disaccharide building block: Towards the stereoselective synthesis of chondroitin sulfates disaccharides.

The chemoselective N-trifluoroacetylation of a chondroitin disaccharide obtained from controlled acid hydrolysis of a commercially available polymeric chondroitin sulfate is reported for the first time. We also described the multi-gram scale synthesis of a donor block having a benzylidene moiety further used for the expeditious and stereocontrolled synthesis of glycosides fitted with various aglycons. Stereocontrolled ß-glycosylation, sulfation and efficient N-TFA deprotection steps afforded the desired disaccharides in good yields.

The myrosinase-glucosinolate system to generate neoglycoproteins: A case study targeting mannose binding lectins.

A convenient strategy for a 'one-pot' synthesis of neoglycoproteins (NGP) was developed using the myrosinase-glucosinolate couple, a natural enzyme-substrate system. This enzymatic reaction allowed us to generate an isothiocyanate in situ which then reacted with the lysine residues of bovine serum albumin protein (BSA) to produce multivalent neoglycoproteins. Using two models, glucomoringin which is a natural glucosinolate bearing a l-rhamnose unit, and an artificial glucosinolate specifically designed for mannose type lectins, an average of up to 17.8 and 28.7 carbohydrate residues could be respectively grafted onto the BSA protein. This process is comparable to commercial approaches using BSA-ManC without the disadvantage of handling harmful chemical reagents. Lectin binding screening (GLYcoPROFILE®) showed that among all NGPs synthesized, BSA-Man 16 gave similar and in some cases better affinities in comparison with commercial BSA-Manc towards various mannose-specific lectins.

Capillary electrophoresis with dual detection UV/C4D for monitoring myrosinase-mediated hydrolysis of thiol glucosinolate designed for gold nanoparticle conjugation.

Capillary electrophoresis (CE) with dual UV and conductivity detection was used for the first time to monitor the functionalization of gold nanoparticles (AuNPs), a process catalyzed by an enzyme, myrosinase (Myr). A thiol glucosinolate (GL-SH) designed by our group was used as substrate. Hydrolysis of free and immobilized GL-SH was characterized using off-line and on-line CE-based enzymatic assays. The developed approaches were validated using sinigrin, a well-referenced substrate of Myr. Michaelis-Menten constant of the synthetized GL-SH was comparable to sinigrin, showing that they both have similar affinity towards Myr. It was demonstrated that transverse diffusion of laminar flow profiles was well adapted for in-capillary Mixing of nanoparticles (AuNPs) with proteins (Myr) provided that the incubation time is inferior to 20 min. Only low reaction volume (nL to few µL) and short analysis time (<5 min) were required. The electrophoretic conditions were optimized in order to evaluate and to confirm the AuNPs stability before and after functionalization by CE/UV based on surface plasmon resonance band red-shifting. The hydrolysis of the functionalized AuNPs was subsequently evaluated using the developed CE-C4D/UV approach. Repeatabilities of enzymatic assays, of electrophoretic analyses and of batch-to-batch functionalized AuNPs were excellent.

Glycerol carbonate in Ferrier reaction: Access to new enantiopure building blocks to develop glycoglycerolipid analogues.

Glycerol carbonate and tri-O-acetyl-D-glucal were used for the synthesis of glycero-functionalized carbohydrates. Ferrier reaction between the two partners afforded the O-glucoside in 84% yield. Spontaneous crystallization yielded 28% of a pure diastereoisomer with the S configuration as determined by X-ray crystallography. Then, the azido-glycerosugar was prepared in two steps: ring opening of the cyclic carbonate with sodium azide and per-acetylation with an excellent yield of 94%. A library of glycoconjugates were prepared using a 1,3-dipolar cycloaddition in yields ranging from 64 to 99%.

Selective targeting of synthetic antioxidants to mitochondria: towards a mitochondrial medicine for neurodegenerative diseases?

Mitochondria are the major source of superoxide, and are responsible for activating apoptosis and oxidative damage during acute neuronal cell death and neurodegenerative disorders like Alzheimer and Parkinson diseases. While the molecular mechanisms by which mitochondrial oxidative stress triggers apoptosis are still investigated, attempts to achieve neuroprotection using antioxidant molecules have already been successful in several models of neuronal cell death. To increase the availability of antioxidant drugs at the mitochondrial level within cells, Michael P. Murphy recently proposed to covalently couple antioxidant molecules to a membrane-permeable lipophilic cation serving as carrier. Since mitochondria maintain at rest a potential of -180 mV, the diffusible cationic moiety drives the accumulation of the complex inside the matrix towards a diffusion equilibrium: for a monovalent cationic carrier, a thousand-fold accumulation of the complex is theoretically achievable; for a divalent cation, a million-fold accumulation is expected. Such mitochondria-targeted versions of natural antioxidants have successfully been synthesized and were found to counteract the pro-apoptotic effects of exogenous oxidative insults, while having no effects in models mimicking physiological apoptosis. Based on these observations, we carried out the synthesis of targeted variants of the artificial free radical scavengers 4-hydroxy-2,2,6,6-tetramethylpiperidin-N-oxide (TEMPOL) and Salen-Mn(III) complex of o-vanillin (EUK-134). Our preliminary results indicate that these targeted compounds, while delaying apoptosis after an exogenous oxidative insult, are not more active than their untargeted variants. This questions the general efficiency of the targeting procedure used and/or suggests that the main pro-apoptotic effector targets of exogenous oxidative insults are not located within mitochondria.

New access to trisubstituted 3-pyrrolines under phosphine catalysis.

Conjugated dienes, properly activated by electron-withdrawing groups on both ends, are shown to be suitable substrates for phosphine-promoted organocatalytic processes. Their reactions with imines, under phosphine catalysis, afford a new and efficient synthetic approach to functionalized 3-pyrrolines.