Bacterial lipid II analogs : novel in vitro substrates for mammalian oligosaccharyl diphosphodolichol diphosphatase (DLODP) activities

Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities

Synthetic Route to Glycosyl β-1C-(phosphino)-phosphonates as Unprecedented Stable Glycosyl Diphosphate Analogs and Their Preliminary Biological Evaluation

International audienceThe synthesis of glycosyl-β-1C-(phosphino)-phosphonates is a challenge since it has not yet been described. In this paper, we report an innovative synthetic method for their preparation from Glc-, Man-, and GlcNAc-lactone derivatives. The proposed original strategy involves the addition of the corresponding δ-hexonolactones onto the dianion of (methylphosphino) phosphonate as a key step, followed by dehydration and stereoselective addition of dihydrogen on the resulting double bond. Final deprotection provides the new glycosyl diphosphate analogs in 35%, 36%, and 10% yield over 6 steps from the corresponding δ-hexonolactones. The synthetized compounds were evaluated as inhibitors of phosphatase and diphosphatase activities and found to have complex concentration-dependent activatory and inhibitory properties on alkaline phosphatase. The synthetized tools should be useful to study other enzymes such as transferases

Synthesis and glycosidase inhibitory activity of new hexa-substituted C8-glycomimetics

BACKGROUND: Glycosidases are involved in several metabolic pathways and the development of inhibitors is an important challenge towards the treatment of diseases, such as diabetes, cancer and viral infections including AIDS. Thus, inhibition of intestinal α-glucosidases can be used to treat diabetes through the lowering of blood glucose levels, and α-glucosidase inhibitors are being marketed against type 2 (non-insulinodependent mellitus) diabetes (i.e.: Glyset(® )or Diastabol(®), Basen(® )and Glucor(® )or Precose(®)). RESULTS: In that context, new C8-carbasugars and related aminocyclitols have been targeted in order to study the effect of the enhanced flexibility and of the new spatial distribution displayed by these structures on their adaptability in the active site of the enzymes. The synthesis of these new C8-glycomimetics is described from enantiomerically pure C(2)-symmetrical polyhydroxylated cyclooctenes. Their obtention notably involved a syn-dihydroxylation, and more extended functionalization through formation of a cis-cyclic sulfate followed by amination and subsequent reductive amination. This strategy involving the nucleophilic opening of a cis-cyclic sulfate by sodium azide is to our knowledge the first example in C8-series. It revealead to be an efficient alternative to the nucleoplilic opening of an epoxide moiety which proved unsuccessful in this particular case, due to the hindered conformation of such epoxides as demonstrated by X-ray cristallographic analysis. CONCLUSION: The biological activity of the synthesized glycomimetics has been evaluated towards 24 commercially available glycosidases. The weak observed activities can probably be related to the spatial disposition of the hydroxy and amino groups which depart too much from that realized in glycomimetics such as valiolamine, voglibose and valienamine. Nevertheless, the synthetic strategy described here is efficient and general, and could be extended to increase the diversity of the glycosidase inhibitors obtained since this diversity is introduced in an ultimate step of the synthesis

Synthesis and biological evaluation of potential new inhibitors of the bacterial transferase MraY with a β-ketophosphonate structure

International audienc

Synthesis and biological evaluation of chemical tools for the study of Dolichol Linked Oligosaccharide Diphosphatase (DLODP).

International audienceCitronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [(3)H]OSP from [(3)H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity

A Diastereoselective Synthesis of 5′-Substituted-Uridine Derivatives

A straightforward strategy for the synthesis of 5′-substituted-uridine derivatives is described. It relies on the introduction of various substituents at C-5′ at the last step of the synthesis by regioselective nucleophilic opening of a unique epoxide that provides access to a small library of compounds. This epoxide results from the diastereoselective epoxidation, performed at a multigram scale, of a uridine-derived alkene. The configuration of the newly created 5′ asymmetric center has been unambiguously assigned by X-ray diffraction analysis

Toward Analogues of MraY Natural Inhibitors: Synthesis of 5′-Triazole-Substituted-Aminoribosyl Uridines Through a Cu-Catalyzed Azide–Alkyne Cycloaddition

A straightforward strategy for the synthesis of triazole-containing MraY inhibitors has been developed. It involves the sequential introduction of a terminal alkyne at the 5′ position of an uridine derivative and <i>O</i>-glycosylation with a protected aminoribose leading to an elaborated alkyne scaffold. An efficient Cu­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) allowed the introduction of chemical diversity toward a small library of inhibitors

A Sub-Micromolar MraYAA Inhibitor with an Aminoribosyl Uridine Structure and a (S,S)-Tartaric Diamide: Synthesis, Biological Evaluation and Molecular Modeling

International audienceNew inhibitors of the bacterial tranferase MraY are described. Their structure is based on an aminoribosyl uridine scaffold, which is known to be important for the biological activity of natural MraY inhibitors. A decyl alkyl chain was introduced onto this scaffold through various linkers. The synthesized compounds were tested against the MraYAA transferase activity, and the most active compound with an original (S,S)-tartaric diamide linker inhibits MraY activity with an IC50 equal to 0.37 µM. Their antibacterial activity was also evaluated on a panel of Gram-positive and Gram-negative strains; however, the compounds showed no antibacterial activity. Docking and molecular dynamics studies revealed that this new linker established two stabilizing key interactions with N190 and H325, as observed for the highly potent inhibitors carbacaprazamycin, muraymycin D2 and tunicamycin