Stereoselective Synthesis of 1-C-Diethylphosphonomethyl and -difluoromethyl Iminosugars from Sugar Lactams.

A strategy allowing the straightforward synthesis of 1-C-phosphonomethyl and 1-C-phosphonodifluoromethyl iminosugars is reported. Conversion of sugar lactams to the corresponding imines with Schwartz's reagent followed by their reaction with LiCH2P(O)(OEt)2 and LiCF2P(O)(OEt)2 stereoselectively afforded the 1,2-cis and 1,2-trans glycosyl phosphonates, respectively, in modest to good yields. Application of this methodology to C-2 orthogonally protected sugar lactams paved the way to 2-acetamido- and 2-deoxy-1-C-phosphonomethyl iminosugars.

Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle.

Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co(2+)-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [(3)H]OSP release from [(3)H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [(3)H]OSP release from [(3)H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction.

Efficient synthesis of both diastereomers of ß,γ-diamino acids from phenylalanine and tryptophan.

We synthesized in a few steps both diastereomers of orthogonally protected ß,γ-diamino acids starting from L-phenylalanine or L-tryptophan. These final compounds are interesting building blocks for peptide synthesis and foldamer chemistry. The key step is a Blaise reaction performed under ultrasound conditions.

Antibody recognition in multiple sclerosis and Rett syndrome using a collection of linear and cyclic N-glucosylated antigenic probes.

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I' ß-turn N-glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients' sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different ß-turn conformations, which were evaluated in inhibition enzyme-linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)-2-amino-4-pentynoic acid (L-Pra) residues Ac-Pra-RRN(Glc)GHT-Pra-NH2 , with an IC50 in the nanomolar range. This peptide was adequately modified for solid-phase ELISA (SP-ELISA) and surface plasmon resonance (SPR) experiments. Pra-RRN(Glc)GHT-Pra-NH2 peptide was modified with an alkyl chain linked to the N-terminus, favoring immobilization on solid phase in SP-ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac-Pra-RRN(Glc)GHT-Pra-NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).

Glaser oxidative coupling on peptides: stabilization of ß-turn structure via a 1,3-butadiyne constraint.

The Glaser-Eglinton reaction between either two C or N propargylglycine (Pra or NPra) amino acids, in the presence of copper(II), led to cyclic hexa- and octapeptides constrained by a butadiyne bridge. The on-resin cyclization conditions were analyzed and optimized. The consequences of this type of constraint on the three dimensional structure of these hexapeptides and octapeptides were analyzed in details by NMR and molecular dynamics. We show that stabilized short cyclic peptides could be readily prepared via the Glaser oxidative coupling either with a chiral (Pra), or achiral (NPra) residue. The 1,3-butadiyne cyclization, along with disulfide bridged and lactam cyclized hexapeptides expands the range of constrained peptides that will allow exploring the breathing of amino acids around a ß-turn structure.

Lipopolysaccharide biosynthesis and traffic in the envelope of the pathogen Brucella abortus.

Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus, smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp. involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify an original class of bifunctional O-antigen ligases.

Sugar-Derived Amidines and Congeners: Structures, Glycosidase Inhibition and Applications.

Glycosidases, the enzymes responsible for the breakdown of glycoconjugates, including di-, oligo- and polysaccharides, are present across all kingdoms of life. The extreme chemical stability of the glycosidic bond combined with the catalytic rates achieved by glycosidases makes them among the most proficient of all enzymes. Given their multitude of roles in vivo, inhibition of these enzymes is highly attractive with potential in the treatment of a vast array of pathologies ranging from lysosomal storage and diabetes to viral infections. Therefore great efforts have been invested in the last three decades to design and synthesize inhibitors of glycosidases leading to a number of drugs currently on the market. Amongst the vast array of structures that have been disclosed, sugars incorporating an amidine moiety have been the focus of many research groups around the world because of their glycosidase transition state-like structure. In this review, we report and discuss the structure, the inhibition profile, and the use of these molecules, including related structural congeners as transition state analogs.

Stereoselective Access to Iminosugar C,C-Glycosides from 6-Azidoketopyranoses.

We report the synthesis of iminosugar C,C-glycosides starting from 6-azidoketopyranoses. Their Staudinger-azaWittig-mediated cyclization provided bicyclic N,O-acetals, which were stereoselectively opened with AllMgBr to afford ß-hydroxyazepanes with a quaternary carbon α to the nitrogen. Their ring contraction via a ß-aminoalcohol rearrangement produced the six-membered l-iminosugars with two functional handles at the pseudoanomeric position. Inversion of the free OH at the azepane level furnished the d-iminosugars.

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

Stable analogs of bacterial transferase MraY substrate or product with a pyrophosphate surrogate in their structure are described. ß-ketophosphonates were designed as pyrophosphate bioisosteres and were investigated as UDP-GlcNAc mimics. The developed strategy allows introduction of structural diversity at a late stage of the synthesis. The biological activity of the synthesized compounds was evaluated on the MraY enzyme.

Multi-Stage Mass Spectrometry Analysis of Sugar-Conjugated ß-Turn Structures to be Used as Probes in Autoimmune Diseases.

Synthetic sugar-modified peptides were identified as antigenic probes in the context of autoimmune diseases. The aim of this work is to provide a mechanistic study on the fragmentation of different glycosylated analogs of a synthetic antigenic probe able to detect antibodies in a subpopulation of multiple sclerosis patients. In particular the N-glucosylated type I' ß-turn peptide structure called CSF114(Glc) was used as a model to find signature fragmentations exploring the potential of multi-stage mass spectrometry by MALDI-LTQ Orbitrap. Here we compare the fragmentation of the glucosylated form of the synthetic peptide CSF114(Glc), bearing a glucose moiety on an asparagine residue, with less or non- immunoreactive forms, bearing different sugar-modifications, such as CSF114(GlcNAc), modified with a residue of N-acetylglucosamine, and CSF114[Lys(7)(1-deoxyfructopyranosyl)], this last one modified with a 1-deoxyfructopyranosyl moiety on a lysine at position 7. The analysis was set up using a synthetic compound specifically deuterated on the C-1 to compare its fragmentation with the fragmentation of the undeuterated form, and thus ascertain with confidence the presence on an Asn(Glc) within a peptide sequence. At the end of the study, our analysis led to the identification of signature neutral losses inside the sugar moieties to characterize the different types of glycosylation/glycation. The interest of this study lies in the possibility of applyimg this approach to the discovery of biomarkers and in the diagnosis of autoimmune diseases. Graphical Abstract .

Synthesis of pyrrolidine-based analogues of 2-acetamidosugars as N-acetyl-D-glucosaminidase inhibitors.

A ring-contraction strategy applied to ß-azido,γ-hydroxyazepanes yielded after functional group manipulation new tetrahydroxylated pyrrolidines displaying an acetamido moiety, one of these iminosugars demonstrating low micromolar inhibition on N-acetylglucosaminidases.

Synthesis of 1,2-cis-homoiminosugars derived from GlcNAc and GalNAc exploiting a ß-amino alcohol skeletal rearrangement.

The synthesis of 1,2-cis-homoiminosugars bearing an NHAc group at the C-2 position is described. The key step to prepare these α-D-GlcNAc and α-D-GalNAc mimics utilizes a ß-amino alcohol skeletal rearrangement applied to an azepane precursor. This strategy also allows access to naturally occurring α-HGJ and α-HNJ. The α-D-GlcNAc-configured iminosugar was coupled to a glucoside acceptor to yield a novel pseudodisaccharide. Preliminary glycosidase inhibition evaluation indicates that the α-D-GalNAc-configured homoiminosugar is a potent and selective α-N-acetylgalactosaminidase inhibitor.

Synthesis of 1,2-trans-2-acetamido-2-deoxyhomoiminosugars.

The first synthesis of 1,2-trans-homoiminosugars devised as mimics of ß-D-GlcNAc and α-D-ManNAc is described. Key steps include a regioselective azidolysis of a cyclic sulfite and a ß-amino alcohol skeletal rearrangement applied to a polyhydroxylated azepane. The ß-D-GlcNAc derivative has been coupled to serine to deliver an iminosugar C-amino acid. The two homoiminosugars demonstrate moderate glycosidase inhibition.