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O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of Vibrio cholerae O100 and studied by component analyses and 1D and 2D NMR spectroscopy. The following structure of the O-polysaccharide was established: →3)-β- d -QuipNAc4N(dHh)-(1 → 3)-α- d -Fucp4N(RHb)-(1 → 3)-α- l -FucpNAc-(1→ where Hb and dHh indicate 3-hydroxybutanoyl and 3,5-dihydroxyhexanoyl, respectively. The O-antigen gene cluster of V. cholerae O100 has been sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the OPS structure.

N-acetylglucosamine 6-phosphate deacetylase (NagA) catalyzes the conversion of N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 A. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artifact using single particle electron cryo-microscopy. Analysis of the determined crystal structure reveals a set of hot-spot residues involved in novel interactions at the dimer-dimer interface. PmNagA binds to one Zn2+ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10-fold the KM ), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation–reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.

WOS: 000403692800006, Four new chiral Schiff base ligands (3a-4a, 3b-4b) have been prepared using aminoisopropylidene derivatives of glucose and aminochloralose derivatives of mannose. The synthesized compounds 1 and 2 were characterized by nuclear magnetic resonance (H-1 and C-13 NMR), Fourier transform infrared spectroscopy (FTIR), UV/visible spectroscopy, and elemental analysis. A total of eight Schiff base compounds (3a-6a, 3b-6b) were tested for antimicrobial activity against Escherichia coli ATCC 12228, Staphylococcus aureus ATCC 6538, Klebsiella pneumoniae CCM 2318, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium CCM 5445, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, and Candida albicans ATCC 10239 and exhibited a range of activities.

Enzymes belonging to the GNAT superfamily are widely distributed in nature where they play key roles in the transfer of acyl groups from acyl-CoAs to primary amine acceptors. The amine acceptors run the gamut from histones to aminoglycoside antibiotics to small molecules such as serotonin. Whereas those family members that function on histones have been extensively studied, the GNAT enzymes that employ nucleotide-linked sugars as their substrates have not been well characterized. Indeed, though the structures of two of these “amino sugar” GNAT enzymes have been determined within the past 10 years, details concerning their active site architectures have been limited because of a lack of bound nucleotide-linked sugar substrates. Here we describe a combined structural and biochemical analysis of FdhC from Acinetobacter nosocomialis O2. On the basis of bioinformatics, it was postulated that FdhC catalyzes the transfer of a 3-hydroxybutanoyl group from 3-hydroxylbutanoyl-CoA to dTDP-3-amino-3,6-dideoxy-d-galactose, to yield an unusual sugar that is ultimately incorporated into the surface polysaccharides of the bacterium. We present data confirming this activity. In addition, the structures of two ternary complexes of FdhC, in the presence of CoA and either 3-hydroxybutanoylamino-3,6-dideoxy-d-galactose or 3-hydroxybutanoylamino-3,6-dideoxy-d-glucose, were solved by X-ray crystallographic analyses to high resolution. Kinetic parameters were determined, and activity assays demonstrated that FdhC can also utilize acetyl-CoA, 3-methylcrotonyl-CoA, or hexanoyl-CoA as acyl donors, albeit at reduced rates. Site-directed mutagenesis experiments were conducted to probe the catalytic mechanism of FdhC. Taken together, the data presented herein provide significantly new molecular insight into those GNAT superfamily members that function on nucleotide-linked amino sugars.

The antibiotic streptothricin (ST) possesses an amino sugar bound to an l -β-lysine (β-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a β-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics. IMPORTANCE The antibiotic streptothricin (ST) possesses an amino sugar bound to an l -β-lysine (β-Lys) side chain via a peptide bond formed by a nonribosomal peptide synthetase (NRPS). BD-12, an analogue of ST, carries a glycine-derived side chain rather than β-Lys, and here, we describe the BD-12-biosynthetic gene cluster from Streptomyces luteocolor NBRC13826, which contains the orf11 gene encoding a novel tRNA-dependent peptide bond-forming enzyme. The unique Fem-like enzyme (Orf11) accepts the amino sugar as a substrate and mediates the peptide formation between the amino sugar intermediate and glycine. Our studies demonstrate that the structural diversity of the side chains of ST-related compounds in nature is generated via two distinct peptide bond-forming mechanisms.

Lipopolysaccharides (LPSs) play key roles in humoral immunity. Recently, the LPS structure of the Psychrobacter cryohalolentis K5T strain was reported. Due to the presence of unnatural amino sugars and branched linkages, its structure is unique. Herein we report the total synthesis of an LPS analogue of P. cryohalolentis K5T. After overcoming the issues like ring conformation changes and elimination of triflate, we were able to develop a strategy for the synthesis of the newly reported 2,3,4-triacetamido-2,3,4-trideoxy-l-arabinose derivative. Coupling of different donors with suitable acceptors from the nonreducing end to the reducing end and further functional group modifications delivered the protected LPS hexasaccharide repeating unit. After functional group modifications, we were unable to oxidize the hindered primary hydroxyl group to synthesize the target molecule. Alternatively, removal of the permanent protecting groups afforded the LPS hexasaccharide repeating unit analogue of Psychrobacter cryoh...

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa-bridged enediyne with two attached sugars, l-mycarose, and l-kedarosamine. The biosynthesis of l-kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N-methyltransferase that utilizes S-adenosylmethionine as the methyl donor and a dTDP-linked C-4' amino sugar as the substrate. Here we describe the three-dimensional architecture of KedS8 in complex with S-adenosylhomocysteine. The structure was solved to 2.0 A resolution and refined to an overall R-factor of 17.1%. Unlike that observed for other sugar N-methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β-strands at the N-termini of its subunits. The structure presented here represents the first example of an N-methyltransferase that functions on C-4' rather than C-3' amino sugars.

A number of novel 2-(N-(2-(5,5-dimethyl-1,3-dioxane-2-yl)ethyl)aminoacyl)amino-2-deoxy- d-glucopyranoside were synthesized from a readily available starting material, glucosamine, 2,2-dimethyl-1,3-propanediol and 1,1,3,3-tetramethoxypropane, and evaluated for their anti-inflammatory activity. Our results showed that all of the compounds tested exhibited a significant inhibition of xylene-induced inflammation in mice.

A series of quinoxaline azido reverse nucleosides 3a-c and their O-regioisomers 4a-c was prepared by reaction of quinoxaline 1a-c with 3-azido-3-deoxy-1,2-O-isopropylidene-5-p-toluenesulfonyl-D-ribofuranose (2) in the presence of sodium hydride. Structure modification of these interesting structures includes reduction and the subsequent acetylation reactions to give quinoxaline amino and acetyl amino reverse nucleosides and their O-regioisomers.

For the synthesis of novel spinosyn analogues, the selective introduction of the 2-deoxyamino sugar, D-forosamine, necessary for their bioactivity, is described. The reactions of different alcohol acceptors with the D-forosamine donor in the presence of iodine and Et3SiH gave the corresponding glycosides in yields of up to 96 % and with an α/β selectivity of 2:3.

A novel sugar adduct, 4,6-dimethoxy-1,3,5-triazin-2-yl β-d-glucosaminide (GlcN-β-DMT), has been prepared by the reaction of d-glucosamine (GlcN) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM). The adduct was recognized by exo-β-d-glucosaminidase (GlcNase) from Amycolatopsis orientalis and transferred the GlcN moiety, giving rise to the corresponding glucosaminides. This chemo-enzymatic process was successfully applied to d-galactosamine (GalN). GalN-β-DMT prepared directly from GalN and DMT-MM behaved as an efficient glycosyl donor for transfer of the GalN moiety catalyzed by the same enzyme. The introduction of the 4,6-dimethoxy-1,3,5-triazin-2-yl leaving group to the anomeric center significantly enhanced transglycosylating ability, resulting in the efficient glycosidase-catalyzed synthesis of glycosaminides.

(−)-α-Conhydrine was synthesized from propargyl alcohol in 20% overall yield in nine steps. The key intermediates were obtained via a regioselective epoxide opening, cross-metathesis, tandem hydrogenation and hydrogenolysis, and stereoselective dihydroxylaton. The side product from the epoxide ring opening was used to synthesize the seven-membered amino sugar DMJ analogue.

Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro sugar, l-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)-l-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-l-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of (18)O(2) establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 Å resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

A stereodivergent synthesis of differently configured C2-branched 4-amino sugar derivatives was accomplished. The Lewis acid mediated rearrangement of phenylthio-substituted 1,2-oxazines delivered glycosyl donor equivalents that can directly be employed in glycosidation reactions. Treatment with methanol provided internally protected amino sugar equivalents that have been transformed into the stereoisomeric methyl glycosides 28, ent-28, 29, ent-29 and 34 in two simple reductive steps. Reaction with natural carbohydrates or bicyclic amino sugar precursors allowed the synthesis of homo-oligomeric di- and trisaccharides 44, 46 and 47 or a hybrid trisaccharide 51 with natural carbohydrates. Access to a bivalent amino sugar derivative 54 was accomplished by reaction of rearrangement product 10 with 1,5-pentanediol. Alternatively, when a protected L -serine derivative was employed as glycosyl acceptor, the glycosylated amino acid 60 was efficiently prepared in few steps. In this report we describe the synthesis of unusual amino sugar building blocks from enantiopure 1,2-oxazines that can be attached to natural carbohydrates or natural product aglycons to produce new natural product analogues with potential applications in medicinal chemistry.

The amino sugar moieties of staurosporine and nogalarol have been stereospecifically synthesized by a strategy utilizing N-sulfinyl Diels-Alder chemistry.

A flexible strategy for the stereoselective synthesis of branched amino sugar analogues is described. It is based on a C 3 -chain elongation of suitable protected aldoses. By using the sequence homoaldol reaction, epoxidation, and methanolysis α-methyl allo-furanosides are obtained. Proximate amination of the corresponding triflates afford the title compounds. All reactions proceed with high yield, high diastereoselectivities, and allow for a broad application.

Two bis-C-aminoglycosyl arenes containing the angolosamine and the vancosamine moieties, which are potentially useful as the D-ring fragments of the pluramycin-type antibiotics, were efficiently synthesized by the O→C-glycoside rearrangement based strategy.

Well concealed: 1,2-oxazines such as 1 rearrange under Lewis acidic conditions to bicyclic products of type 2, which can be incorporated into oligosaccharides as protected amino sugar equivalents. Subsequent reductive steps provide unusual oligosaccharides 3 having C2-branched 4-amino sugar units. Most of the reactions proceed with excellent stereocontrol and allow the synthesis of a collection of stereoisomers.

In alcohols and esters, a neighbouring dialkylamino group can enhance the reactivity towards acylation and deacylation, respectively, that is, such amino alcohols can act as transacylation catalysts like DMAP. This effect is dependent on the number of (carbon) spacer atoms, flexibility of the molecule and the presence and position of further heteroatoms. Based on this effect, the site selective acylation and deacylation of desmycosin, a macrocycle antibiotic possessing an amino sugar moiety, is described.

有機合成において 30 年以前に開発された 2-アジド糖供与体が，cis-選択的アミノ糖グリコシル化反応において，現在においても第一選択として用いられるが，選択性，調製に問題点がある。我々は trans-カーバメート糖がグリコシル化反応において非常に高い cis-選択性を示すことを見出した。trans-カーバメート糖はその調製も簡便，高収率である。また，この糖供与体を用いて抗ピロリ菌活性を持つ糖鎖合成を行い，trans-カーバメート糖がオリゴ糖鎖合成に実践的に有用であることを示した。

The acylation of unprotected vancomycin or eremomycin with activated esters of N(alpha)-protected amino acids or N(alpha)-alkyl-N(alpha)-Fmoc-amino acids is directed selectively to the amino group of the disaccharide branch (N') and after Fmoc-group removal leads to the corresponding N'-alpha-aminoacyl derivatives. A series of N'-alpha-aminoacyl and N'-alpha-(N(alpha)-alkylamino)acyl derivatives of eremomycin and vancomycin containing hydrophobic moieties has been synthesized. The structures of all derivatives were confirmed by Electrospray Ionization mass-spectral (ESI MS) analysis, and by chemical degradation methods. Position of the introduced N'-alpha-aminoacyl- and N-(N(alpha)-alkylamino)acyl groups were determined after Edman degradation and acidic hydrolysis. The structures of the synthesized starting reagents (N(alpha)-alkylamino acids or N(alpha)-alkyl-N(alpha)-Fmocamino acids) were confirmed by NMR-spectra data. In general, N'-(N-alkylglycyl)-derivatives were more active than the corresponding N'-alpha-(N(alpha)-alkylamino)acylated derivatives containing other amino acids (L-Lys, L-Met, L-Orn, L- and D-Ala, L- and D-Phe and benzyl-O-L-Tyr).

Semicarbazide-sensitive amine oxidase (SSAO) also functions as a vascular-adhesion protein (VAP-1). The nature of the target site on lymphocytes to which endothelial-cell SSAO/VAP-1 binds is unknown. We have shown that amino sugars (galactosamine, glucosamine and mannosamine), which are not SSAO substrates, can bind to the enzyme as reversible inhibitors. Thus, they serve as a model system in which to study the interaction process. Binding occurred during substrate (benzylamine) oxidation but not when the amino sugar was incubated, for extended periods, with SSAO alone. These results suggest that one, or more of the products of the SSAO-catalysed amine oxidation might be necessary for the inhibitory process to occur. Two of the reaction products of benzylamine oxidation, benzaldehyde and ammonia were found to have no effect on the inhibition of SSAO by galactosamine. Preincubation of the enzyme with galactosamine plus H(2)O(2) was, however, found to result in time-dependent inhibition. This is not a result of the non-enzymic reaction between H(2)O(2) and the amino sugar, since preincubation of galactosamine with H(2)O(2) alone, for extended periods, did not give rise to an inhibitory species. The amount of exogenously added H(2)O(2) necessary for inhibition was very much greater than that formed during substrate oxidation. These results suggest that the H(2)O(2) formed as a product of the SSAO-catalysed oxidation reaction is more efective in promoting the binding of amino sugars.

Lemonose is a component of the antibiotic lemonomycin and other antibiotics and natural products. Three routes to the synthesis of the title compound, a protected, desmethyl derivative of lemonose, from l-rhamnose or its glycal, were investigated based on electrophilic cyclization, epoxidation-ring opening, and deoxygenation of an intermediate that was used in the synthesis of the amino sugar callipeltose. The deoxygenation route was successful and it provided the title compound, which was then converted to a phenyl thioglycoside.

An acidic O-specific polysaccharide (PS) of the agar-digesting bacterium Shewanella japonica with the type strain KMM 3299 T was obtained by mild acid hydrolysis of the lipopolysaccharide. The polysaccharide was studied by component analysis, methylation analysis, 1 H and 13 C NMR spectroscopy, including 2D NMR experiments. The PS was determined to have the following structure involving three unusual amino sugars: → 3 ) - α - D - Fuc p 4 NAc - ( 1 → 4 ) - α - D - Gal p A - ( 1 → 3 ) - α - L - Fuc p NAc - ( 1 → 3 ) - β - D - Qui p NAc 4 NAc - ( 1 →

The conversion of uridine diphosphate N-acetyl-D-glucosamine into uridine diphosphate N-acetyl-L-fucosamine was demonstrated with enzymes from cytoplasmic fraction of Salmonella arizonae O:59 cells in the presence of NAD+ (NADP+) and NADPH. The reaction product was identified by ion-pair, reverse-phase HPLC with the use of synthetic nucleoside diphosphate sugar standards under conditions specially developed for separation of uridine diphosphate 2-acetamido-2,6-dideoxyhexoses. L-Fucose dehydrogenase from porcine liver was shown to be applicable for determination of N-acetyl-L-fucosamine, this enzyme being used to confirm L-configuration of the amino sugar residue in the sugar nucleotide formed.

5 pages, 1 figure, 2 schemes.-- Available online Dec 8, 2004.-- Taken in part from the Ph.D. thesis of AM., A stereocontrolled synthesis of an unsaturated sugar bearing two amino groups (one of them masked as an azide), using an Overman rearrangement as key step, is described. This scaffold is used to prepare two peptides having aromatic fragments, which have shown activity as calpain inhibitors., Financial support from Janssen-Cilag and Spanish Ministry of Science and Technology (project # BQU2001-2270) is gratefully acknowledged. A.M. thanks CSIC for an EPO-fellowship.

No orthologs are present in the genomes of the archaea encoding genes for the first two steps in the biosynthesis of the aromatic amino acids leading to 3-dehydroquinate (DHQ). The absence of these genes prompted me to examine the nature of the reactions involved in the archaeal pathway leading to DHQ in Methanocaldococcus jannaschii. Here I report that 6-deoxy-5-ketofructose 1-phosphate and l-aspartate semialdehyde are precursors to DHQ. The sugar, which is derived from glucose 6-P, supplies a "hydroxyacetone" fragment, which, via a transaldolase reaction, undergoes an aldol condensation with the l-aspartate semialdehyde to form 2-amino-3,7-dideoxy-D-threo-hept-6-ulosonic acid. Despite the fact that both hydroxyacetone and hydroxyacetone-P were measured in the cell extracts and confirmed to arise from glucose 6-P, neither compound was found to serve as a precursor to DHQ. This amino sugar then undergoes a NAD dependent oxidative deamination to produce 3,7-dideoxy-d-threo-hept-2,6-diulosonic acid which cyclizes to 3-dehydroquinate. The protein product of the M. jannaschii MJ0400 gene catalyzes the transaldolase reaction and the protein product of the MJ1249 gene catalyzes the oxidative deamination and the cyclization reactions. The DHQ is readily converted into dehydroshikimate and shikimate in M. jannaschii cell extracts, consistent with the remaining steps and genes in the pathway being the same as in the established shikimate pathway.

A synthesis of the amino sugar 2-amino-2,3-didesoxyribose is described. Starting from D-glucosamine, β-methylfuranoside was obtained in eight steps in 20% yield. This carbohydrate is a novel building block for nucleosides and for backbone modified antisense oligonucleotides with 2′–5′ amide linkages.

The synthesis of azido analogues 2a,2b of the pyranonaphthoquinone antibiotic medermycin 1 has been achieved in eight steps from naphthol 8 and azido glycosyl sugar 7 in 9.3% overall yield. Key steps include the direct BF 3 .Et 2 O promoted C-glycosylation of naphthol 8, introduction of an acetyl group onto a bromonaphthoquinone via Stille coupling with (α-ethoxy-vinyl)tributyltin, furofuran annulation of a naphthoquinone to a furonaphthofuran using 2-trimethylsilyloxyfuran 5 and oxidative rearrangement of a furonaphthofuran to a furonaphthopyran.

There is a growing interest on glycol-split low-molecular weight heparins (gs-LMWHs), obtained by periodate oxidation of LMWHs, optionally followed by borohydride reduction, as potential anticancer and anti-inflammatory drugs. However, their structural characterization is still a challenging task, mainly because of the high microheterogeneity of the starting material. In addition, susceptibility to oxidation of some end-groups of LMWHs induces additional heterogeneity, making analysis of gs-LMWHs more complex. In our previous study we showed that 1,6-anhydro-d-mannosamine N-sulfate was affected by periodate, while its epimer 1,6-anhydro-d-glucosamine N-sulfate was resistant. In order to understand the apparently anomalous behavior of terminal 1,6-anhydro-d-mannosamine N-sulfate residues, in the present work we have studied by NMR spectroscopy and LC/MS the behavior of the reducing end amino sugar residues of the tetrasaccharides, isolated from the LMWH enoxaparin, in the presence of periodate. Their molecular mechanics conformational characterization has been also performed. We have shown that the C(2)-C(3) bond of the 1,6-anhydro-d-mannosamine residue can be split by periodate despite the N-substitution. Moreover, we have found that both terminal d-mannosamine N-sulfate and d-glucosamine N-sulfate, lacking the 1,6-anhydro-bridge, can be also oxidized by periodate but with a significantly lower rate. The present results suggest that the cis-e-/a-position of OH and NHSO3(-) groups of N-sulfated 1,6-anhydro-d-mannosamine is not the only factor that makes these end residues susceptible to the oxidation. The 1,6-anhydro-bridge that 'blocks' the ring conformation appears another crucial factor for oxidation to occur. Moreover, we have shown that controlling the reaction time could permit to selectively split non-sulfated iduronic acids of enoxaparin chains without oxidizing terminal amino sugar residues, a finding that may be useful to obtain more structurally homogeneous gs-LMHWs.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss−pmrA C mutant of E. coli and assign its structure as undecaprenyl phosphate-α-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M − H]− at m/z 977.5, consistent with undecaprenyl phosphate-α-l-Ara4N (M r = 978.41). 31P NMR spectroscopy showed a single phosphorus atom at −0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional 1H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IVA to lipid IIA, which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-α-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

A high-molecular-mass O-specific polysaccharide was obtained by mild acid degradation of Proteus vulgaris O4 lipopolysaccharide followed by GPC. The polysaccharide was studied by chemical methods along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, H-detected 1H,13C HMQC, and 1H,13C HMBC experiments. Solvolysis of the polysaccharide with trifluoromethanesulfonic (triflic) acid resulted in a GlcpA-(1→3)-GlcNAc disaccharide and a novel amino sugar derivative, 4,6-dideoxy-4-{N-[(R)-3-hydroxybutyryl]- l -alanyl}amino- d -glucose [Qui4N(HbAla)]. On the basis of the data obtained, the following structure of the tetrasaccharide repeating unit of the O-specific polysaccharide was established: →4 )- β - d - Glc p A -(1 →3 )- β - d - Glc p NAc -(1 →2 )- β - d - Qui p4 N ( HbAla )-(1 →3 )- α - d - Gal p-(1 → This structure is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied in a separate Proteus serogroup.

The structure of methyl 4-amino-4-cyano-4,6-dideoxy-2,3- O -isopropylidene-α- L -talopyranoside was established by X-ray analysis confirming a talo configuration at C-4 andsuggesting a 1 C 4 conformation of the pyranose ring. The values of relevant torsion anglesand calculated puckering parameters revealed a distortion into the direction of 5 E con-formation, thus indicating a flattening at C-2. Keywords: Amino nitrile, methyl talopyranoside, amino sugar, X-ray analysis. Introduction The amino sugars represent a very important class of organic compounds primarily because of theirbiological role in living organisms. Their presence in bacteria and all tissues and fluids of pluricellularorganisms as well as their association with many proteins and lipids suggests a great medicinalimportance. The rapid increase of knowledge in the field of relationship between structure of aminosugar-containing compounds and biological activity requires availability many of suitable syntheticallyprepared model compounds with well established structures.

The synthesis of methyl (β- d -glucopyranosyluronic acid)-(1→3)-(2-acetamido-2-deoxy-6- O -sulfonato-β- d -galactopyranosyl)-(1→4)-(β- d -glucopyranosid)uronate trisodium salt, a chondroitin 6-sulfate trisaccharide derivative, is described. Loss of stereocontrol in glycosylation reactions involving activated 4,6- O -benzylidene derivatives of the 2-deoxy-2-trichloroacetamido- d - galacto series and d -glucuronic acid-derived acceptors was highlighted. This drawback was overcome through the use of phenyl 3,4,6-tri- O -acetyl-2-deoxy-1-thio-2-trichloroacetamido-β- d -galactopyranoside, which afforded the desired β-linked disaccharide derivative in high yield with an excellent stereoselectivity. This later was submitted to acid-catalyzed methanolysis, followed by benzylidenation, and condensed with methyl 2,3,4-tri- O -benzoyl-1- O -trichloroacetimidoyl-α- d -glucopyranuronate to afford the expected trisaccharide derivative. Subsequent transformation of the N -trichloroacetyl group into N -acetyl, mild acid hydrolysis, selective O-sulfonation at C-6 of the amino sugar moiety, and saponification afforded the target molecule as its sodium salt in high yield.

An extension of the latent-active glycosylation strategy is reported whereby 3-buten-2-yl 2-deoxy-2-azidoglycosides and 3-buten-2-yl 2-deoxy-2-phthalimidoglycosides are used as building blocks for the preparation of amino sugar containing oligosaccharides. The allyl moieties of the latent substrates 5, 16 and 19 can be conveniently isomerised by treatment with a catalytic amount of (Ph3P)3RhCl/BuLi to give the active vinyl glycosides 6, 17 and 20 in high yield. These glycosyl donors were successfully used in glycosylations with acceptors 7, 9 and 11. In the case of glycosyl donor 6, the disaccharides 8, 10 and 12 could be obtained as anomeric mixtures or with high α-or β-selectivities depending on the reaction conditions selected. Glycosylations with glycosyl donors 17 and 20 in each case gave solely the β-linked products only in high yields.

Strategies and tactics associated with the total synthesis of hybrid natural products are discussed. The target is ravidomycin (2), one of the gilvocarcin-class antitumor antibiotics with an aryl C-glycoside structure. The first total synthesis of 2, which was achieved along similar lines of that of gilvocarcin V (1), served for the determination of the relative as well as the absolute stereochemistry of 2. Also revealed was a limitation of the synthetic scheme so long as the amino sugar congener was concerned. A preliminary result is discussed on the [2+2+2] approach that relies on the ready availability of various benzocyclobutene derivatives via regioselective [2+2] cycloaddition of α-alkoxybenzynes and ketene silyl acetals.

A novel approach for asymmetric synthesis of amino sugars is developed, starting from readily available chiral building blocks 1 and 2,3- O -isopropylideneglyceraldehyde 2 , via the Julia olefination and subsequent dihydroxylation as key steps.

The objective of the investigation was to identify the most important organic N-containing fractions extracted from soils by electroultrafiltration (EUF) or a CaCl2 solution, respectively, and their importance for nitrogen mineralization. The investigation comprised 19 agricultural and one forest top soil. Net N mineralization was tested in Mitscherlich pot experiments with three treatments: (1) fallow soil without N fertilizer, (2) soil cultivated with rye grass without N fertilizer, (3) soil cultivated with rye grass with N fertilizer. The highest proportion of N in the extracts was the amino N fraction (amino acids + peptides) amounting to approximately 60% of the total N extracted by CaCl2 and to about 40% of the total N extracted by EUF. The proportion of amino sugars from total N extracted was in average 10% for the CaCl2 and 5.2% for the EUF extracts. The proportion of heterocyclic N bases derived from nucleic acids amounted in average to 4.8% and 3.6% for the CaCl2 and EUF extract, respectively. Amino N (amino acids + peptides) were correlated best with net N mineralization (EUF, r = 0.81***, CaCl2, r = 0.86***). The correlation between amino sugars and net N mineralization was r = 0.55* for the EUF extract and r = 0.49* for the CaCl2 extract. The heterocyclic N bases did not correlate with net N mineralization. Correlations between Norg extracted by CaCl2versus net N mineralization were higher than those obtained by the EUF extract. Net N mineralization was about four times higher in the fallow soils than in the treatment with grass and no N fertilizer. In the treatment with grass + N fertilizer on average no net N mineralization occurred, moreover there was a tendency of N immobilization. It is assumend that in the treatments with grass cultivation, organic C released by roots stimulated the assimilation of mineral N and amino acids by soil microorganisms resulting in a low net N mineralization. Net N mineralization led to a highly significant depletion in the Norg pools and particularly in the amino N and amino sugar pools in the treatment with grass and without N fertilizer. This depletion was particularly evident in the CaCl2 extracts. The results justify the conclusion that the Norg obtained with both extraction methods originates from a dynamic N pool into which N flows in and out. The amino N extractable with EUF or CaCl2 is a reliable indicator for the net N mineralization potential of soils. Stickstoffverbindungen, mittels Elektroultrafiltration (EUF) oder mittels CaCl2-Losung extrahiert, und ihre Beziehungen zur Stickstoffmineralisation in Boden Ziel der Untersuchung war es, die wichtigsten N-haltigen Stoffgruppen, die mittels Elektroultrafiltration (EUF) oder CaCl2-Losung extrahierbar sind, zu identifizieren und deren Bedeutung fur die Netto-N-Mineralisation in Boden zu ermitteln. Die Untersuchungen umfasten 19 landwirtschaftlich genutzte Boden und einen Waldboden. Ihre Netto-N-Mineralisation wurde in einem Mitscherlich-Gefasversuch mit nachstehenden drei Varianten untersucht: (1) unbewachsener Boden ohne N-Dungung, (2) Boden mit Weidelgras ohne N-Dungung, (3) Boden mit Weidelgras mit N-Dungung. Die Stoffgruppe mit hochstem Anteil am N der Extrakte war der Amino-N (Aminosauren + Peptide). Ihr Anteil betrug im Durchschnitt 60% des gesamten mit der CaCl2-Losung und 40% des gesamten mit EUF extrahierten N. Der Anteil an Aminozuckern betrug durchschnittlich 10% im CaCl2-Extrakt und 5,2% im EUF-Extrakt vom gesamten extrahierten N. Die Anteile an heterocyclischen Basen der Nukleinsauren beliefen sich im Durchschnitt auf 4,8% im CaCl2- und 3,6% im EUF-Extrakt. Von diesen drei N-haltigen Stoffgruppen korrelierte der Amino-N am engsten mit r = 0.81*** (EUF-Extrakt) und mit r = 0.86*** (CaCl2-Extrakt) mit der Netto-N-Mineralisation. Die Korrelationskoeffizienten fur die Beziehung Aminozucker versus Netto-N-Mineralisation betrugen 0.55* fur den EUF-Extrakt und 0.49* fur den CaCl2-Extrakt. Die heterocyclischen N-Basen korrelierten nicht mit der Netto-N-Mineralisation. Norg des CaCl2-Extraktes war enger mit der Netto-N-Mineralisation korreliert als Norg des EUF-Extraktes. Im Durchschnitt war die Netto-N-Mineralisation in der unbewachsenen Variante etwa vierfach hoher als in der Variante mit Gras und ohne N-Dungung. In der Variante mit Gras + N-Dungung lag im Durchschnitt keine Netto-N-Mineralisation, sondern die Tendenz zur N-Immobilisierung vor. Es wird angenommen, das in den Varianten mit Grasbewuchs organischer C, der von den Wurzeln freigesetzt wurde, zur Assimilation von mineralischem N und Aminosauren durch Mikroorganismen beitrug, wodurch die Netto-N-Mineralisation vermindert wurde. In der bewachsenen Variante ohne N-Dungung bewirkte die Netto-N-Mineralisation eine hoch signifikante Abnahme des N in den Norg-Pools und besonders eine Abnahme des N in den Amino-N und Aminozucker-Pools. Diese Abnahme war am starksten im N des CaCl2-Extraktes ausgepragt. Die Ergebnisse berechtigen zu dem Schlus, das der Norg beider Extraktionsmethoden einen dynamischen Pool darstellt, in den N hinein- und herausfliest, und das die mittels EUF oder CaCl2-Losung extrahierbaren Amino-N-Verbindungen einen zuverlassigen Indikator fur das N-Mineralisationspotential von Boden darstellen.

Two novel heptadecaglycoside antibiotics, saccharomicins A (1) and B (2), were isolated from a new species of Saccharothrix. Their structures were determined by a combination of chemical degradation and spectroscopic methods. Saccharomicin B (2) was identified as N-(m,p-dihydroxycinnamoyl) taurine p-O-[α-4-epi-vancosaminopyranosyl(1→4)-α-4-epi-vancosaminopyranosyl(1→4)-α-digitoxopyranosyl(1→4)-α- 4-epi-vancosaminopyranosyl(1→4)-α-digitoxopyranosyl(1→3)-β-fucopyranosyl(1→4)-β-saccharosaminopyranosyl(1→4)-α-digitoxopyranosyl(1→3)]-[α-4-epi-vancosaminopyranosyl(1→4)]-β-fucopyranosyl(1→4)-β- saccharosaminopyranosyl(1→4)-α-rhamnopyranosyl(1→3)-β-fucopyranosyl(1→4)-β-saccharosaminopyranosyl(1→4)-β-fucopyranosyl(1→4)-β-saccharosaminopyranosyl(1→4)-β-2-sulfate-fucopyranoside. Saccharomicin A (1) differs from saccharomicin B (2) only in the 10th sugar residue, where α-digitoxopyranosyl(1→3) is replaced with α-rhamnopyranosyl(1→3). The novel amino sugar residue, designated saccharosamine, was identified by NMR spec...

N-Phthaloyl, N-acetyl, N-benzyl, N-acetyl-N-methyl, N,N-dimethyl, N-benzoyl, and N,N-dibenzoyl derivatives of the trisaccharide β-D-glucopyranosyl-(1-3)-O-(2-deoxy-2-amino-β-D-glucopyranosyl)-(1-4)-O-β-D-glucopyranose were synthesized and analyzed by FAB MS. The intensity ratios of the peaks resulting from cleavage of the anomeric bond of the glucosamine residue and the respective molecular ion peaks turned out to be high for the N-acyl derivatives and up to two orders of magnitude lower for the N-alkyl compounds. These results show that fragmentation at the anomeric carbon of the amino sugar may be assisted by the carbonyl group and the resulting cation is stabilized by delocalization of the positive charge.

Preparative routes for anomerically specific 1- C -allylation of 2-amino-2-deoxy sugars have been evaluated in a comparative study of various N -substituents and aglycons as precursors for glycosyl radicals that effectively capture an allyl group from allyltributyltin. The crystalline triacetate 4 of 3-(2-acetamido-2-deoxy- α - d -glucopyranosyl)-1-propene ( 6 ) was obtained in 70% yield when 2-acetamido-3,4,6-tri- O -acetyl-2-deoxy- α - d -glucopyranosyl chloride ( 1 ) was treated with allyltributyltin under free-radical conditions, whereas the corresponding bromide 3 led only to an oxazolidine derivative; the β -1-ethylxanthate analogue of 1 gave 4 , but in only 25% yield. The 2-trifluoroacetamido 1-bromide analogue of 1 was also an effective radical source, giving the 2-trifluoroacetamido analogue 8 of 4 in 60% yield. The free amino analogue 7 of 4 was conveniently obtained via the 2- p -methoxybenzylideneamino 1-bromide analogue of 1 . Use of 3,4,6-tri- O -acetyl-2-deoxy-2-phthalimido- β - d -glucopyranosyl bromide as radical precursor allowed stereospecific access to β -1- C -allyl derivatives of the amino sugar. The crystalline galactosamine analogue 12 of 4 was obtained by using the galacto analogue of chloride 1 , but the corresponding manno chloride gave only an oxazoline product. The 1- C -allylated amino sugar derivatives are conformationally more mobile than derivatives not having a 1- C -linked substituent.

The synthesis of callipeltose, a novel amino sugar with the proposed structure, 4-amino-4,6-dideoxy-2, O -3- C -dimethyl- l -talopyranosyl-3,4-urethane, from l -rhamnose is described. Oxime reduction, carbamate cyclization and selective methylation are key steps. The synthesis supports the assignment of the relative stereochemistry of callipeltose.

Chain-extended amino sugars are synthesized via stereocontrolled Lewis acid-catalyzed aza-Cope rearrangement of N-glycosyl homoallylamines in high yield and stereoselectivity.

Derivatives of the branched amino sugar methyl 3-amino-2,3,6-trideoxy-3-C-methyl-α-L-ribo-hexopyranoside and its 3-nitro analog were coupled with glycosyl donors of 3,4-di-O-acetyl-2,6-dideoxy-L-fucopyranose. Successful glycosylations were developed using the fucosyl bromide, activated with silver triflate, and the fucosyl oxysilane, activated with trimethylsilyl triflate. High stereoselectivities for the desired α-1,4 linkage were observed in both cases. The N-trifluoroacetamido disaccharide was deacylated and the amino group oxidized to nitro with dimethyldioxirane. An alternate route based on coupling of the fucosyl bromide with the nitro sugar methyl α-L-decilonitroside also gave the α-linked disaccharide, which is related to oligosaccharides found in the antibiotics cororubicin and arugomycin.

Concise synthesis of L-vancosamine, the amino sugar constituent of vancomycin and other antibiotics, has been achieved. The key steps include (I) stereoselective addition of methylcerium reagent to oximino ether, and (2) stereoselective hydrogenation of 5,6-unsaturated glycoside with C(5) inversion to give L-vancosamine derivative. C-Glycosidation of vancosaminyl acetate with iodoresorcinol proceeded in the presence of Sc(OTf) 3 in high yield.

The monosaccharide component (α and β-anomer) of the E Ring of calicheamicin γ1I and esperamicin A1 has been synthetized by an efficient and improved stereoselective procedure starting from methyl 2-deoxy-α- and β-D-ribopyranoside. The synthetic procedure makes use, in each case, of a cyclic sulphate and of the regioselective ring opening of an intermediate activated aziridine.

An asymmetric aldol strategy has been developed for the synthesis of l-daunosamine and d-ristosamine derivatives starting from noncarbohydrate precursors. Lithium and boron enolate mediated aldol reactions of 12 with O-TBS lactaldehyde gave non-Evans syn and Evans syn aldol products, respectively, with high selectivity. The chemical efficiency of the lithium enolate reactions were higher than the corresponding reactions with the boron enolates. Curtius rearrangement of lactone acids 23 and 26 gave the corresponding N-BOC amino lactones 30 and 32 in 64% and 62%, respectively, with complete retention of configuration. Lactone 30 was converted by a two-step sequence to N-benzoyldaunosamide 40. The overall yield for the amino sugar 40 was 18% over six steps. Similarly, lactone 32 was converted to N-benzoylristosamide 42 with an overall yield of 18% starting from 12.

A capsular polysaccharide was obtained from Alteromonas nigrifaciens IAM 13010T by saline extraction. On the basis of 1H and 13C NMR spectroscopy, including one-dimensional (1D) NOE spectroscopy, 2D rotating-frame NOE spectroscopy (ROESY), and 1H-detected heteronuclear 1H,13C multiple-quantum coherence (HMQC), it was concluded that the polysaccharide contained inter alia an acidic sugar, 3-deoxy-D-manno-octulosonic acid (Kdo), and a rare amino sugar, 2-acetamido-2,6-dideoxy-L-talose (L-6dTalNAc, N-acetylpneumosamine), and has a pentasaccharide repeating unit of the following structure: [equation: see text]

Baumycins are coproduced with the clinically important anticancer secondary metabolites daunorubicin and doxorubicin, which are glycosylated anthracyclines isolated from Streptomyces peucetius. The distinguishing feature of baumycins is the presence of an unusual acetal moiety appended to daunosamine, which is hydrolyzed during acidic extraction of daunorubicin from fermentation broth. The structure of the baumycin acetal suggests that it is likely derived from an unknown C3″-methyl deoxysugar cleaved between the C3″ and C4″ positions. This is supported by analysis of the baumycin/daunorubicin biosynthetic gene cluster (dox), which also encodes putative proteins consistent with production of an anthracycline dissacharide containing a branched sugar. Notably, the dnmZ gene in the dox gene cluster possesses high translated sequence similarity to nitrososynthases, which are flavin-dependent amine monooxygenases involved in the four-electron oxidation of amino sugars to nitroso sugars. Herein we demonstrate that DnmZ is an amino sugar nitrososynthase that initiates the conversion of thymidine-5'-diphosphate-l-epi-vancosamine to a ring-opened product via a previously uncharacterized retro oxime-aldol reaction.