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Synthesis of the bacterial cell envelope requires a regulated partitioning of resources from central metabolism. Here, we consider the key metabolic junctions that provide the precursors needed to assemble the cell envelope. Peptidoglycan synthesis requires redirection of a glycolytic intermediate, fructose-6-phosphate, into aminosugar biosynthesis by the highly regulated branchpoint enzyme GlmS. MurA directs the downstream product, UDP-GlcNAc, specifically into peptidoglycan synthesis. Other shared resources required for cell envelope synthesis include the isoprenoid carrier lipid undecaprenyl phosphate and amino acids required for peptidoglycan cross-bridges. Assembly of the envelope requires a sharing of limited resources between competing cellular pathways and may additionally benefit from scavenging of metabolites released from neighboring cells or the formation of symbiotic relationships with a host.

Eight new trichothecene derivatives, trichodermarins G-N (1-8), and two new cuparene derivatives, trichocuparins A (9) and B (10), as well as six known trichothecenes (11-16) were isolated from the fungal strain Trichoderma brevicompactum A-DL-9-2 obtained from the inner tissue of the marine red alga Chondria tenuissima. The structures and relative configurations of 1-10 were assigned by NMR and MS data, and the absolute configurations of 1, 2, and 9 were established by X-ray diffraction. Compound 8 features an aminosugar unit bond to the trichothecene framework for the first time, while 9 and 10 represent the first occurrence of cuparene sesquiterpenes in Trichoderma. All the isolates were assayed for growth inhibition of five phytopathogenic fungi (Botrytis cinerea, Cochliobolus miyabeanus, Fusarium oxysporum f. sp. cucumerium, Fusarium oxysporum f. sp. niveum, and Phomopsis asparagi) and four marine phytoplankton species (Amphidinium carterae, Heterocapsa circularisquama, Heterosigma akashiwo, and Prorocentrum donghaiense). Several of them exhibited significant inhibitory activities against the fungi and phytoplankton tested of which trichodermin (12) showed the highest antifungal and antimicroalgal activities with MIC and IC50 values being 4.0 and 0.82 μg/mL, respectively.

3- and 6-aminosugars are common motifs in bioactive compounds playing pivotal roles in the bioactivity of the core molecules to which they are attached. However, de novo synthesis of 3- and 6-amino...

RNAs play critical roles in diverse catalytic and regulatory biological processes and are emerging as important disease biomarkers and therapeutic targets. Thus, developing chemical compounds for targeting any desired RNA structures has great potential in biomedical applications. The viral and cellular RNA sequence and structure databases lay the groundwork for developing RNA-binding chemical ligands through the recognition of both RNA sequence and RNA structure. Influenza A virion consists of eight segments of negative-strand viral RNA (vRNA), all of which contain a highly conserved panhandle duplex structure formed between the first 13 nucleotides at the 5' end and the last 12 nucleotides at the 3' end. Here, we report our binding and cell culture anti-influenza assays of a short 10-mer chemically modified double-stranded RNA (dsRNA)-binding peptide nucleic acid (PNA) designed to bind to the panhandle duplex structure through novel major-groove PNA·RNA2 triplex formation. We demonstrated that incorporation of chemically modified PNA residues thio-pseudoisocytosine (L) and guanidine-modified 5-methyl cytosine (Q) previously developed by us facilitates the sequence-specific recognition of Watson-Crick G-C and C-G pairs, respectively, at physiologically relevant conditions. Significantly, the chemically modified dsRNA-binding PNA (dbPNA) shows selective binding to the dsRNA region in panhandle structure over a single-stranded RNA (ssRNA) and a dsDNA containing the same sequence. The panhandle structure is not accessible to traditional antisense DNA or RNA with a similar length. Conjugation of the dbPNA with an aminosugar neamine enhances the cellular uptake. We observed that 2-5 μM dbPNA-neamine conjugate results in a significant reduction of viral replication. In addition, the 10-mer dbPNA inhibits innate immune receptor RIG-I binding to panhandle structure and thus RIG-I ATPase activity. These findings would provide the foundation for developing novel dbPNAs for the detection of influenza viral RNAs and therapeutics with optimal antiviral and immunomodulatory activities. Ministry of Education (MOE) Ministry of Health (MOH) Nanyang Technological University National Medical Research Council (NMRC) This work was supported by National Science Centre Grant UMO-2015/19/B/NZ1/02803 to E.K. and Grant UMO-2016/21/N/NZ1/00565 to J.K., the Polish Ministry of Science and Higher Education under the KNOW program, Singapore Ministry of Education (MOE) Tier 1 Grants RGT3/13 and RG42/15 to G.C., MOE Tier 2 Grants MOE2013-T2-2-024 and MOE2015-T2-1-028 to G.C., NTU start-up grant and MOH NMRC Grant OFIRG17nov084 to D.L., Temasek Life Sciences Laboratory, Singapore (to M.P.), and Fondation pour la Recherche Med́icale and Agence Nationale de Recherche Programme Labex (ARCANE, ANR-11-LABX-003 to J.-L.D.).

Streptothricins are components of the natural product, nourseothricin; each containing identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked β-lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940’s, generating intense interest because of excellent Gram-negative activity. However, the natural product mixture was associated with toxicity, and subsequent exploration was limited. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50and MIC90for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes inin vitrotranslation assays. There was >10-foldin vitroselectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines.In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressingKlebsiella pneumoniaeNevada strain at dosing levels without observable or minimal toxicity. Resistance mutations obtained in single ribosomal operonE. coliidentify novel interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, gram-negative pathogens.IMPORTANCEStreptothricins are a historic class of antibiotics discovered by Waksman and colleagues in the 1940’s. Toxicities associated with the streptothricin natural product mixture, also known as nourseothricin, discouraged further development. However, we found that a component of nourseothricin, streptothricin-F, retained potent activity against contemporary carbapenem-resistant Enterobacterales with significant selectivity inin vitroandin vivoassays. This included demonstration of rapid bactericidal activityin vitroand substantial therapeutic effect in the murine thigh model against a pandrug-resistantKlebsiella pneumoniaeisolate at non-toxic concentrations. Through resistance mutation analysis, we identified helix 34 of 16S rRNA in the prokaryotic ribosome, and specifically bases C1054 and A1196, as critical for streptothricin’s activity. The mechanism of action is distinct from other known translation inhibitors. Based on promising and unique activity, we believe the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram-negative pathogens.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae . Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli . Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C-C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.

Infections with Streptococcus pneumoniae are a major health burden. Glycoconjugate vaccines based on capsular polysaccharides (CPSs) successfully protect from infection, but not all pneumococcal serotypes are covered with equal potency. Marketed glycoconjugate vaccines induce low levels of functional antibodies against the highly invasive serotype 1 (ST1), presumably due to the obscuring of protective epitopes during chemical activation and conjugation to carrier proteins. Synthetic oligosaccharide antigens can be designed to carry linkers for site-selective protein conjugation while keeping protective epitopes intact. Here, we developed an efficacious semisynthetic ST1 glycoconjugate vaccine candidate. A panel of synthetic oligosaccharides served to reveal a critical role of the rare aminosugar, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (d-AAT), for ST1 immune recognition. A monovalent ST1 trisaccharide carrying d-AAT at the nonreducing end induced a strong antibacterial immune response in rabbits and outperformed the ST1 component of the multivalent blockbuster vaccine Prevenar 13, paving the way for a more efficacious vaccine., A synthetic trisaccharide antigen confers antibacterial immunity against the highly invasive S. pneumoniae serotype 1 that is insufficiently covered by marketed vaccines.

Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells lose cell polarity and cell–cell adhesion and gain migratory and invasive properties to become mesenchymal cells that are very vital for development, wound healing and stem cell behavior and contribute pathologically to fibrosis and cancer progression. miR21, a potent regulator of the tumor suppressor gene PTEN, can be silenced to reverse EMT, thereby providing an attractive target for abrogating the malignant behavior of breast cancer. Here, we report the design, synthesis and binding of a peptidic-aminoglycoside (PA) based chemical library against pre-miR21 that led to the identification of a group of small molecules that bind to pre-miR21 with high affinities and antagonize miR-21 maturation and function, thereby reversing EMT. The approach described here offers a promising miRNA targeting platform where such aminosugar conjugates can be similarly used to target other oncogenic miRNAs. Minor changes in the amino acid sequence allow us to tailor the binding effectiveness and downstream biological effects, thus making this approach a potentially tunable method of regulation of miRNA function.

Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Streptococcus pneumoniae. Some components of the S. pneumoniae glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using S. pneumoniae serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar l-PneuNAc and a branched l-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.

The relevance of the study is determined by the development of the fishing industry and, as a result, aquaculture farms in the Far East of the Russian Federation. Far Eastern sea cucumber (tre-pang) is considered as one of the objects of aquaculture, which is of a great demand in the market of the countries of the Asia-Pacific region. The aim of the work is to substantiate the expedi-ency of obtaining food products for healthy nutrition from the Far Eastern sea cucumber (Stichopus japonicus). The paper presents the assessment results of the physical, chemical, biological and microbiological properties of sea cucumbers grown in the North-ern bay of Khasansky district of Primorsky Krai. The mass com-position of cucumbers produced in the spring and autumn periods is given. In addition to the general chemical composition, the con-tent of such physiologically valuable substances as triterpene gly-cosides, aminosugar (glucosamine, galactosamine) in the sea cu-cumber's muscle tissues was studied. The high biological potency of these components makes it possible to assign the Far Eastern sea cucumber to valuable biological objects. In general, the results of experimental studies on the evaluation of the mass, chemical composition and safety parameters of the Far Eastern sea cu-cumber confirm the expediency of obtaining physiologically useful food products from it.

A diversified synthesis of 3-aminosugar analogues of digitoxin and digoxin with potent anticancer activities was explored. The synthesis was based on a highly efficient gold-catalyzed glycosylation reaction with alkynylbenzoate 3-aminoglycosides as glycosyl donors. According to this strategy, a small library containing digitoxin and digoxin analogues with various 3-aminosugar scaffolds was successfully constructed.

The strained epoxide ring of oxirane-derived furanoside and pyranoside of d-fructose are regioselectively opened by various primary and secondary amines as well as azide to yield C4-alkylamino-C4-deoxy and the corresponding azido derivatives. The epoxide ring of a furanoside derived from l-sorbose, a C-5 epimer of d-fructose also afforded C4-amino and C4-azido analogues. All three epoxides generated piperazine linked disaccharides. These epoxides are also easily opened by p-tolylthiol to access thiosugars. One such thiosugar was converted to vinylsulfone-modified fructofuranoside and subjected to nucleophillic addition. A suitably designed vinyl sulfone-modified fructofuranoside having a leaving group at C-6, act as an efficient substrate for MIRC (Michael Initiated Ring Closure) reactions to construct cyclopropane skeleton in d-fructose. The strategy is general in nature and provides an easy access to cyclopropanated sugar derivatives. Thus, the easily accessible "spring loaded" epoxides of d-fructose and l-sorbose have been used as pivotal starting point for the synthesis of hitherto unknown modified carbohydrates.

RNA targeted high-throughput assays that allow for rapid detection of high affinity binding ligands are important in RNA recognition studies. A number for fluorescent dyes have been reported that can assist in rapidly identifying nucleic acid (RNA) binding elements without the need for immobilization of RNA or the ligand. A number of these dyes are planar aromatic molecules that bind non-specifically to nucleic acids and often distort their parent nucleic acid structures leading to ambiguity in the interpretation of results. In this light, we report here, the use of an aminoglycoside (neomycin) based fluorescent probe (F-Neo) which can reversibly bind to different RNA motifs and help identify ligands with needed affinity and selectivity, without any immobilization of the probe or the target. In this chapter, we provide the details of the assay development, experimental considerations and data analysis to use the probe and identify novel ligands. We then provide a brief introduction to calorimetry (ITC) and circular dichroism (CD) spectroscopy based methods in validating the binding of such identified compounds.

Method of electrical stimulation of cells on bioengineering nanocomposite substrates with electrical pulses with amplitude from 60 mV to 120 mV and pulse duration of 2.5 ms for 24 h is developed. Nanocomposite substrates consist of a polymer matrix based on natural components - protein albumin, aminosugar chitosan and reinforcing filler - carbon nanotubes have a three-dimensional structure with an internal electrically conductive frame created by laser formation. A developed device for electrical stimulation of cells allows the transmission of stimulating signals to cells through a conducting substrate consisted of bioengineer nanocomposite. As a result, the dependence of the proliferation factor on the electric signal amplitude was obtained and characteristics of the spreading of cells on the substrate was investigated.

This paper presents the results of a study on the effect of water soluble aminosugar chitosan on the components binding in biocompatible composite materials that are intended to replace tissue defects. The effect on the binding of single-walled carbon nanotubes with a protein filler, albumin and collagen, was evaluated. The experimental samples were formed by evaporation of the water component from the dispersion by ytterbium laser radiation with a wavelength of 1064 nm, a pulse duration of 100 nm. Binding studies were carried out using the methods of vibrational spectroscopy (IR and Raman), and the data on the scaffold morphology were supplemented with the results of confocal microscopy.

Background/Aim: Resistance against cisplatin is a problem for the success of gastric cancer chemotherapy. Herein, we evaluated the antitumor effect of a new aminosugar-conjugated, mono-functional platinum complex (Pt-Oqn), which forms a single covalent bond with DNA. Materials and Methods: We compared the cytotoxicity of Pt-Oqn to that of cisplatin (CDDP), oxaliplatin (L-OHP) and carboplatin (CBDCA). We also compared Pt-Oqn and cisplatin for DNA double-strand breaks based on phosphorylated histone H2AX levels in cancer cells and antitumor effects in xenograft models. Results: The resistance factor (RF) for Pt-Oqn was low among the four drugs, indicating the potential of Pt-Oqn for overcoming CDDP-induced resistance. In MKN45-R cells, γ-H2AX protein increased following treatment with Pt-Oqn, but not with cisplatin. Finally, Pt-Oqn, but not cisplatin, showed significant antitumor effects in MKN45-R xenografts. Conclusion: This new aminosugar-conjugated platinum complex is a promising candidate agent for overcoming the drug resistance of cisplatin-resistant stomach cancer.

Pathogens encounter and metabolize a range of host-derived metabolites while proliferating inside the host. Our understanding of these metabolites and their metabolic processes has remained largely incomplete. We investigated the role of the Magnaporthe oryzae N-acetylglucosamine (GlcNAc) catabolic pathway during rice infection. The catabolic pathway is composed of a GlcNAc transporter (MoNgt1), hexokinase(s), a GlcNAc-6-phosphate deacetylase (MoDac) and a GlcN-6-phosphate deaminase (MoDeam). A detailed characterization of the Δmongt1, Δmodac and Δmodeam null mutants revealed that a defect in GlcNAc catabolism impairs the pathogenicity of M. oryzae. These mutants showed severely reduced virulence in susceptible rice cultivar due to their inability to neutralize host-derived reactive oxygen species and their failure to develop invasive hyphal growth within the host tissue. Interestingly, during oxidative stress, M. oryzae proliferated efficiently in GlcNAc-containing media compared with other sugars, and the expression of fungal antioxidant genes was upregulated following GlcNAc treatment. However, GlcNAc inhibited the growth of the Δmodac and Δmodeam mutants, and this growth inhibition was enhanced during oxidative stress. These results suggest that GlcNAc helps fungus to overcome oxidative stress inside its host, perhaps by activating an antioxidant defence. In the absence of a functional catabolic pathway, GlcNAc becomes toxic to the cells.

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...

A stereodivergent approach to creation of a specific 14-membered macrolide library is described. We designed a new 14-membered macrolide template possessing 10 sterogenic centers, and established a new library of fully-synthesized 14-membered ring compounds. The designed macrodiolides were synthesized through a convergent approach using an Evans aldol reaction, Keck esterification, and Yamaguchi macrolactonization, or Mitsunobu macrolactonization, as key steps. Moreover, introduction of an aminosugar to a macrodiolide aglycone by Schmidt glycosylation also afforded a new macrodiolide. Comparison between the conformation analysis of the macrolides, which we designed, and NMR analysis of the synthetic macrolides, indicated our stereoisomer library has significant diversity.

The third generation of glycoconjugated azo dyes (GADs) was prepared linking monoazo dyes to 6-amino-6-deoxy-d-galactose or 6′amino-6′-deoxylactose through mixed amido-ester connections. The complementary conjugation reactions were studied using the succinyl derivative of either the acetal protected aminosugar or the azo dye. Target “naturalized” GADs were obtained after acid hydrolysis of the acetal protecting groups present on the sugar moiety.

The synthesis of two tetrafluorinated 4-aminosugars, 4-amino-2,3,4-trideoxy-2,2,3,3-tetrafluoro- d - erythro -hexopyranose hydrochloride ( 7•HCl ) and 4-amino-2,3,4-trideoxy-2,2,3,3-tetrafluoro- d - threo -hexopyranose hydrochloride ( 8•HCl ), is described. The amino group in α-position of a CF 2 (CF 2 ) group is proposed as a mimic for the hydrogen bond accepting capacity of an alcohol group in an unfluorinated sugar. The synthesis of the two sugars was achieved in 4 steps each from the sulfinylimine diastereoisomers of d -glyceraldehyde.

A 215-member mono- and diamino acid peptidic-aminosugar (PA) library, with neomycin as the model aminosugar, was systematically and rapidly synthesized via solid phase synthesis. Antibacterial activities of the PA library, on 13 bacterial strains (seven Gram-positive and six Gram-negative bacterial strains), and binding affinities of the PA library for a 27-base model of the bacterial 16S ribosomal A-site RNA were evaluated using high-throughput screening. The results of the two assays were correlated using Ribosomal Binding-Bacterial Inhibition Plot (RB-BIP) analysis to provide structure-activity relationship (SAR) information. From this work, we have identified PAs that can discriminate the E. coli A-site from the human A-site by up to a 28-fold difference in binding affinity. Aminoglycoside-modifying enzyme activity studies indicate that APH(2″)-Ia showed nearly complete removal of activity with a number of PAs. The synthesis of the compound library and screening can both be performed rapidly, allowing for an iterative process of aminoglycoside synthesis and screening of PA libraries for optimal binding and antibacterial activity for lead identification.

A convergent strategy was developed for the stereoselective synthesis of four unusual N-acylated monosaccharides (5-8), which are fragments of lipooligosaccharide IV (LOS-IV) from Mycobacterium marinum. A critical substrate-controlled asymmetric cyclization of an amino acid derived oxazolidine provided a key lactam intermediate 11, which was successfully converted to targets 5-7. The key step in the synthesis of 8 was a one-pot cascade oxidation-cyclization-oxidation reaction of a Boc-protected amino alcohol, prepared from 3-butynol, which led to the formation of lactam 15. The five-membered ring lactam intermediates in these synthetic routes were sensitive to elimination side reactions, but careful manipulation of the reaction sequence allowed for the stereoselective synthesis of the targets. This work represents the first synthesis of these unusual motifs, which have been shown to be essential to the bioactivity of LOS-IV.

Diversity in eukaryotic rRNA structure and function offers possibilities of therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescence displacement assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 × 10–9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 7 μM. The fluorescence displacement assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for E...

Several different approaches have been explored for conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues. Single and double conjugation of 2-(2-(2-aminoethoxy)ethoxy)ethanol was obtained using carbonyldimidazole. Using a post PNA-assembly coupling procedure the building block 2-(2-(2-(benzoyloxy)ethoxy)ethoxy)acetic acid multiple attachment of 2-(2-(2-hydroxyethoxy)ethoxy)acetyl groups to both N-terminal and β-amino groups of inserted diaminopropionic acids residues was achieved. Use of a new oligoether functionalized amino acid allows inclusion of oligoether conjugates during on-line machine assisted synthesis which also allowed combination of methods for attachment of different oligoethers and co-conjugation of neocuproine as well as conjugation of an aminosugar.

Streptothricin-F (STT-F), one of the early-discovered antibiotics, consists of three components, a β-lysine homopolymer, an aminosugar D-gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long-lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized in vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high-resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an Fe(II) -dependent double hydroxylation reaction converting L-Arg into (3R,4R)-(OH)2 -L-Arg via (3S)-OH-L-Arg, while OrfR catalyzes an unusual PLP-dependent elimination/addition reaction cyclizing (3R,4R)-(OH)2 -L-Arg to the six-membered (4R)-OH-capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT-F by a feeding experiment.

Kanosamine is an aminosugar antibiotic, and component of complex antibiotics such as kanamycin. The biosynthesis of kanosamine varies among different bacteria; best known is a pathway starting from UDP-glucose, but Bacillus subtilis can produce kanosamine in a three-step pathway from glucose 6-phosphate. A set of genes proposed to encode a kanosamine pathway has previously been identified within the zwittermicin A gene cluster of Bacillus cereus UW85. These genes, designated kabABC, are similar to the B. subtilis kanosamine pathway genes (ntdABC), but have never been studied experimentally. We have expressed each of the kab genes, and studied the in vitro substrate scope and reaction rates and kinetic mechanisms of all three enzymes. The kab genes encode enzymes that catalyze a route similar to that found in B. subtilis from glucose 6-phosphate to kanosamine, passing through an unusual and unstable 3-keto intermediate. Kinetic studies show the first step in the pathway, the KabC-catalyzed oxidation of glucose 6-phosphate at carbon-3, is very slow relative to the subsequent KabA-catalyzed aminotransferase and KabB-catalyzed phosphatase reactions. KabC differs from its homolog, NtdC, in that it is NADP- rather than NAD-dependent. The KabA kinetic study is the first such report for a kanosamine 6-phosphate aminotransferase, revealing an extremely efficient PLP-dependent reaction. These results show that this kanosamine biosynthesis pathway occurs in more than one organism, and that the reactions are tuned in order to avoid any accumulation of the unstable intermediate.

Diastereoselective aza-Wacker cyclization of O-allyl hemiaminals under aerobic conditions enables efficient access to 1,2-aminoalcohol derivatives from allylic alcohols. The scope of this method is presented and its utility is highlighted in a streamlined synthesis of the biologically important aminosugar (–)-acosamine.

The increasing interest in urea compounds as self-assembling molecules, ion transporters and organocatalysts prompted several efforts towards synthetic urea-linked glycomimetics. In this frame we studied in details a novel two steps dimerization reaction of sugar vicinal aminoalcohol building blocks, opening a synthetic path to a series of urea-linked neosaccharides. Glucosamine neodisaccharide possessing an oxazolidinone–urea–oxazolidinone system could be transformed into both cyclic and higher linear neosaccharides. Furthermore, a series of six urea-linked glucosamine and galactosamine neodisaccharides was tested for self-assembling properties by measuring NMR spectra at different temperatures and concentrations as well as by gelation of several organic solvents.

In the last decade aminosugars, especially d-glucoamine based organocatalysts, have been applied to catalyze various asymmetric reactions such as aldol reactions, Michael addition, Strecker reactions, Biginelli reactions, epoxidation, fluorination, and imine reduction, and for the synthesis of various biologically important molecules such as 3-alkylnitro-2-hydroxynaphthoquinones, trans-dihydrobenzofurans etc. Immense growth has been also observed in the structural modification of aminosugar based organocatalysts to obtain the best results from them. This review sheds light on such organocatalytic transformations reported in last the decade including the effect of the structural modification of sugar amines on their catalytic efficiency and the stereoselectivity of the reaction.

Nucleic acids adopt a broad array of hydrogen-bonded structures that enable their diverse roles in the cell; even the familiar DNA double helix displays subtle architectural nuances that are sequence dependent. While there have been many approaches for recognition of B-form nucleic acids, A-form DNA recognition has lagged behind. Here, using a tight binding fluorescein-neomycin (F-neo) conjugate that can probe the electrostatic environment of A-form DNA major groove, we developed a fluorescent displacement assay to be used as a screen for DNA duplex-binding compounds. As opposed to intercalating dyes that can significantly perturb DNA structure, the groove binding F-neo allows the probing of native DNA conformation. In combination with the assay development and probing of DNA grooves, we also report the synthesis and binding of a series of neomycin-anthraquinone conjugates, two units with a known preference for binding GC rich DNA. The assay can be used to identify duplex DNA-binding compounds, as well as probe structural features of a target DNA duplex, and can easily be scaled up for high throughput screening of compound libraries.

The substrate specificity was studied for the metabolic degradation of N-acetyl-D-glucosamine (GlcNAc) derivatives by Rhodococcus rhodochrous IFO 15564 which possesses N-acetyl-D-glucosamine deacetylase as a key-step enzyme. This microorganism degraded a wide range of substrates with modified N-acyl groups. The metabolizing activity of this strain became low to the substrates substituted at 1,3,4,6-positions of GlcNAc, and GlcNAc itself was suggested to be metabolized via an open-chain aldehyde form. Based on these results, a simplified procedure for the isolation of allyl α-N-acetyl-D-glucosaminide from an α, β-anomeric mixture was developed by selectively hydrolyzing the β-anomer with Jackbean β-N-acetyl-D-glucosaminidase and subsequently degrading the resulting N-acetyl-D-glucosamine in the reaction mixture with this microorganism.

Previous research has shown that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This report further explores the role of N-sulfation of intact heparan chains in podocyte-matrix interactions. For the purposes of this study, a murine model in which the enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) was specifically deleted in podocytes and immortalized podocyte cell lines lacking NDST1 were developed and used to explore the effects of such a mutation on podocyte behavior in vitro. NDST1 is a bifunctional enzyme, ultimately responsible for N-sulfation of heparan glycosaminoglycans produced by cells. Immunostaining of glomeruli from mice whose podocytes were null for Ndst1 (Ndst1−/−) showed a disrupted pattern of localization for the cell surface proteoglycan, syndecan-4, and for α-actinin-4 compared with controls. The pattern of immunostaining for synaptopodin and nephrin did not show as significant alterations. In vitro studies showed that Ndst1−/− podocytes attached, spread, and migrated less efficiently than Ndst1+/+ podocytes. Immunostaining in vitro for several markers for molecules involved in cell-matrix interactions showed that Ndst1−/− cells had decreased clustering of syndecan-4 and decreased recruitment of protein kinase-Cα, α-actinin-4, vinculin, and phospho-focal adhesion kinase to focal adhesions. Total intracellular phospho-focal adhesion kinase was decreased in Ndst1−/− compared with Ndst1+/+ cells. A significant decrease in the abundance of activated integrin α5β1 on the cell surface of Ndst1−/− cells compared with Ndst1+/+ cells was observed. These results serve to highlight the critical role of heparan sulfate N-sulfation in facilitating normal podocyte-matrix interactions.

RESEARCH ARTICLE Molecular Characterization of the N- Acetylglucosamine Catabolic Genes in Candida africana, a Natural N-Acetylglucosamine Kinase (HXK1) Mutant Maria Rosa Felice 1 , Megha Gulati 2 , Letterio Giuffre 1 , Domenico Giosa 1 , Luca Marco Di Bella 1 , Giuseppe Criseo 1 , Clarissa J. Nobile 2 , Orazio Romeo 1,3 *, Fabio Scordino 3 1 Department of Biological and Environmental Sciences, University of Messina, Messina, Italy, 2 Department of Molecular and Cell Biology, University of California Merced, Merced, California, United States of America, 3 Scientific Institute for Research, Hospitalization and Health Care (IRCCS)—Centro Neurolesi Bonino-Pulejo , Messina, Italy * oromeo@unime.it Abstract Background OPEN ACCESS Citation: Felice MR, Gulati M, Giuffre L, Giosa D, Di Bella LM, Criseo G, et al. (2016) Molecular Characterization of the N-Acetylglucosamine Catabolic Genes in Candida africana, a Natural N- Acetylglucosamine Kinase (HXK1) Mutant. PLoS ONE 11(1): e0147902. doi:10.1371/journal.pone.0147902 Editor: Valerio Pazienza, IRCCS Casa Sollievo della Sofferenza Hospital, ITALY Received: August 12, 2015 Accepted: January 11, 2016 Published: January 25, 2016 Copyright: © 2016 Felice et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In this study we report the genetic characterization, including expression analysis, of the genes involved in the uptake (NGT1) and catabolism (HXK1/NAG5, DAC1/NAG2, NAG1) of the aminosugar N-acetylglucosamine (GlcNAc) in Candida africana, a pathogenic biovar- iant of Candida albicans that is naturally unable to assimilate the GlcNAc. Results DNA sequence analysis of these genes revealed a number of characteristic nucleotide sub- stitutions including a unique and distinctive guanine insertion that shifts the reading frame and generates a premature stop codon (TGA) 154 bp downstream of the ATG start codon of the HXK1 gene encoding the GlcNAc-kinase, a key enzyme of the GlcNAc catabolic path- way. However, all examined genes produced transcripts even though different levels of expression were observed among the Candida isolates examined. In particular, we found an HXK1-idependent relationship of the NGT1 gene and a considerable influence of the GlcNAc-kinase functionality on the transcription of the DAC1 and NAG1 genes. Additional phenotypic analysis revealed that C. africana isolates are hyperfilamentous in the first 24- 48h of growth on filament-inducing media and revert to the yeast morphological form after 72h of incubation on these media. Data Availability Statement: All relevant data are within the paper. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. Conclusions Our results show that C. africana is a natural HXK1 mutant, displaying a number of pheno- typic characteristics distinct from typical C. albicans isolates. PLOS ONE | DOI:10.1371/journal.pone.0147902 January 25, 2016

A series of novel modified 5-O-desosamine-ketolides were synthesized. The 5-O-desosamine fragment was removed from ketolide by an efficient and mild manipulation. 4-O-substituted desosamine was introduced into the ketolide aglycon and various coupling methods were essayed for the glycosylation. Three novel ketolides were tested for in vitro antibacterial activity against a panel of susceptible and resistant pathogens. Compound 26 showed potent activity against all the methicillin-sensitivity and resistant pathogens.

N-acetylglucosamine (GlcNAc), the monomer of chitin and constituent of bacterial peptidoglycan, is a preferred carbon and nitrogen source for streptomycetes. Recent studies have revealed new functions of GlcNAc in nutrient signaling of bacteria. Exposure to GlcNAc activates development and antibiotic production of Streptomyces coelicolor under poor growth conditions (famine) and blocks these processes under rich conditions (feast). Glucosamine-6-phosphate (GlcN-6P) is a key molecule in this signaling pathway and acts as an allosteric effector of a pleiotropic transcriptional repressor DasR, the regulon of which includes the GlcNAc metabolic enzymes N-actetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase (NagA) and GlcN-6P deaminase (NagB). Intracellular accumulation of GlcNAc-6P and GlcN-6P enhanced production of the pigmented antibiotic actinorhodin. When the nagB mutant was challenged with GlcNAc or GlcN, spontaneous second-site mutations that relieved the toxicity of the accumulated sugar phosphates were obtained. Surprisingly, deletion of nagA also relieved toxicity of GlcN, indicating novel linkage between the GlcN and GlcNAc utilization pathways. The strongly enhanced antibiotic production observed for many suppressor mutants shows the potential of the modulation of GlcNAc and GlcN metabolism as a metabolic engineering tool toward the improvement of antibiotic productivity or even the discovery of novel compounds.

Poly-N-acetyllactosamine extensions on N-and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial β3-N-acetylglucosaminyltransferase (β3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1,-2,-3 and-4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galβ1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in β1-3 linkage to type-1 (Galβ1-3GlcNAc) or type-3/4 (Gal1-3GalNAc/) sequences. Thus, by alternating the use of the H. pylori β3GlcNAcT with galactosyltransferases that make the 1-4 or 1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the β3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori β3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans. © 2012 The Author.

Structural factors behind erm macrolide resistance were studied through synthesis of new macrolide derivates possessing truncated desosamine sugar moieties and subsequent determination of their antibacterial activity. Synthesized compounds with 2'-deoxy and 3'-desmethyl desosamine rings demonstrated decreased antibacterial activity on the native Staphylococcus aureus strain and were inactive against constitutively resistance S. aureus. The obtained results indicate that steric repulsion between the dimethylated A2058 and desosamine ring cannot be considered as a primary reason for erm-resistance.

The azidonitration of di-O-acetyl-l-fucal has previously been shown to be an efficient route to the bacterial aminosugar N-acetyl-l-fucosamine. Upon repeating this sequence, with updated versions of the glycal formation and amide installation steps, we have obtained X-ray crystal structures of several of the addition products, which are now reported along with the solid-state structure of N-acetyl-l-fucosamine itself.

Major increases of the selectivity and/or reactivity in aminosugar and sugar O-alkylation were observed in the presence of tetrabutylammonium fluoride (TBAF) in comparison to TBAI under phase-transfer conditions or in solution. The presence of TBAF allowed the selective and rapid alkylation of the 6-hydroxyl function of neamine and efficient preparation of protected intermediates useful in synthesis and potent or potential antimicrobial O-alkylated derivatives of neamine and paromamine. In regard to the observed strong effects of TBAF, the alkylation and acylation of carbohydrates merit to be studied in the presence of TBAF under phase-transfer conditions.

A series of neomycin dimers have been synthesized using 'click chemistry' with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a 59 base pair stem loop structure located at 5'-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (T(m)) of the HIV TAR RNA up to 10°C. Ethidium bromide displacement titrations revealed nanomolar IC(50) between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC(50) in the micromolar range is observed.

2-NBDG is a widely used fluorescent tracer for monitoring d-glucose uptake into single living cells. However, 2-NBDG alone is not sufficient for monitoring the net stereoselective uptake of d-glucose, unless its possible non-stereoselective uptake is properly evaluated. l-Glucose derivatives, which emit fluorescence distinct from that of 2-NBDG, should provide valuable information on the stereoselective uptake, when used with 2-NBDG in combination. In the present study, we synthesized Texas Red (sulforhodamine 101 acid)-coupled and [2-(benz-2-oxa-1,3-diazol-4-yl)amino]-coupled 2-deoxy-D-glucose, referred to as [2-TRG] and [2-BDG], respectively. These derivatives showed emission wavelength longer and shorter than that of 2-NBDG, respectively. 2-TRLG, an antipode of 2-TRG, proved to be an effective tracer for evaluating the extent of non-stereoselective uptake of 2-NBDG when used simultaneously with 2-NBDG. On the other hand, 2-BDG exhibited very weak fluorescence, but the application of a novel cross coupling in the presence of a benzoxadiazole group may be useful for the future development of effective glucose tracers.

Various C -pyrrolyl glycosides were obtained through tandem condensation of aminosugars ( d -glucosamine and d -galactosamine) and 1,3-dicarbonyl compounds under green conditions in moderate to good yields. The reactions were carried out in the presence of 10 mol % InCl 3 in water at 50 °C. A reaction mechanism through carbocation intermediates was proposed.

Amino sugars are one of the important microbial residue biomarkers which are associated with soil organic matter cycling. However, little is known about their transformation kinetics in response to available substrates because living biomass only contributes a negligible portion to the total mass of amino sugars. By using 15 N tracing technique, the newly synthesized (labeled) amino sugars can be differentiated from the native portions in soil matrix, making it possible to evaluate, in quantitative manner, the transformation pattern of amino sugars and to interpret the past and ongoing changes of microbial communities during the assimilation of extraneous 15 N. In this study, laboratory incubations of soil samples were conducted by using 15 NH 4 + as nitrogen source with or without glucose addition. Both the 15 N enrichment (expressed as atom percentage excess, APE) and the contents of amino sugars were determined by an isotope-based gas chromatography–mass spectrometry. The significant 15 N incorporation into amino sugars was only observed in glucose plus 15 NH 4 + amendment with the APE arranged as: muramic acid (MurN) > glucosamine (GlcN) > galactosamine (GalN). The dynamics of 15 N enrichment in bacterial-derived MurN and fungal-derived GlcN were fitted to the hyperbolic equations and indicative for the temporal responses of different soil microorganisms. The APE plateau of MurN and fungal-derived GlcN represented the maximal extent of bacterial and fungal populations, respectively, becoming active in response to the available substrates. The different dynamics of the 15 N enrichment between MurN and GlcN indicated that bacteria reacted faster than fungi to assimilate the labile substrates initially, but fungus growth was dominant afterward, leading to integrated microbial community structure over time. Furthermore, the dynamics of labeled and unlabeled portions of amino sugars were compound-specific and substrate-dependent, suggesting their different stability in soil. GlcN tended to accumulate in soil while MurN was more likely degraded as a carbon source when nitrogen supply was excessive.

Knowledge of the soil nitrogen (N) supply and the N mineralization potential of the soil combined with an understanding of weed-crop competition in response to soil nutrient levels may be used to optimize N fertilizer rates to increase the competitive advantage of crop species. A greenhouse study (2006) and field studies (2007 to 2008) in Illinois and Nebraska were conducted to quantify the growth and interference of maize and velvetleaf in response to varying synthetic N fertilizer rates in soils with high and low N mineralization potential. Natural soils were classified as having ‘‘low mineralization potential’’ (LMP), while soils amended with composted manure were classified as having ‘‘high mineralization potential’’ (HMP). Maize and velvetleaf were grown in monoculture or in mixture in both LMP and HMP soils and fertilized with zero, medium, or full locally recommended N rate. In the greenhouse, velvetleaf interference in maize with respect to plant biomass increased as N rate increased in the HMP soil, whereas increasing N rate in the LMP soil reduced velvetleaf interference. In contrast, velvetleaf interference in maize decreased as N rate increased regardless of soil class in the field experiment. With respect to grain yield, velvetleaf interference in maize was unaffected by N rate or soil class. In both greenhouse and field experiments, velvetleaf biomass was greater in the HMP soil class, whereas maize interference in velvetleaf was generally greater in the LMP soil class. While soil N levels influenced weed-crop interference in the greenhouse, the results of the field study demonstrate the difficulty of controlling soil nutrient dynamics in the field and support a maize fertilization strategy independent of weed N use considerations. Nomenclature: Velvetleaf, Abutilon theophrasti Medic. ABUTH; maize, Zea mays L.

Five new streptothricin derivatives with a carbamoyl group substituted at C-12 (1-5) and three known analogues have been isolated from the culture broth of Streptomyces sp. I08A 1776 by ion exchange and hydrophilic interaction chromatographic techniques. Their structures were determined by spectroscopic and chemical methods. Compound 3 was a streptothricin derivative possessing a cis-streptolidine moiety. Its absolute configuration was defined by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Compound 5 and streptothricin E (6) displayed antibacterial and antifungal activity with MIC values in the range 1-64 μg/mL.

A number of fluorinated versatile building blocks (synthons) were developed for the synthesis of trifluoromethylated and gem-difluoromethylenated biologically interesting compounds. Extensive studies have demonstrated that fluorine atoms have profound stereoelectronic effects on neighboring groups, leading to special performance of the fluorine-containing compounds in reactions. It was found that, in comparison with their nonfluorinated counterparts, a C―O bond adjacent to a CF 2 group was more stabilized which made it hard to be substituted in a S N 2 way; double bond next to fluorinated groups was too electron deficient to participate in ring-closing-metathesis (RCM) reactions, and the carbocation that was strongly destabilized by neighboring CF 2 group reacted immediately with nucleophilic molecules to produce unexpected product and so on. Thus improved methodologies were developed to achieve the desired transformations. The conformational analysis of difluorothiouridine suggested that the gem-difluoro atoms governed the overall conformation of the sugar ring and nucleobase. The biological evaluation of the synthetic difluoromethylenated azasugars towards eight glycosidases showed that the CF 2 group decreased the pK a of azasugars which enhanced their binding affinity and selectivity to glycosidases. 1 Introduction 2 Synthesis of Trifluoromethylated Building Blocks and their Application for the Preparation of Biologically Interesting Molecules 2.1 Improved FSO 2 CF 2 CO 2 Me/CuI Method for the Synthesis of Trifluoromethylated Retinoid 2.2 Preparation of α-Trifluoromethyl-α,β-Unsaturated Esters for the Synthesis of Trifluoromethylated Nucleosides and Prolines 2.3 Development of Trifluoropropenyl-Containing Synthons for the Preparation of Trifluoromethylated Analogues of Amino Acids, Sugars, and Macrolactones 3 Preparation of Difluoromethylenated Building Blocks and their Application for the Synthesis of Difluoromethylenated Nucleosides, α,β-Uunsaturated-β-lactones and Azasugars 3.1 Novel Route to Difluorohomoallyl Alcohol and its Wide Utility in the Synthesis of Difluoromethylenated Nucleosides 3.2 Construction of Difluoromethylenated Synthons via Silicon-Induced Reformatskii―Claisen Rearrangement and their Application for the Preparation of Difluoromethylenated Nucleosides 3.3 Synthesis of Azanucleosides Bearing Exocyclic CF 2 H Group Using a Wittig-Type Olefination Approach 3.4 Conformational Analysis of D -2'-Deoxy-2',2'-difluoro-4'-dihydro-4'-thiouridine 3.5 Synthesis of Difluoromethylenated Six-Membered α,β-Unsaturated-β-lactones and other Related Compounds 3.6 Synthesis and Biological Evaluation of Difluoromethylenated Azasugars 4 Conclusion.

Triazole linked heparosan and chondroitin disaccharide and tetrasaccharide building blocks were synthesized in a stereoselective manner by applying a very efficient copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction of appropriately substituted azido-glucuronic acid and propargyluted N-acetyl glucosamine and N-acetyl galactosamine derivative, respectively. The resulting suitably substituted tetrasaccharide analogues can be easily converted into azide and alkyne unit for further synthesis of higher oligosaccharide analogues.

A sensitive, selective and efficient liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of glucosamine in healthy human urine. Urine samples were extracted by acetonitrile and derivatized with o-phthalaldehyde/3-mercaptopropionic acid. Analysis was then carried out using ESI source and methanol/0.2% ammonium acetate-0.1% formic acid mobile phase gradient elution, with tolterodine tartrate as the internal standard. The linear calibration curve ranged from 0.41 μg/ml to 82.7 μg/ml. The intra-day and inter-day precisions were less than 3.93% and 10.0%, respectively. The extraction recoveries determined at three concentration levels were higher than 88.6%. The method was successfully applied for determining the urine concentration of glucosamine up to 24 h after oral administration of 1 g glucosamine sulfate dispersible table (containing 785.08 mg glucosamine) from a clinical pharmacokinetic study in healthy volunteers.