Synthesis of fused bicyclic thioglycosides of N-acylated glucosamine as analogues of mycothiol

The synthesis of a fused bicyclic thioglycoside analogue of mycothiol, (3R)-3-acetylamino-4-one-6,7-dihydro-(1',2'-dideoxy-beta-D-glucopyranoso)[2',1'-f]-1,5-thiazepane (5), is reported. Treatment of phthalimido-protected peracetylated glucosamine with N-acetyl-cysteine and boron trifluoride-etherate gave the beta-linked thioglycoside, which was deprotected and cyclized, using HOBt and EDCl to form the lactam and giving the target structure. This mycothiol mimic and its tri-O-acetate will be investigated as potential inhibitors of enzymes involved in the biosynthesis of mycothiol. The protected derivative also has the potential to be an alpha-selective N-cysteinyl glucosamine donor; however, initial glycosylation attempts failed due to the apparent stability of the fused bicyclic system.     

Interactions of substrate with calreticulin,an endoplasmic reticulum chaperone

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc(1)Man(7-9)GlcNAc(2) present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc(1)Man(7-9)GlcNAc(2), determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K(b)) and changes in enthalpy of binding (Delta H(b) degrees ) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 x 10(4) m(-1) at 279 K is obtained when glucose occurs in alpha-1,3 linkage to Man alpha Me as in Glc alpha 1-3Man alpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed.     

Binding of multivalent carbohydrates to concanavalin A and Dioclea grandiflora lectin. Thermodynamic analysis of the "multivalency effect"

Binding of a series of synthetic multivalent carbohydrate analogs to the Man/Glc-specific lectins concanavalin A and Dioclea grandiflora lectin was investigated by isothermal titration microcalorimetry. Dimeric analogs possessing terminal alpha-D-mannopyranoside residues, and di-, tri-, and tetrameric analogs possessing terminal 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues, which is the core trimannoside of asparagine-linked carbohydrates, were selected in order to compare the effects of low and high affinity analogs, respectively. Experimental conditions were found that prevented precipitation of the carbohydrate-lectin cross-linked complexes during the isothermal titration microcalorimetry experiments. The results show that the value of n, the number of binding sites on each monomer of the lectins, is inversely proportional to the number of binding epitopes (valency) of each carbohydrate. Hence, n values close to 1.0, 0.50, and 0.25 were observed for the binding of mono-, di-, and tetravalent sugars, respectively, to the two lectins. Importantly, differences in the functional valency of a triantennary analog for concanavalin A and D. grandiflora lectin are observed. The enthalpy of binding, DeltaH, is observed to be directly proportional to the number of binding epitopes in the higher affinity analogs. For example, DeltaH of a tetravalent trimannoside analog is nearly four times greater than that of the corresponding monovalent analog. Increases in K(a) values of the multivalent carbohydrates relative to monovalent analogs, known as the "multivalency effect," are shown to be due to more positive entropy (TDeltaS) contributions to binding of the former sugars. A general thermodynamic model for distinguishing binding of multivalent ligands to a single receptor with multiple, equal subsites versus binding to separate receptor molecules is given.     

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.     

Relations between uptake and utilization of NO−3 in Pisum growing exponentially under nitrogen limitation

The utilization and translocation of nitrogen was investigated in exponentially growing, nitrogen-limited Pisum sativum L. cv. Marma. The plants were given N daily at exponentially increasing, although suboptimal, relative nitrogen addition rates (R_N) calculated to yield a relative increment in N of 0.06 day^?1 and 0.12 day^?1. After 10 days of NO^?₃ additions (26 days after sowing), the relative growth rate more or less equaled R_N. Uptake of NO^?₃ was several-fold higher than the N requirement for the growth rate set by R_N. The daily addition of NO^?₃ was taken up after 7 to 8 h, resulting in a cyclic behaviour in the NO^?₃ utilization. During the phase of net NO^?₃ influx, the filling phase (0 to 8 h), in vitro nitrate reductase activity (NR activity) and intracellular levels of soluble N in the root increased. In the phase of no net influx of NO^?₃ the depletion phase (8 to 24 h), the plants were entirely dependent on stored N. During this phase both in vitro NR activity and intracellular levels of soluble N decreased. Also the calculated actual rate of NO^?₃ reduction was high in the filling phase, while it was close to zero in the depletion phase. The pattern of these fluctuations indicates that the regulation of NO^?₃ utilization involves an interplay between transmembrane fluxes of NO^?₃, the cytosolic NO^?₃ concentration and NR activity. Cyclic fluctuations in N-containing compounds were also found in the xylem. Nitrogen was mainly transported as amino acids. The pattern of NO^?₃ transport in the xylem and the fluctuations in the shoot of in vitro NR activity indicate that a reasoning similar to that for the regulation of NO^?₃ assimilation in the root also applies for the shoot. The results also indicate a substantial supply of amino acids to the xylem through recirculation from the shoot.     

A conformational study of alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->O)-L-Ser by NMR 1H,1H T-ROESY experiments and molecular-dynamics simulations

The conformational preference of alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->O)-L-Ser has been investigated by one-dimensional (1)H,(1)H T-ROESY experiments and molecular-dynamics simulations with CHARMM22 type of force fields and water as explicit solvent. Proton-proton distances were obtained from the simulations and subsequently experimentally determined distances could be derived. Measurements were performed on the title compound as well as on selectively deuterium-substituted analogues synthesized as part of this study to alleviate possible NMR spectroscopic difficulties. A very good agreement was present between the separate NMR experiments. In the subsequent analysis a key nuclear Overhauser effect between the anomeric protons in the two sugar residues was used to assess the conformational dynamics revealed by the molecular simulations. The combined results support a model in which two states are significantly populated as a result of flexibility around the bond defined by the glycosidic torsion angle psi.     

The tyrosine gate as a potential entropic lever in the receptor-binding site of the bacterial adhesin FimH

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections. During infection, UPEC adhere to mannosylated glycoreceptors on the urothelium via the FimH adhesin located at the tip of type 1 pili. Synthetic FimH antiadhesives such as alkyl and phenyl α-D-mannopyranosides are thus ideal candidates for the chemical interception of this crucial step in pathogenesis. The crystal structures of the FimH lectin domain in its ligand-free form and in complexes with eight medium- and high-affinity mannopyranoside inhibitors are presented. The thermodynamic profiles of the FimH-inhibitor interactions indicate that the binding of FimH to α-D-mannopyranose is enthalpy-driven and has a negative entropic change. Addition of a hydrophobic aglycon influences the binding enthalpy and can induce a favorable entropic change. The alleviation of the entropic cost is at least in part explained by increased dynamics in the tyrosine gate (Tyr48 and Tyr137) of the FimH receptor-binding site upon binding of the ligand. Ligands with a phenyl group directly linked to the anomeric oxygen of α-D-mannose introduce the largest dynamics into the Tyr48 side chain, because conjugation with the anomeric oxygen of α-D-mannose forces the aromatic aglycon into a conformation that comes into close contact (≈2.65 ?) with Tyr48. A propargyl group in this position predetermines the orientation of the aglycon and significantly decreases affinity. FimH has the highest affinity for α-D-mannopyranosides substituted with hydrophobic aglycons that are compatible in shape and electrostatic properties to the tyrosine gate, such as heptyl α-D-mannose.     

Nitrogen redistribution from the roots in post-anthesis plants of spring wheat

The aim of this study was to investigate post-anthesis N redistribution from the roots to other organs in the wheat plant. Two cultivars of spring wheat (Triticum aestivum L.) Sport and WL were grown to maturity in culture solution with stepwise-decreasing N supply and ¹⁵N before sampling. The N concentration of dry weight in the roots decreased from anthesis to full maturity. At maturity the root N concentration was 5.5 mg g⁻¹ dry weight for Sport and 4.9 mg g⁻¹ dry weight for WL, while the main shoot N concentration was 2.1 mg g⁻¹ dry weight for Sport and 2.2 mg g⁻¹ dry weight for WL. The roots were a major sink for N taken up post-anthesis and contained the main proportion of the ¹⁵N during the whole post-anthesis period. The N redistributed from the roots contributed 24.3% of the total N amount in the grains at maturity in cv Sport and 8.7% in cv WL. For cv Sport, 46.3% of the root N amount was redistributed to other parts of the plant and for WL 25.4%. The tiller grains were supplied with N from the ¹⁵N applied post-anthesis to a higher degree than the main shoot grains. The transport capability was maintained until 12 days after complete yellowness. We concluded that wheat has a great ability to redistribute nitrogen from the root system to the grains.     

A new route for the synthesis of Streptococcus pneumoniae 19F and 19A capsular polysaccharide fragments avoiding the β-mannosamine glycosylation step

The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F. Carbohydr. Res. 2008, 343, 2545–2556.] β-d-MaNAcp-(1→4)-β-d-Glcp thiophenyl glycosyl donor 3 was used in α-glycosylation reactions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl α-l-rhamnopyranoside acceptors 7 and 8. Glycosylation of the axial OH-2 of 7 took place in high yield (76%) and with acceptable stereoselectivity (α/β = 3.4) leading to the protected trisaccharide α-11, corresponding to the repeating unit of Streptococcus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide α-15, a constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselection (α/β = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-4″, the trisaccharide α-11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated oligomers.