d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

β-d-glucosidase-catalysed transfer of the glycosyl group from aryl β-d-gluco- and β-d-xylo-pyranosides to phenols

The effect of phenols on the hydrolysis of substituted phenyl β-d-gluco- and β-d-xylo-pyranosides by β-d-glucosidase from Stachybotrys atra has been investigated. Depending on the glycon part of the substrate and on the phenol substituent, the hydrolysis is either inhibited or activated. With aryl β-d-xylopyranosides, transfer of the xylosyl residue to the phenol, with the formation of new phenyl β-d-xylopyranosides, is observed. With aryl β-d-glucopyranosides, such transfer does not occur when phenols are used as acceptors, but it does occur with anilines. A two-step mechanism, in which the first step is partially reversible, is proposed to explain these observations. A qualitative analysis of the various factors determining the overall effect of the phenol is given.     

Synthesis of monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside for epitope mapping of anti-Candida albicans antibodies

A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation–reduction sequence at C-2′. The β-manno configurations of the final deprotected congeners 2–7 were confirmed by measurement of ¹J_C1,H1 heteronuclear and ³J_1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the protective epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site.     

Enzymatic synthesis of d-xylulose 5-phosphate from hydroxypyruvate and d-glyceraldehyde-3-phosphate

An enzymatic method for obtaining d-xylulose 5-phosphate has been developed, based on the irreversible reaction catalyzed by transketolase: hydroxypyruvate + d-glyceraldehyde-3-phosphate → d-xylulose 5-phosphate. The preparations of sodium d-xylulose 5-phosphate, obtained using this approach, were 88% pure and contained no aldehyde admixtures.     

Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum

Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Unique transglycosylation potential of extracellular α-d-galactosidase from Talaromyces flavus

The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Enzymatic resolution of methyl dl-β-acetylthioisobutyrate and dl-β-acetylthioisobutyramide using a stereoselective esterase from Pseudomonas putida IFO12996

Esterase (PpEST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-β-acetylthioisobutyrate (DL-MATI) and dl-β-acetylthioisobutyramide (DL-ATIA) to give d-β-acetylthioisobutyric acid (DAT). DAT is a key intermediate for the synthesis of a series of angiotensin converting enzyme inhibitors. To use enzyme for the DAT production, the PpEST gene of P. putida IFO12996 was cloned and expressed in Escherichia coli. PpEST with a molecular weight of 33 kDa could hydrolyze DL-MATI and DL-ATIA to give DAT with enantiometric excess value (e.e. value) about 97% and enantioselectivity value (E-value) >150, respectively. The kinetic constants of PpEST for DL-MATI and DL-ATIA were examined and they showed that DL-ATIA was a poorer substrate than DL-MATI for PpEST. However, DL-ATIA was 20-fold more soluble in water than DL-MATI, it was more stable than DL-MATI and it did not show substrate inhibition of the PpEST up to 780 mM. This result suggested that PpEST is an esterase but with amidase activity, which can kinetically resolve DL-ATIA to yield DAT and DL-ATIA is a better choice than DL-MATI for industrial production of DAT by the enzymatic resolution method.     

2′-Azido-2′-deoxy- and 2′-amino-2′-deoxy-β-d-arabino-furanosyl purine nucleosides

A general method for the preparation of 2′-azido-2′-deoxy- and 2′-amino-2′-deoxyarabinofuranosyl-adenine and -guanine nucleosides is described. Selective benzoylation of 3-azido-3-deoxy-1,2-O-isopropylidene-α-d-glucofuranose afforded 3-azido-6-O-benzoyl-3-deoxy-1,2-O-isopropylidene-α-d-glucofuranose (1). Acid hydrolysis of 1, followed by oxidation with sodium metaperiodate and hydrolysis by sodium hydrogencarbonate gave 2-azido-2-deoxy-5-O-benzoyl-d-arabinofuranose (3), which was acetylated to give 1,3-di-O-acetyl-2-azido-5-O-benzoyl-2-deoxy-d-arabinofuranose (4). Compound 4 was converted into the 1-chlorides 5 and 6, which were condensed with silylated derivatives of 6-chloropurine and 2-acetamido-hypoxanthine. The condensation reaction gave α and β anomers of both 7- and 9-substituted purine nucleosides. The structures of the nucleosides were determined by n.m.r. and u.v. spectroscopy, and by correlation of the c.d. spectra of the newly prepared nucleosides with those published for known purine nucleosides.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

Unsaturated sugars I. Decarboxylative elimination of methyl 2,3-di-O-benzyl-α-D-glucopyranosiduronic acid to methyl 2,3-di-O-benzyl-4-deoxy-β-L-threo-Pent-4-enopyranoside

Decarboxylative elimination of methyl 2,3-di-O-benzyl-α-D-glucopyranosiduronic acid (1) with N,N-dimethylformamide dineopentyl acetal in N,N-dimethylformamide gave methyl 2,3-di-O-benzyl-4-deoxy-β-L-threo-pent-4-enopyranoside (3). Debenzylation of 3 was effected with sodium in liquid ammonia to give methyl 4-deoxy-β-L-threo-pent-4-enopyranoside (4). Hydrogenation of 3 catalyzed by palladium-on-barium sulfate afforded methyl 2,3-di-O-benzyl-4-deoxy-β-L-threo-pentopyranoside (5), whereas hydrogenation of 3 over palladium-on-carbon gave methyl 4-deoxy-β-L-threo-pentopyranoside (6). An improved preparation of methyl 4,6-O-benzylidene-α-D-glucopyranoside is also described.     

E versus Z geometry in β-d-arabino-hexopyranosidulose oximes

Koenigs–Knorr-type glycosidations of peracylated 2Z-benzoyloxyimino-glycopyranosyl bromides invariably proceed with retention of the Z-geometry. Accordingly, the many β-d-hexosidulose oximes in literature which were prepared in this way and for which the oxime geometry has not been addressed explicitly, are the Z-oximes throughout. By contrast, oximation of β-d-hexopyranosid-2-uloses leads to mixtures of E and Z oximes readily separable and structurally verifiable by ¹H and ¹³C NMR. Configurational assignments rested on comparative evaluation of NMR data of E and Z isomers, and, most notably on an X-ray structural analysis of the pivaloylated isopropyl 2E-benzoyloxyimino-2-deoxy-β-d-arabino-hexopyranoside revealing the unusual ¹S₅^1,4B conformation for the pyranoid ring.     

The crystal and molecular structure of 1,6-anhydro-β-d-mannopyranose

The crystal structure of 1,6-anhydro-β-d-mannopyranose, C₆H₁₀O₅, is orthorhombic, P2₁2₁2₁, with a = 10.971(2), b = 13.935(3), c = 9.012(1) Å, V = 1377.76 »³ (MoKα, λ = 0.7107 Å), Z = 8, D_x = 1.563 M.gm⁻³, D_m = 1.565 M.gm⁻³. the structure was solved by MULTAN and refined to R(F) = 0.043 for 2355 reflections. The two symmetry-independent molecules in the unit cell have similar conformations, except for the orientation of one of the three hydroxyl groups. The conformation of the pyranose rings is ¹C₄ distorted towards E_o, and that of the anhydro rings is E. There are significant differences between the two molecules in two of the four C---O bond-lengths. These occur where there are important differences in the hydrogen-bonding environment of the oxygen atoms. The molecules are hydrogen-bonded by three linear and three bifurcated O---H···O interactions which form four-membered loops linked into infinite chains. Empirical force-field calculations with MMI-CARB reproduced the geometry of the molecules within the variations observed experimentally between the two molecules, except for a C---O bond in one of the molecules. The effect of excluding the anomeric effect from the theoretical calculations was not significant. Calculations for an intramolecularly hydrogen-bonded molecule were also carried out as a model for the molecules in a non-polar solvent.     

Chain conformation and anti-tumor activities of phosphorylated (1→3)-β-d-glucan from Poria cocos

(1→3)-β-d-Glucan isolated from Poria cocos was phosphorylated to obtain a series of phosphorylated derivatives. Their structures, weight-average molecular weights (M_w), and chain conformation were studied by ¹³C NMR, ³¹P NMR, static laser light scattering and viscometry. The experimental results revealed that the phosphorylated glucan existed as relatively extended flexible chain in 0.15 M NaCl aqueous solution, and exhibited relatively strong inhibition against S-180 tumor cell in vitro and in vivo. In vivo, the fractions with relatively high molecular weight at low dosage exhibited stronger anti-tumor activities. The results revealed that the molecular weights and molecular conformation could influence the anti-tumor activities. The molecular weight ranging from 2.6 × 10⁴ to 26.8 × 10⁴ and the extended chain conformation were beneficial to enhance the anti-tumor activity, as a result of the increasing of the interaction between polysaccharide and immune system.     

N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide

A complex trisaccharide β-d-GalpNAcA-(1 → 4)-β-d-GlcpNAc-(1 → 4)-d-ManpNAc (3) was prepared in a good yield (35%) in a transglycosylation reaction catalyzed by β-N-acetylhexosaminidase from Talaromyces flavus using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galacto-hexodialdo-1,5-pyranoside (1) as a donor followed by the in situ oxidation of the aldehyde functionality by NaClO₂. The disaccharide β-d-GlcpNAc-(1 → 4)-d-ManpNAc (2) was used as galactosyl acceptor. A disaccharide β-d-GalpNAcA-(1 → 4)-d-GlcpNAc (4; 39%) originated as a by-product in the reaction. Oligosaccharides comprising a carboxy moiety at C-6 are shown to be very efficient ligands to natural killer cell activation receptors, particularly to human receptor CD69. Thus, oxidized trisaccharide 3 is the best-known oligosaccharidic ligand to this receptor, with IC₅₀ = 2.5 × 10⁻⁹ M. The presented method of introducing a β-d-GalpNAcA moiety into carbohydrate structures is versatile and can be applied in the synthesis of other complex oligosaccharides.     

The synthesis of P-(2-acetamido-2-deoxy-α-D-glucopyranosyl) P-ficaprenyl pyrophosphate

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α,β-D-glucopyranosylammonium phosphate was prepared by the action of crystalline phosphoric acid on 2-acetamido-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose. The α-D anomer (3) was the main product, and was isolated pure by preparative thin-layer chromatography or by removal of the β-D anomer (6) by selective acid hydrolysis. Ficaprenyl phosphate was prepared from ficaprenol, obtained as an isomeric mixture (mainly C₅₅) from an extract of Ficus elastica. Compound 3 was converted into the free acid and then into the tributyl-ammonium salt, which was treated with P¹-diphenyl P²-ficaprenyl pyrophosphate to give the acetylated pyrophosphate diester 8, characterized by analytical, spectral, and hydrogenolytic studies. Deacetylation of 8 gave the synthetic “lipid intermediate”, P¹-(2-acetamido-2-deoxy-D-glucopyranosyl) P²-ficaprenyl pyrophosphate (9), the properties of which were compared with those of natural substances considered to be active in the biosynthesis of teichoic acids.