Synthesis of derivatives of methyl β-sophoroside

Selective tritylation of methyl β-sophoroside (1) and subsequent acetylation gave the 3,4,2′,3′,4′-penta-O-acetyl-6,6′-di-O-trityl derivative, which was O-detritylated, and the product p-toluenesulfonylated, to give methyl 3,4,2′,3′,4′-penta-O-acetyl-6,6′-di-O-p-tolylsulfonyl-β-sophoroside (4) in 63% net yield. Compound 4 was also obtained in 69% yield by p-toluenesulfonylation of 1, followed by acetylation. Several, 6,6′-disubstituted derivatives of 1 were synthesized by displacement reactions of 4 with various nucleophiles. Treatment of 4 with sodium methoxide afforded methyl 3,6:3′,6′-dianhydro-β-sophoroside. Several 6- and 6′-monosubstituted derivatives of 1 were prepared, starting from the 4,6-O-benzylidene derivative of 1.     

Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl–methyl nuclear overhauser enhancement spectroscopy

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl–methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.     

Metal-mediated allylation of enzymatically oxidized methyl α-D-galactopyranoside

The C-6 unit of methyl α-d-galactopyranoside was selectively modified by combining enzymatic oxidation with an indium-mediated allylation reaction. The Barbier-Grignard type reaction, where a carbonyl group reacts with an allyl halide, proceeds in aqueous solution, even with water as the only solvent; thus carbohydrates can be modified without the need for drying or protection-deprotection steps. The corresponding homoallyl alcohols are produced in high yields of >90% in the reactions with allyl bromide and cinnamyl chloride. The main products were isolated and characterized by GC-MS and NMR spectroscopy.     

The oxidation of methyl α-D-glucopyranoside with methyl sulproxide and acetic anhydride

The relative proportions of carbonyl, O-acetyl, and O-(methylthio)methylsugars resulting from the partial oxidation of methyl α-D-glucopyranoside with methyl sulphoxide and acetic anhydride have been investigated@ the preparation of the 2- and 6-(methylthio)methyl ethers of methyl α-D-glucopyranoside is described.     

Getting old: an endangered seahorse (Hippocampus whitei) lives for up to 7 years in the wild

A long-term monitoring programme from 2005 to 2021 has allowed the assessment of age and longevity in an endangered seahorse Hippocampus whitei in the wild. Seahorses were marked using visible implant fluorescent elastomer (VIFE) which allows for individual identification. The longest period from marking to last sighting was 6 years 8 months and 17 days for a female. Using a von Bertalanffy growth function model for the species, this individual would have been approximately 7 years 7 months old on last sighting. These observations suggest that seahorses in the wild can live for over 7 years and demonstrate the benefits of using VIFE in long-term movement, population abundance and life-history studies of seahorses.     

Crystal and molecular structure of methyl L-glycero-α-D-manno-heptopyranoside, and synthesis of 1→7 linked L-glycero-D-manno-heptobiose and its methyl α-glycoside

Methyl l-glycero-α-d-manno-heptopyranoside was synthesized in good yield by a Fischer-type glycosylation of the heptopyranose with methanol in the presence of cation-exchange resin under reflux and microwave conditions, respectively. The compound crystallized from 2-propanol in an orthorhombic lattice of space group P2(1)2(1)2 showing a comparatively porous structure with a 2-dimensional O-H?O hydrogen bond network. As model compounds for the side chain domains of the inner core structure of bacterial lipopolysaccharide, l-glycero-α-d-manno-heptopyranosyl-(1→7)-l-glycero-d-manno-heptopyranose and the corresponding disaccharide methyl α-glycoside were prepared. The former compound was generated via glycosylation of a benzyl 5,6-dideoxy-hept-5-enofuranoside intermediate followed by catalytic osmylation and deprotection. The latter disaccharide was efficiently synthesized in good yield by a straightforward coupling of an acetylated N-phenyltrifluoroacetimidate heptopyranosyl donor to a methyl 2,3,4,6-tetra-O-acetyl heptopyranoside acceptor derivative followed by Zemplén deacetylation.     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

A concise synthesis of methyl 2,6-dideoxy-2-fluoro-β-l-talopyranoside

The 4,6-benzylidene acetal of methyl 2-deoxy-2-fluoro-α,β-d-glucopyranoside underwent inversion at C-3 via an oxidation–reduction sequence, and treatment of the derived 3-acetate with N-bromosuccinimide in carbon tetrachloride gave methyl 3-O-acetyl-4-O-benzoyl-6-bromo-2,6-dideoxy-2-fluoro-α-d-allopyranoside (6). Dehydrobromination of 6 and reduction of the resultant 5,6-ene gave the 5-epimer of 6, which after removal of the ester substituents, afforded the title compound in good overall yield.     

Reduced methyl group acceptance of 1-β-d-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-β-d-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.     

Ectomycorrhizal fungi: A new source of atmospheric methyl halides?

Incomplete source budgets for methyl halides – compounds that release inorganic chlorine and bromine radicals which, in turn, catalyze atmospheric ozone depletion – limit our ability to predict the fate of the stratospheric ozone layer. We report here the first measured emissions of methyl chloride, methyl bromide, and methyl iodide from ectomycorrhizal fungi. We grew nine fungal isolates on growth media containing halide concentrations similar to those found in soils and plant tissues. The observed range of emissions was 0.003–65 μg methyl chloride, 0.001–3 μg methyl bromide, and 0.02–12 μg methyl iodide g^?1 dry weight fungi day^?1. Species varied in production rates of methyl chloride vs. methyl bromide vs. methyl iodide. Cenococcum geophilum, a widespread ectomycorrhizal fungus, was further tested to investigate the effects of halide substrate concentration in growth media. Emissions from this species increased linearly with increasing concentrations of both bromide and iodide. In addition, a subset of four fungi was studied with two media concentrations each of chloride, bromide, and iodide (0.2 or 20 mm ). These fungi had similar responses to halide concentration, despite 1000‐fold differences in baseline emission rates between isolates. Finally, high chloride concentrations (20 mm ) in media did not appear to inhibit emissions of methyl bromide or methyl iodide. Overall, ectomycorrhizal fungi might be an important source of methyl halides to the atmosphere, and substrate concentrations and community composition may influence production levels in ecosystems.     

A combined nuclear magnetic resonance and molecular dynamics study of the two structural motifs for mixed-linkage β-glucans: methyl β-cellobioside and methyl β-laminarabioside

The conformational and hydration properties of the two disaccharides methyl β-cellobioside and methyl β-laminarabioside were investigated by NMR spectroscopy and explicit solvation molecular dynamics simulations using the carbohydrate solution force field (CSFF). Adiabatic maps produced with this force field displayed 4 minima A: (Φ = 300°, Ψ = 280°), B: (Φ = 280°, Ψ = 210°), C: (Φ = 260°, Ψ = 60°), and D: (Φ = 60°, Ψ = 260°) for methyl β-cellobioside and 3 minima A: (Φ = 290°, Ψ = 130°), B: (Φ = 270°, Ψ = 290°), and C: (Φ = 60°, Ψ = 120°) for methyl β-laminarabioside. Molecular dynamics simulations were initiated from all minima. For each disaccharide, the simulation started from the A minimum was conducted for 50 ns, while the other minima were explored for 10 ns. The simulations revealed two stable minima for both compounds. For methyl β-cellobioside, the simulation minima in aqueous solution were shifted from their adiabatic map counterparts, while the simulation minima for methyl β-laminarabioside coincided with the corresponding adiabatic map minima. To validate the simulation results, NMR-derived NOEs and coupling constants across the glycoside linkage, ³J_HC and ³J_CH, were compared with values calculated from the MD trajectories. For each disaccharide, the best agreement was obtained for the simulations started at the A minimum. For both compounds, inter-ring water bridges in combination with the direct hydrogen bonds between the same groups were found to be determining factors for the overall solution structure of the disaccharides which differed from solid-state structures. Comparison with helical parameters showed that the preferred glycosidic dihedral configurations in the methyl β-cellobioside simulation were not highly compatible with the structure of cellulose, but that curdlan helix structures agreed relatively well with the methyl β-laminarabioside simulation. Polymers generated using glycosidic dihedral angles from the simulations revealed secondary structure motifs that that may help to elucidate polymer associations and small-molecule binding.     

Monomolar acetalations of methyl α-d-mannosides—synthesis of methyl α-d-talopyranoside

Methyl α-d-mannopyranoside (1 mole) reacts with 2,2-dimethoxypropane (1 mole), to give the 4,6-O-isopropylidene derivative (2) which rearranges to the 2,3-O-isopropylidene derivative (4). Compound4 can also be prepared by graded hydrolysis of methyl 2,3:4,6-di-O-isopropylidene-α-d-mannopyranoside. Successive benzoylation, oxidation, and reduction of4 provides a useful route to a number ofd-talopyranoside compounds. Methyl α-d-mannofuranoside (1 mole) reacts with 1–2 moles of 2,2-dimethoxypropane to give the 5,6-O-isopropylidene derivative (16) in 90% yield.     

Synthesis and anti-tumor activities of methyl 2-O-aryl-6-O-aryl′-d-glucopyranosides

A synthetic method of introducing bulky aryl groups at the 2-O- and 6-O-positions on glucopyranosides was developed. A total of 37 new compounds of this class were obtained successfully. These compounds were tested on several tumor cell lines by MTT assays, and some of them exhibited encouraging inhibitory activities. The most potent compound, CAB-SHZH-27, exhibited EC₅₀ values of 14, 12, and 10 μmol/L on A549, MDA-MB-231 and HeLa cells, respectively. A preliminary structure–activity relationship analysis indicates that the two free hydroxyl groups on the d-glucose core are indispensable for the biological activities of this class of compounds, and the aryl group at the 6-O-position has a more obvious impact than the one at the 2-O-position. An interesting ‘on–off’ mechanism of this class of compounds was also observed in our MTT assays, which remains to be explored.     

An improved regioselective preparation of methyl 2,3-di-O-acetyl-α,β-d-xylofuranoside

Abstract

Methyl 2,3-di-O-acetyl-α,β-d-xylofuranoside was prepared as the sole regioisomer in 63–72% yield, according to the applied mass of substrate, through a Candida antarctica lipase B catalysed alcoholysis of methyl 2,3,5-tri-O-acetyl-α,β-d-xylofuranoside. The product is a potential synthetic precursor for 5-modified xylofuranosides and 5′-modified xylonucleosides.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Accessing ns–μs side chain dynamics in ubiquitin with methyl RDCs

This study presents the first application of the model-free analysis (MFA) (Meiler in J Am Chem Soc 123:6098–6107, 2001; Lakomek in J Biomol NMR 34:101–115, 2006) to methyl group RDCs measured in 13 different alignment media in order to describe their supra-τ _c dynamics in ubiquitin. Our results indicate that methyl groups vary from rigid to very mobile with good correlation to residue type, distance to backbone and solvent exposure, and that considerable additional dynamics are effective at rates slower than the correlation time τ _c. In fact, the average amplitude of motion expressed in terms of order parameters S ² associated with the supra-τ _c window brings evidence to the existence of fluctuations contributing as much additional mobility as those already present in the faster ps-ns time scale measured from relaxation data. Comparison to previous results on ubiquitin demonstrates that the RDC-derived order parameters are dominated both by rotameric interconversions and faster libration-type motions around equilibrium positions. They match best with those derived from a combined J-coupling and residual dipolar coupling approach (Chou in J Am Chem Soc 125:8959–8966, 2003) taking backbone motion into account. In order to appreciate the dynamic scale of side chains over the entire protein, the methyl group order parameters are compared to existing dynamic ensembles of ubiquitin. Of those recently published, the broadest one, namely the EROS ensemble (Lange in Science 320:1471–1475, 2008), fits the collection of methyl group order parameters presented here best. Last, we used the MFA-derived averaged spherical harmonics to perform highly-parameterized rotameric searches of the side chains conformation and find expanded rotamer distributions with excellent fit to our data. These rotamer distributions suggest the presence of concerted motions along the side chains.     

Mass-spectral fragmentation of methyl 2,3,4-tri-O-methyl-α-D-glucopyranosiduronamide and its analytical application

Fragmentation pathways for methyl 2,3,4-tri-O-methyl-α-D-glucopyranosiduronamide are proposed, based on 70- and 12-eV spectra of the compound specifically labelled with CD₃ and ND₂ groups. The presence of the NH₂ group in the molecule gives rise to new fragmentation series. The number and positions of CD₃ groups can be unequivocally determined from the mass spectra. Partially methylated derivatives of hexuronic acids, obtained by methylation analysis of hexuronic acid-containing substances, can be identified by exhaustive trideuteriomethylation and conversion into readily obtainable crystalline amides.