Transport of 3-O-methyl d-glucose and β-methyl d-glucoside by rabbit ileum

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereospecific affinity and (b) are common.The apparent affinity constants at the brush-border indicate that sugars are selected in the order, $\text{β-methyl glucose >}\text{d}\text{-galactose}\text{> 3-O-}\text{methyl}$ glucose, (the K_m values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the K_t values for Na⁺ activation of sugar entry across the brush-border are: 27.25, and 140 mequiv. for β-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na⁺, water, sugar and membrane-binding groups are all factors which determine selective affinity.In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer [Na] or [sugar] is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)². This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion.Theophylline reduces the serosal border permeability to β-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increases the intracellular accumulation of these sugars.The permeability of the serosal border to β-methyl glucose entry is lower than permeability of the serosal border to β-methyl glucose exit, which suggests that β-methyl glucose may be convected out of the cell across the lateral serosal border.     

The synthesis of the α-d-anomers of “homonucleosides”: derivatives of 2,5-anhydro-d-altritol

Reaction of 2,3,5-tri-O-benzyl-d-ribofuranosyl bromide with mercuric cyanide afforded an anomeric mixture of cyanides (3) and 1,4-anhydro-2,3,5-tri-O-benzyl-d-erythro-pent-1-enitol (6). Reduction of 3 with lithium aluminum hydride gave a pair of epimeric amines (4 and 5), which were separated by chromatography and characterized by conversion into the known 2,5-anhydro-3,4,6-tri-O-benzyl-1-deoxy-1-ureido-d-allitol (7) and its epimer, 2,5-anhydro-3,4,6-tri-O-benzyl-1-deoxy-1-ureido-d-altritol (8). Compound 8 and its precursor were used for the synthesis of various “α-homonucleosides”.     

The synthesis of 1,6-anhydro-β-d-glucopyranose and d-glucosyl oligosaccharides from maltose by a fungal glucosyltransferase

The formation of 1,6-anhydro-β-d-glucopyranose and several d-glucosyl oligosaccharides has been observed during the action of a purified, fungal glucosyltransferase (EC 2.4.1.24) on maltose. Such products are synthesized by a transglucosylation mechanism involving the formation of a d-glucosyl-enzyme complex and the displacement of the d-glucosyl group by appropriate acceptor-substrates. The formation of the 1,6-anhydro bond is a novel type of transfer reaction and occurs by displacement of the enzyme from the d-glucosyl-enzyme complex by the proton of the primary hydroxyl group of the same glucosyl group. This reaction is characterized by inversion of configuration at the position of glucosidic bond-cleavage of the substrate. Synthesis of the d-glucosyl oligosaccharides occurs by displacement of the d-glucosyl groups from the enzyme by suitable acceptor-substrates. In these cases, the reactions are characterized by retention of configuration of the d-glucosidic bonds of the substrate. The list of oligosaccharides produced from maltose includes nigerose, kojibiose, isomaltose, maltotriose, panose, isomaltotriose, and 6-O-d-glucosyl-panose. The identity of these compounds has been established by methylation analysis and enzymic hydrolysis. d-Glucose is also a product of the reaction and arises from both the reducing and the non-reducing groups of maltose.     

The use of 1-O-tosyl-d-glucopyranose derivatives in α-d-glucoside synthesis

1-O-Tosyl-d-glucopyranose derivatives having a nonparticipating benzyl group at O-2 have been shown to react rapidly in various solvents with low concentrations of alcohols, either methanol or methyl 2,3,4-tri-O-benzyl-α-d-glucopyranoside. The stereospecificity of the glucoside-forming reaction could be varied from 80% of β to 100% of α anomer by changing the solvent or modifying the substituents on the 1-O-tosyl-d-glucopyranose derivative. 2,3,4-Tri-O-benzyl-6-O-(N-phenylcarbamoyl)-1-O-tosyl-α-d-glucopyranose in diethyl ether gave a high yield of α-d-glucoside. Kinetic measurements of reaction with various alcohols (methanol, 2-propanol, and cyclohexanol) show a high rate even at low concentrations of alcohol, and give some insight into the reaction mechanism. The high rate and stereoselectivity of their reaction suggest that the 1-O-tosyl-d-glucopyranose derivatives may be used as reagents for oligosaccharide synthesis.     

The acid-catalyzed hydrolysis of β-d-xylofuranosides

The rate constants for the hydrolysis of six alkyl and four aryl β-d-xylofuranosides in aqueous perchloric acid at various temperatures have been measured. The effects of varying the aglycon structure on the hydrolysis rate are interpreted in terms of two concurrent reactions. Either, the substrate, protonated on the glycosidic oxygen atom, undergoes a rate-limiting heterolysis to form a cyclic oxocarbonium ion, or, an initial rapid protonation of the ring oxygen is followed by a unimolecular cleavage of the five-membered ring, all subsequent reactions being fast. It is suggested that xylofuranosides having strongly electron-attracting aglycon groups react mainly by the former pathway, whereas the latter is more favourable for substrates having electron-repelling aglycon groups. The negative entropies of activation obtained with the latter compounds are attributed to the rate-limiting opening of the five-membered ring. The rate variations of the hydrolyses of alkyl β-d-xylofuranosides in aqueous perchloric acid-methyl sulfoxide mixtures are interpreted as lending further support for the suggested chance in mechanism.     

Synthesis of 5-O-α-and-β-d-glucopyranosyl-d-glucofuranose and 5-O-α-d-glucopyranosyl-d-fructopyranose (leucrose)

Reaction of 1,2-O-cyclopentylidene-α-d-glucofuranurono-6,3-lactone (2) with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide (1) gave 1,2-O-cyclopentylidene- 5-O-(2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl)-α-d-glucofuranurono-6,3-lactone (3, 45%) and 1,2-O-cyclopentylidene-5-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-α-d-glucofuranurono-6,3-lactone (4, 38%). Reduction of 3 and 4 with lithium aluminium hydride, followed by removal of the cyclopentylidene group, afforded 5-O-α-(9) and -β-d-glucopyranosyl-d-glucofuranose (12), respectively. Base-catalysed isomerization of 9 yielded crystalline 5-O-α-d-glucopyranosyl-d-fructopyranose (leucrose, 53%).     

d-Xylose,an anomer-specific inhibitor of α-galactosidase

d-xylose is a highly anomer-specific, powerful and competitive inhibitor of plant α-galactosidases. The apparent inhibition constant (K_i) for the interaction of d-xylose with chick pea (Cicer arietinum) α-galactosidase and the apparent number of inhibitor molecules (n) bound per enzyme molecule, using p-nitrophenyl-α-d-galactopyranoside as substrate, were found to be 0.4 × 10^?2M and 0.8, respectively.     

The ineffectiveness of analogs of d-galactal as competitive inhibitors of,and substrates for, β-d-galactosidase from Escherichia coli

2,6-Anhydro-3-deoxy-aldehydo-d-lyxo-hept-2-enose (7) 2,6-anhydro-3-deoxy-d-lyxo-hept-2-enitol (8) were synthesized as half-chair analogs of d-galactal (1). As 1 is a strong inhibitor of, as well as a substrate for, β-d-galactosidase from Escherichia coli, the same properties were expected for 7 and 8; however, both were ineffective. This result, together with those of other authors, allows speculative conclusions on the tight binding of 1 to the enzyme only, when water or an alcohol is bound as a co-substrate.     

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.     

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.     

X-ray crystal structure of maltitol (4-O-α-d-glucopyranosyl-d-glucitol

Maltitol, crystallised from aqueous solution, has m.p. 146.5–147°, [α]_d + 106.5° (water), and is orthorhombic with the space group P2₁2₁2₁ and Z = 4, and with cell dimensions a = 8.166(5), b = 12.721(9), and c = 13.629(6) Å. The molecule shows a fully extended conformation with no intramolecular hydrogen-bonds. All nine hydroxyl groups are involved in intermolecular hydrogen-bond networks and in bifurcated, finite chains. The d-glucopyranosyl moiety has the ⁴C₁ conformation, and the conformation about the C-5–C-6 bond is gauche-gauche. The d-glucitol residue has the bent [ap, Psc, Psc (APP)] conformation. The empirical formula for the solubility in water is C = 119.1 + 1.204 T + 4.137 × 10^?2 T² ? 7.137 × 10^?4 T³ + 7.978 × 10^?6 T⁴. The thermal properties are as follows: ΔH_f = 13.5 kcal.mol^?1, and Q = ?5.57 kcal.mol^?1.     

Development and other characteristics of α-methyl-d-glucoside transport by rat kidney cortex slices

α-Methyl-d-glucoside has been shown to be a non-metabolizable sugar which is accumulated against a concentration gradient by a Na⁺-dependent and phlorizin inhibited process by adult rat renal cortical slices incubatedin vitro at 37 °C. (2) The velocity of accumulation increased linearly with substrate concentrations up to 1.5 mM, but at higher concentrations obeyed saturable kinetics with an apparentK_m of about 6 mM. (3) Uptake was enhanced as Na⁺ was increased from 0 to 100 mequiv/l. Higher Na⁺ concentrations caused no further effect. (4) A pH maximum of transport occurred between 7.35 and 8.0. (5) Glucoside uptake was inhibited byd-glucose,d-galactose,d-fructose,d-mannose andd-ribose. The inhibition byd-glucose andd-galactose was competitive with apparentK_t of 24 and 53 mM, respectively. (6) Bothd-glucose andd-galactose accelerated the efflux of α-methyl-d-glucoside from preloaded cells. (7) Kidney cortex slices from 1-day-old rats were unable to accumulate α-methyl-d-glucoside to form a concentration gradient. The ability to concentrate the glucoside increased progressively after birth, reaching near normal in tissue from 15-day-old animals. The data indicate that the transport process in the newborn is rudimentary, failing also to display accelerated efflux phenomenon. (8) α-Methyl-d-glucoside is transported in rat kidney cortex by a mechanism similar in many ways to that ofd-galactose.     

Inhibition of protein kinase activity and amino acid and α-methyl-d-glucoside transport by diamide

Dizene dicarboxylic acid $\text{bis}\text{-(N,N-}\text{dimethylamide}\text{)}$, commonly called diamide, is known to oxidize stoichiometrically intracellular pools of reduced glutathione and inhibit the accumulation of sugars and amino acids by rat kidney slices. Incubation of rat renal cortical slices in diamide also leads to a significant decrease in the level of endogenous protein kinase activity. The inhibition of sugar and amino acid transport and protein kinase activity by diamide is partially reversible by the addition of exogenous glutathione or other thiols. A comparison of protein kinase activity with amino acid and sugar transport at various concentrations of diamide indicates that there is a high degree of correlation between these two processes.     

Crystal structure of 2-deoxy-β-d-lyxo-hexose (2-deoxy-β-d-galactose)

2-Deoxy-β-d-lyxo-hexose (2-deoxy-β-d-galactose, C₆H₁₂O₅), M_r = 164.16, is monoclinic, P2₁ with a = 9.811(1), b = 6.953(1), c = 5.315(1) Å, β = 91.58(2)°, V = 362.5(1) ų, Z = 2, and D_x = 1.504 g.cm^?3. The structure was solved by direct methods (MULTAN 79) and refined to R = 0.032 for 800 observed reflections. Each hydroxyl oxygen, acting both as donor and acceptor, is involved in a hydrogen-bonding system, which consists of infinite helical chains around the crystallographic screw axes. Moreover, weak interactions allow the incorporation of the ring-oxygen atoms into an interconnected network.     

The synthesis of methyl 4-O-(4-O-α-d-galactopyranosyl-β-d-galactopyranosyl)-β-d-glucopyranoside: The methyl β-glycoside of the trisaccharide related to Fabry's disease

As part of a program to synthesize the ceramide trisaccharide (1) related to Fabry's disease, methyl 4-O-(4-O-α-$\text{d}$-galactopyranosyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (12) was prepared. Methyl β-lactoside (2) was converted into methyl 4-O-(4,6-O-benzylidene-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (4). Methyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (7) was synthesized from 4 through the intermediates methyl 2,3,6-tri-O-benzoyl-4-O-(4,6-O-benzylidene-2,3-di-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (5) and methyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (6). The halide-catalyzed condensation of 7 with 2,3,4,6-tetra-O-benzyl-$\text{d}$-galactopyranosyl bromide (8) gave methyl 2,3,6-tri-O-benzoyl-4-O-[2,3,6-tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzyl-α-$\text{d}$-galactopyranosyl)- β-$\text{d}$-galactopyranosyl]-β-$\text{d}$-glucopyranoside (10). Stepwise deprotection of 10 led to 12, the methyl β-glycoside of the trisaccharide related to Fabry's disease.     

The effect of pH on α-methyl-d-glucoside accumulation by rat kidney cortex slices

The kinetics of α-methyl-d-glucoside accumulation by rat kidney cortex slices under conditions of varying extracellular pH are compared with values obtained at pH 7.4. At pH below 7.4 there is a diminished initial uptake and reduced influx of the sugar which results in a decrease in the steady-state intracellular pool. This was associated with a decrease in the $\text{V}$ of the entry process without affecting the apparent $\text{K}{}_{\mathrm{m}}$ of transport. At pH 8 there is no change in the rate of glucoside entry. The efflux of the glucoside, however, is impaired and the steady-state concentration gradient becomes greater than that observed at pH 7.4.