An enzymic synthesis of purine d-Arabinonucleosides

A method is described for the synthesis of purine d-arabinonucleosides that uses purine bases and 2,2′-anhydro-(1-β-d-arabinofuranosylcytosine), AraC-an, as the starting materials. AraC-an was chosen as the precursor to the d-arabinosyl donor, because it is more readily available than any of the products that may be sequentially derived from it, namely, 1-β-d-arabinofuranosylcytosine (AraC), 1-β-d-arabinofuranosyluracil (AraU), and α-d-arabinofuranosyl-1-phosphate (Araf 1-P), a d-arabinofuranosyl donor. Four reactions were involved in the overall process; (a) AraC-an was nonenzymically hydrolyzed at alkaline pH to AraC which was then (b) deaminated by cytidine deaminase to AraU, a nucleoside, (c) phosphorylyzed by uridine phosphorylase to Araf 1-P, and (d) this ester caused to react with a purine base to afford a purine d-arabinonucleoside, the reaction being catalyzed by purine nucleoside phosphorylase. All four reactions occurred in situ, the first and second being performed sequentially, whereas the third and fourth were combined in a single step. The three enzyme catalysts were purified from Escherichia coli. The efficiency of the method is exemplified by the synthesis of the d-arabinonucleosides of 2,6-diaminopurine and adenine; the overall yields, based on AraC-an, were 60 and 80%, respectively.     

Enzymic determination of d-galactose, d-arabinose,and their homologs

The enzymes d-galactose dehydrogenase and d-arabinose dehydrogenase were demonstrated to be applicable to the quantitative determination of d-galactose (and homologs) and d-arabinose (and homologs), respectively. The enzymic reactions were quite specific. When coupled with β-galactosidase, d-galactose dehydrogenase could be used in the quantitative determination of β-galactosides.     

Nature of the uptake of d-galactose, d-glucose and α-methyl-d-glucoside by Saccharomyces cerevisiae

Both glucose-grown baker's yeast after induction and galactose-grown yeast appear to take up d-galactose by a system not requiring phosphorylation and only up to a diffusion equilibrium, as shown by pulse labelling, sampling at very short intervals and chromatographic analysis of extracts. Part of the sugar taken up is transformed into trehalose which is present in substantially greater amounts in cells than the transported sugar itself. The effect of 2,4-dinitrophenol and of iodoacetamide, as well as the nature of the efflux of sugars from preloaded cells, support the results. d-Glucose and α-methylglucoside are also taken up without phosphorylation.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

Acetalation of d-glucitol: 2,3:5,6-Di-O-isopropylidene-d-glucitol

The reaction of d-glucitol with acetone-zinc chloride gave a mixture of isopropylidene derivatives, from which the 2,3:5,6-diacetal (12) could be separated as its 1,4-dimesylate (13) or 1,4-ditosylate (14). The structure of 12 was proved by converting 14, via the 1-mono-iodide, into the known 1-deoxy-d-glucitol, and by mass-spectrometric investigation of the 1-deoxy-4-O-methyl diacetal. The terminally situated acetal group in 12 can be selectively hydrolyzed, and, on treatment with base, the 5,6-dihydroxy derivative obtained gives a d-galactitol 4,5-epoxide derivative.     

Isomerization of d-fructose by base: Liquid-chromatographic evaluation and the isolation of d-psicose

Several bases have been evaluated as catalysts for the production of d-psicose (d-ribo-2-hexulose) from d-fructose. The hexose levels in the isomerized mixtures were quantified by l.c. on a μBondapak/Carbohydrate column. The most effective and convenient base was found to be pyridine, and mixtures produced by boiling concentrated solutions (1 g/mL) of d-fructose in pyridine under reflux contained 12.4% of psicose, lesser proportions of glucose and mannose, and 25.8% of the starting material. Following removal of solvent, fermentation with bakers' yeast removed most hexoses other than d-psicose, which was isolated by chromatography on cellulose. The entire procedure required three days, and d-psicose was obtained in gram quantities in 6.8% of the theoretical yield.     

Sodium binding to d-glucuronate residues: Crystal structure of sodium d-glucuronate monohydrate

Three-dimensional X-ray diffraction data were used to determine the crystal structure of sodium β-d-glucuronate monohydrate, a model system for investigating the factors involved in the binding of sodium ions to d-glucuronate residues of glycosaminoglycans. Crystals of the salt are monoclinic, space group P2₁, with a = 9.206(3) Å, b = 7.007(2) Å, c = 7.378(3) Å, β = 96.84(3)°, and Z = 2. Intensity data for 858 reflections were measured with an automated diffractometer. A trial structure, obtained by direct methods, was refined by least squares to R = 0.035. An outstanding feature of the crystal packing is the interaction of d-glucuronate anions with sodium ions. The sodium ion is coordinated to three symmetry-related $\text{d}$-glucuronate anions and to one water molecule. The d-glucuronate anion binds sodium cations through the three following sites: one that involves a carboxyl oxygen atom combined with ring oxygen O-5; one that includes a single carboxyl oxygen atom, and one composed of the O-3–O-4 pair of hydroxyl groups.     

Effects of various conditions on the alkaline degradation of d-fructose and d-glucose

Dilute solutions of d-fructose and d-glucose undergo alkaline degradation, and, at temperatures in the range of 30–70°, almost two moles of alkali are consumed per mole of the carbohydrate. The degradation is partly guided by the dielectric constant of the medium; such additives as acetone and urea have specific effects where the reactions are not essentially guided by the medium dielectric. Acetone and urea presumably form complexes with the carbohydrates; this is revealed for the former by the formation of a dark red solution having a spectral band at 320 nm, like that observed earlier in the presence of ethylenediamine.     

Laser-raman study of solute-solvent interactions in aqueous solutions of d-fructose, d-glucose,and sucrose

The solute-solvent interactions of d-fructose, d-glucose, and sucrose in aqueous solution were studied by comparison of characteristic, Raman of the water and the sugar components. Shifts in frequency and intensity were observed in both the bending and the stretching regions of CH₂ and H₂O. The ratios of integrated, Raman intensities I(CH₂)/I(H₂O) of the CH₂ peak and the H₂O bending band, and I(CH)/I(OH) of the C-H stretching line to O-H stretching band were determined. Their evolutions in terms of mass-concentration display discontinuities at specific concentrations for each of the three sugars. These breaks were interpreted as changes in the hydrogen bonding of the various species.     

The effect of areneboronic acids on the alkaline conversion of d-glucose into d-fructose

Benzeneboronic acid, 4-methoxybenzeneboronic acid, 3-nitrobenzeneboronic acid, and sulphonated benzeneboronic acid have been used to displace the pseudo-equilibria established in aqueous alkali between d-glucose, d-fructose, and d-mannose to give greatly increased yields of d-fructose. The effect of reaction temperature, pH, overall concentration, and molar ratio of acid:sugar on the yield of d-fructose has been investigated by using an automated assay for d-fructose.     

Reactions of 4,6-disulphonates of methyl d-glucopyranosides and methyl d-galactopyranosides with thio-nucleophiles

The reactions of some 4,6-disulphonates of methyl 2,3-di-O-acyl-(and di-O-methyl)-d-glucopyranosides and -galactopyranosides, with thiocyanate, thioacetate, and thiobenzoate anions, have been studied under a variety of conditions. In the glucoside series, somewhat similar reactivity is shown by isomers differing only in anomeric configuration, and there is no very great difference between the reactivities of a 2,3-dibenzoate and its 2,3-di-O-methyl analogue. In contrast to the situation in the β-d-galactoside series, the presence of O-benzoyl groups in an α-d-galactoside does not have an unfavourable effect on displacement at C-4. Two hexose derivatives containing the novel 4,6-epithio bridge are described.     

Convenient,laboratory procedure for producing solid d-arabino-hexos-2-ulose (d-glucosone)

The production of solid d-arabino-hexos-2-ulose (d-glucosone) from d-glucose by use of an enzyme, pyranose-2-oxidase (EC 1.1.3.10), is described. The enzyme is extracted from the mycelia of Polyporus obtusus, partially purified, and then immobilized on activated CH-Sepharose 4B. The enzymic conversion of d-glucose into d-glucosone is simple and convenient, and provides a product free from residual d-glucose. Lyophilization of the filtered reaction-solution yields the product, solid d-glucosone. Assay methods have been developed for monitoring the enzymic reaction and evaluating the purity of the final product.     

X-ray diffraction study of the molecular association in aqueous solutions of d-fructose, d-glucose,and sucrose

X-ray diffractograms of aqueous solutions of d-fructose, d-glucose, and sucrose, and of two amorphous “solid” forms of these carbohydrates are recorded in the angular range of O 2-16°. The formation, in these diffractograms, of one or two intensity maxima as the concentration is varied is interpreted in terms of modification of the molecular association. A model is proposed that takes account of the state of organization of the solutions of the three sugars as a function of the concentration. Sucrose shows an “anomaly”, as it is the only sugar to establish intramolecular bonds. The number of these intramolecular bonds also depends on the concentration.     

Synthesis and growth regulator activity of fatty acyl derivatives of d-glucose and d-galactose

In an attempt to gain information about one or more components of the brassin complex, fatty acid esters of d-glucose and d-galactose were prepared and tested for growth regulator activity in a bean hypocotyl bioassay. 4-O-Acyl-d-glucoses and, perhaps, 1-O-acyl- d-galactoses had a similar qualitative activity to that of the brassin complex. 3-O-Acyl- d-galactoses inhibited elongation of bean hypocotyls and stimulated rooting. 3- And 6-O- acyl-d-glucoses both stimulated and inhibited elongation, depending on the source of fatty acids; in both cases, stimulation was observed when safflower oil was used as the source of fatty acids and inhibition was observed when peanut oil was used as the source of fatty acids. Fatty alkyl β-d-galactopyranosides were inactive.     

The synthesis of antigenic determinants for yeast d-mannan,and a linear (1→6)-α-d-gluco-d-mannan,and their protein conjugates

2-O-Benzoyl-3,4,6-tri-O-benzyl-1-O-tosyl-d-mannopyranose and 2,3,4-tri-O- benzyl-6-O-(N-phenylcarbamoyl)-1-O-tosyl-d-glucopyranose were allowed to react with partially blocked 2-[4-(p-toluenesulfonamido)phenyl]ethyl α-d-manno- and -gluco-pyranosides. Disaccharides having α-d-Manp-(1→2)-α-D-Manp, α-d-manp-(1→6)-α-d-Manp, α-d-Manp-(1→6)-α-d-Manp, and α-d-Glcp-(1→6)-α-d-Manp structures, and a branched trisaccharide having the structure α-d-Manp-(1→2)-[α-d-Manp-(1→6)]-α-d-Manp were synthesized. The oligosaccharides were deblocked with sodium in liquid ammonia to give glycopyranosides having a free primary aromatic amine which were converted into isothiocyanate derivatives with thiophosgene. The functionalized oligosaccharides were then coupled to bovine serum albumin to give protein conjugates.     

An aminotransferase specific for the d-enantiomorph of methionine

An enzyme was isolated from germinating peanut seed and shown to be an aminotransferase specific for the d-enantiomorph of methionine. The keto acid of methionine, α-keto-γ-methylthiobutyrate, was isolated from the reaction mixture and identified. Of the keto acids tested pyruvic acid was the most effective acceptor for the amino group of methionine. A small amount of enzyme was isolated which gave only one band on disc gel electrophoresis.     

2,5-anhydro-d-hexitols: syntheses of 2,5-anhydro-d-altritol and 2,5-anhydro-d-iditol

2,5-Anhydro-d-altritol (2a) and the previously-unknown 2,5-anhydro-d-iditol (3a) have been prepared from 2,5-anhydro-d-mannitol (1a). The preparation of 3a from the intermediate epoxide 7b is particularly sensitive to pH, and a mechanism is proposed to explain this. Attention is drawn to the limitations of the trifluoroperacetic acid-disodium hydrogenphosphate procedure for the epoxidation of alkenes of diminished reactivity.     

Conformational studies on synthetic polypeptides: poly (d-phenylglycine) and poly(d-cyclohexylglycine)

Poly(d-phenylglycine) and poly(d-cyclohexylglycine) containing phenyl and cyclohexyl rings bound to the α-carbon of the polypeptide chain, have been synthesized. Circular dichroism measurements show that both polymers undergo a conformational transition from the random-coil form to an ordered form, upon addition of water, ethanol or trifluoroethanol to sulphuric acid solutions. Solid state measurements indicate that the ordered structures of poly(d-phenylglycine) and poly(d-cyclohexylglycine) are of the β-type. While for the former the antiparallel arrangement is predominant, for the latter there seems to be a greater tendency towards the parallel form. The ordered form of poly(d-cyclohexylglycine) is slightly more stable than the corresponding form of poly(d-phenylglycine) in all the above solvent systems. This can be interpreted in terms of stronger non covalent bond formation in the former polypeptide. Our results have been compared with literature on poly(l-phenylalanine) and poly(l-cyclohexylalanine).     

Rapid and sensitive,colorimetric determination of the anomers of d-glucose with d-glucose oxidase,peroxidase, and mutarotase

A modification, utilising mutarotase, of an enzymic, colorimetric system for determining d-glucose with d-glucose oxidase, peroxidase, and ABTS was satisfactory for the assay of the anomers of d-glucose in aqueous solution. The time required for a single assay is ≈ 10 min, and the lower limit is 0.4 μg of d-glucose. The method is applicable to the anomer analysis of d-glucose released by enzymic hydrolysis of d-glucosides.