Harriet L. Frush

Mechanisms for hydroperoxide degradation of disaccharides and related compounds

Abstract The reactions of disaccharides with alkaline hydrogen peroxide were studied under diverse conditions. Treatment of cellobiose, lactose, and maltose with aqueous sodium peroxide afforded, in each instance, the corresponding aldobionic acid, the next lower aldobionic acid 2- O - d -glucopyranosyl- d -erythronic acid, and formic acid. On the other hand, melibiose and gentiobiose afforded the corresponding aldobionic acid, the next lower aldobionic acid, and a 2- O - d -glycopyranosylglycolic acid. The yields of products varied widely with the experimental conditions, especially with the proportions of alkali peroxide and hydrogen peroxide. The reactions with alkali peroxide were slow, but rapid in the presence of hydrogen peroxide with the gradual addition of alkali. The results indicate that degradation of carbohydrates by alkaline hydrogen peroxide takes place by five reaction paths. These are designated the alpha-hydroxy hydroperoxide cleavage-mechanism, the Baeyer-Villiger mechanism, the ester mechanism, the dihydroxy-epoxide mechanism, and a newly proposed peroxy-radical mechanism. The last-named mechanism is more rapid than the others. With an excess of hydrogen peroxide and slow addition of alkali, it results in rapid, stepwise conversion of both reducing and nonreducing saccharides into formic acid. The process begins with formation of a hydroperoxide adduct of the carbohydrate. Reaction of the adduct with hydrogen peroxide affords a peroxy radical and a hydroxyl radical. The peroxy radical decomposes, affording formic acid, the next lower aldose and hydroxyl radical. Hydroxyl radical produced in a chain reaction oxidizes alditols and aldonic acids by reactions analogous to those of the Fenton reagent.

Oxidation of sodium salts of alduronic and glyculosonic acids by sodium peroxide

Abstract Because, in the presence of a large excess of alkaline hydroperoxides, aldoses are oxidized stepwise to formic acid, it was expected that alduronic acids would be degraded to formic acid and carbon dioxide by the mechanism previously proposed. However, in addition to the products expected, substantial yields of oxalic acid were found, indicating a second mechanism, proposed here. With alkali-metal hexuronates, one mechanism yields five moles of formic acid and one mole of carbon dioxide per mole of substrate, whereas the second mechanism yields four moles of formic acid and one mole of oxalic acid. The results show that, under highly alkaline conditions, the two mechanisms are of approximately equal importance. Oxidation of a D - xylo -5-hexulosonate begins with the cleavage of glycolic acid, and this is followed by degradation of the resulting tetruronic acid by the two mechanisms described for the degradation of hexuronic acids. With 2-hexulosonates, also, two reaction-mechanisms appear to be necessary to account for the products formed under highly alkaline conditions; the main reaction (80%) yields one mole each of carbon dioxide and pentonic acid per mole, whereas the other yields one mole of oxalic acid and four moles of formic acid. Under moderately alkaline conditions, both the alduronic acids and the 2-keto acids react almost entirely by the mechanism that yields carbon dioxide; no detectable amount of oxalic acid was found. In all cases, small amounts of unknown products, not further investigated, are formed. The following compounds were studied: sodium D -galacturonate, sodium D -glucuronate, potassium D -mannuronate, sodium D - lyxo -2-hexulosonate, sodium D - arabino -2-hexulosonate, calcium D - xylo -5-hexulosonate, and glyoxylic acid.

TRITIUM-LABELED COMPOUNDS. III. ALDOSES-1-t

In a new method for preparing aldoses labeled with tritium at carbon 1, the aldonic lactone is reduced with lithium borohydride-t in pyridine solution. The method is suitable for preparing aldoses-1-t having high specific activities. The crude product contains a substantial proportion of the corresponding alditol-1-t, but the pure aldose-1-t is readily separated by fractional recrystallization or paper chromotography. By means of an isotope-dilution technique, yields were determined for the following aldoses-1-t and the corresponding alditols-1-t: d-arabinose-1-t, d-xylose-1-t, d-ribose-1-t, d-glucose-1-t, d-galactose-1-t, d-mannose-1-t, l-rhamnose-1-t, maltose-1-t, and lactose-1-t. These labeled materials were also prepared by reducing the corresponding lactones with sodium amalgam in tritiated water. Although this latter method is not suitable for preparing labeled aldoses of high specific activity, the products are more readily purified than those obtained by reducing the lactones with lithium borohydride-t. d-Glucose-1-t, obtained by each of these reduction methods, was oxidized with bromine, and the resulting d-gluconic acid was found to be nonradioactive. Hence, in the samples oxidized, tritium was present only at C1. An apparatus used tor reclaiming tritiated water by freeze-drying is depicted; it incorporates an efficient device for trapping entrained solids or liquids.

Degradation of 2-deoxyaldoses by alkaline hydrogen peroxide

Abstract Reaction of 2-deoxy- D - arabino -hexose, 2-deoxy- D - lyxo -hexose, and 2-deoxy- D - erythro -pentose with alkaline hydrogen peroxide in the presence of magnesium hydroxide afforded the corresponding 2-deoxyaldonic acid, the 1,4-lactone, and the 1- O -formyl derivative of the next lower alditol. The 2-deoxyaldonic acids were separated in 60–80% yields, as new, crystalline lithium salts. The 1,4-lactones were obtained under conditions that precluded intermidiate formation of the free acids: presumably, the reaction proceeded by way of an intermediate, furanosyl hydroperoxide, which was converted into the lactone by elimination of water. With an excess of alkaline hydrogen peroxide, in the absence of magnesium hydroxide, the substrates were degraded to formic acid, with concurrent decomposition of hydrogen peroxide. It is shown that decomposition of hydrogen peroxide is catalyzed by hydroperoxide anion, and that it takes place by both a chain, and a non-chain, process. The decomposition reactions afford an abundant source of hydroxyl radical capable of oxidizing a wide variety of compounds.

Tritium-labeled compounds IV. D-glucose-6-t, d-xylose-5-t, and d-mannitol-1-t

Methods are presented for the preparation of d-glucose-6-t, d-xylose-5-t, and d-mannitol-1-t by the reduction of suitable compounds with lithium borohydride-t in anhydrous tetrahydrofuran, followed by hydrolysis of the products. The starting materials for the reductions are, respectively, 1,2-O-isopropylidene-d-glucurono-6,3-lactone, 5-aldo-1,2-O-isopropylidene-d-xylo-pentofuranose, and 2,3:5,6-di-O-isopropylidene-d-mannofuranose. The apparatus and procedure for carrying out the reductions in a closed system are described.

TRITIUM-LABELED COMPOUNDS. I. RADIOASSAY OF TRITIUM-LABELED COMPOUNDS IN "INFINITELY THICK" FILMS WITH A WINDOWLESS, GAS-FLOW, PROPORTIONAL COUNTER

A simple, sensitive, and reliable technique has been devised for the radioassay of nonvolatile, water-soluble tritium compounds. The substance to be analyzed is dissolved in an aqueous solution of a thickening agent, preferably sodium O-(carboxymethyl) cellulose or sodium alginate. The solution is placed in a shallow planchet, and after evaporation of the water, the resulting film, which is “infinitely thick” to tritium beta particles, is counted with a 2π, windowless, gas-flow, proportional counter. By means of an empirical factor, determined with a substance of known radioactivity, the counts are converted to microcuries. In a film having a thickness of 0.7 mg/cm2, the counting efficiency is about 4 percent; the standard deviation from the mean, obtained in a series of routine measurements, was less than 2 percent. An assay can readily be made with tritium-containing material having 0.01 microcurie of radioactivity. The method, which is applicable to nonvolatile, water-soluble solids, solutions, or liquids, is suitable for routine analyses.

Tritium-labeled compounds VII. Isotope effects in the oxidation of D-mannitols-C14 and D-mannitols-t to D-fructoses

d-Mannitols, labeled either with carbon-14 at C1, C2, or C3, or with tritium attached to C1, C2, or C3, were prepared. After oxidation by Acetobacter suboxydans, the distribution of radioactivity in each of the resulting labeled d-fructoses was determined. Labeled d-mannitol is unique among the hexitols in that it may be oxidized by A. suboxydans in either the labeled or the unlabeled part of the molecule. Except in the oxidation of d-mannitol-2-t, the competing reactions result in the formation of a mixture of d-fructoses, each having radioactivity in one of two different positions. Hence, the isotope effect, k*/k, (where k* and k are, respectively, the rate constants for oxidation in the labeled and in the unlabeled part of the labeled d-mannitol molecule) is the ratio of the activities at the two positions of the product, d-fructose. The following isotope effects were found for the bacterial oxidation of labeled d-mannitols: (1) for d-mannitol-2-C14, k*/k=0.93; (2) for d-mannitol-2-t, k*/k=0.23; and (3) for d-mannitol-3-t, k*/k=0.70. For d-mannitols labeled at other positions, no isotope effect was detected, since k*/k was unity. The large isotope-effect for d-mannitol-2-t is indicative of rupture of the C2–H bond in the rate-determining process. It is suggested that the secondary isotope-effect for tritium at C3 indicates hyperconjugation of the C3 hydrogen atom in the activated enzyme—substrate complex; the lack of such effect for tritium at C1 may be due to unfavorable steric conditions for hyperconjugation of the C1 hydrogen atoms in the complex. The following substances were prepared and their isotopic distributions determined: d-fructose-1,6-C14 and d-fructose-1,6-t (from 1-labeled d-mannitols); d-fructose-2,5-C14 and d-fructose-5-t (from 2-labeled d-mannitols); and d-fructose-3,4-C14 and d-fructose-3,4-t (from 3-labeled d-mannitols). A procedure, employing d-fructose-1,6-C14 as an internal standard, was devised for the analysis of d-fructose-3,4-t.

Tritium-labeled compounds V. Radioassay of both carbon-14 and tritium in films, with a proportional counter

A convenient procedure is described for the radioassay of both carbon-14 and tritium in water-soluble, nonvolatile compounds by means of a windowless, gas-flow, proportional counter. The materials are counted in uniform films of sodium O-(carboxymethyl) cellulose that are “infinitely thick” to the radiation of tritium but not to the radiation of carbon-14. Films of uniform thickness are obtained by new techniques which are described in detail. If only carbon-14 is present, its absolute activity can be calculated conveniently by means of an empirically established curve for the counting-efficiency. If both carbon-14 and tritium are present, the films are counted in the proportional counter and are then recounted in the presence of a screen that stops all radiation from tritium but only a portion of that from carbon-14. From a film with a thickness of 0.8 mg/cm2, approximately 43 percent of the radiation of carbon-14 is counted. Of this emerging radiation, approximately 50 percent passes through a screen of ¼-mil double-aluminized “Mylar.” By use of suitable calibration curves for counting-efficiency, carbon-14 and tritium in the same sample can be calculated from the counts with, and without, the screen. Satisfactory analyses can be made with samples containing less than 0.001 microcurie of carbon-14 and 0.005 microcurie of tritium. The method is suitable for the radioassay of a wide variety of labeled materials.