No abstract

The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.

The 1H and 13C NMR parameters of the anomeric pairs of aldopyranosyl phosphates and their rigid 1,2-phosphate derivatives are reported.The derivatives of D-glucose, D-galactose, and D-mannose exist in the 4C1 conformation while the L-fuco derivatives are in the C4 conformation. As judged by 31P--1H and 31P--13C coupling constants, all of the alpha anomers of the aldopyranosyl phosphates have the phosphate moiety predominantly trans to C(2) while in the beta anomers other rotamers make significant contributions. This relationship remains the same for the biologically important nucleoside diphosphate sugars (UDPGlc, UDPGal, GDPMan, and GDPFuc). From the pH dependence of 13C chemical shifts, observed in 0.5 M solutions, the pK'a2 of the alpha anomers is 6.1 while the pK'a2 of the beta anomers is 0.6--0.8 pH unit lower. In the 1.2-phosphates, the chair conformation of the parent aldose is retained while an envelope conformation is formed by the cyclic phosphate. In the alpha anomers, the plane is formed between C(2), C(1), O(1), and P while O(2) is above the plane. In the beta anomers, O(1) is out of the plane formed by the other atoms. The beta anomers have phosphorus coupled to C(3) with coupling constants of 10.8--11.7 Hz, approximately 2 Hz greater than the maximum reported for trans coupling (Lapper, R. D., & Smith, I. C. P. (1973) J. Am. Chem. Soc. 95, 2880).

. Can. J. Chem. 59,2086Chem. 59, (1981. Guanosine diphosphate fucose (GDPFuc) has been synthesized chemically in an overall yield of 40% from fucose. This synthesis was made possible by increasing the yield of the key intermediate, P-L-fucopyranosyl phosphate, five-fold over that previously reported (Prihar and Behrman. Biochemistry, 12, 997 (1973)), by utilizing guanosine 5'-phosphoric di-n-butylphosphinothioic anhydride (Furusawa et al. J. Chem. Soc. Perkin Trans I, 171 (1976)) or GMP morpholdate for the synthesis of GDPFuc, and by improving the method of isolating intermediates and the final product. At pH 3, GDPFuc is degraded to GDP with a half-life of 7 h at 37°C and 52 h at 4OC. At pH values between 5 and 8, less than 10% is degraded after 7 days. Neither 20 mM Mn2+ nor Mg2+ in 0.2 M PIPES buffer at pH 7.5 stimulate the hydrolysis of GDPFuc. Heteronuclear, C-P and H-P coupling constants indicate that the preferential position of the phosphate moiety in both P-L-fucopyranosyl phosphate and GDPFuc is trans to C-2. In the ribose moiety of GDPFuc, the 5'-phosphate is trans to C-4. The ' H and I3C nmr parameters of intermediates and products are reported. Chem. 59,2086Chem. 59, (1981. On a synthetise chimiquement le fucose diphosphate de guanosine (FucDPG) avec un rendement global de 40% a partir du fucose. Cette synthkse a pu Stre realisee en preparant I'intermediaire principal, le phosphate de P-L-fucopyrannosyle, avec un rendement 5 fois superieur a celui obtenu anterieurement (Prichar et Behrmann. Biochemistry, 12,997 (1973) [Traduit par le journal1

The classical Kiliani (cyanohydrin) reaction was studied by ' % NMR and GLC. 13C NMR studies were facilitated by the use of [13C]cyanide and/or I3C-enriched aldoses. The effects of aldose configuration, carbon-chain length, and derivatization on the rate and extent of cyanide reaction and on the overall rate of aldononitrile disappearance were investigated. For the condensation of cyanide with D-erythrose, the reaction sequence at pH 10.5 or 12.7 appears to be cyanide + D-erythrosealdononitrilesimido-1,4-lactonescarbinolaminesaldonamides.Aldonamides hydrolyze via carbinolamines and aldonolactones to aldonates. At pH 7 or 8.5, the direct conversion of imido-l,4.-lactones to aldono-1,4-lactones becomes appreciable. Ammonia, which is released in this reaction, can react with imido-1,4-lactones to yield amidines. A reaction between imidolactones and aldononitriles is proposed. 13C NMR pammeters (6 and J) and GLC retention times for the reactants, intermediates, and products are tabulated. Experimental SectionMaterials. Glycolaldehyde, DL-glyceraldehyde, calcium DLglycerate, D-arabinose, D-lyXOSe, D-ribose. D-xylose, D-ribono-1,4-lactone, sodium ~~-2-hydroxybutyrate, D-gUlOnO-1,4-laCtOne, 2-deoxy-D-glucose, ion-exchange resins, potato acid phosphatase (EC 3.1.3.2) and palladium barium sulfate (5%) were purchased from Sigma Chemical Co. and used without further purification. L-Threonine (allo free) was obtained from the United States Biochemical Corp. Calcium D-gdaCtonate and calcium Dgluconate were purchased from Pfanstiehl Laboratories, Inc.Potassium [Wlcyanide (K13CN) was supplied by the Los Alamos Scientific Laboratory, University of California, New Mexico, with 99.6% purity and 90.7 atom 70 13C enrichment. Potassium [14C]cyanide (K14CN) was purchased from New England Nuclear with a specific activity of 46 mCi/mmol. N,O-Bis(trimethylsily1)trifluoroacetamide (BSTFA) with 1 % chloromethylsilane (TMCS) was obtained from Pierce Chemical Co. Pyridine for GLC was distilled from barium oxide and stored over 4-A molecular sieves. Other chemicals and solvents were reagent grade and were used without further purification.Preparations. D-Glyceraldehyde was prepared by oxidation of D-fructose with lead tetraacetate.* D-Lactaldehyde was prepared from t threonine.^ 2,4-O-Ethylidene-~-erythrose was prepared by the method of Perlin." D-Erythrose was prepared by hydrolysis of 2,4-O-ethylidene-~-erythrose monomer." Dilute aqueous solutions (0.14.5 M) contained approximately 5% dimers and/or higher order structures.l* 2,4-O-Ethylidene-~-threose was prepared according to BalP3 and was hydrolyzed as described for 2,4-O-ethylidene-~-erythrose." Tetroses were estimated to be greater than 95% pure by I3C NMR." Sodium D-xylonate was prepared by hypoiodite oxidation of D-Xy10Se.14 D-[ l-'3C]Arabinonamide and D-[ l-'3C]ribonamide were prepared from the respective [ l-13C]lactones.15 (8) Perlin, A. S. Methods Carbohydr. C h e m . 1962, I , 61. (9) Zagalak, B.; Frey, P. A,; Karabatsos, G. L.; Abeles, R. H. J . Biol. (10) Perlin, A. S. M e t h ...