Preparation of the tritiated molecular forms of gangliosides with homogeneous long chain base composition

Gangliosides G_M2, G_M1 and G_D1b were radiolabelled at C-6 of the terminal galactose orN-acetylgalactosamine by the galactose oxidase/[³H]NaBH₄ method; gangliosides G_M2, G_M1, Fuc-G_M1 and G_D1a were radiolabelled at C-3 of the long chain base by the 2,3-dichloro-5,6-dicyanobenzoquinone/[³H]NaBH₄ method.By application of an original HPLC procedure, eight different molecular species were prepared from each labelled ganglioside. Each of these species was characterized by the presence of one of the following long chain bases:erythro C18 sphingosine,threo C18 sphingosine,erythro C18 sphinganine,threo C18 sphinganine,erythro C20 sphingosine,threo C20 sphingosine,erythro C20 sphinganine andthreo C20 sphinganine.From G_D1b only the species containing theerythro forms of long chain bases were obtained.The individual molecular species were more than 99% homogeneous and had a radiopurity better than 99%. The molecular species of the same ganglioside, radiolabelled at C-3 of the long chain base, had identical specific radioactivity, namely 1.17, 1.25, 0.85 and 1.28 Ci/mmol for G_M2, G_M1, Fuc-G_M1 and G_D1a respectively. The molecular species of the same ganglioside, radiolabelled at C-6 of terminal galactose orN-acetylgalactosamine, had similar specific radioactivity, namely 1.34–1.40, 1.44–1.51, 1.37–1.44 Ci/mmol for G_M2, G_M1 and G_D1b respectively.     

The kinetics of lumiflavin reduction by N-methyl-1,4-dihydronicotinamide: direct evidence for a preequilibrium complex between oxidized lumiflavin and N-methyl-1,4-dihydronicotinamide

The rates of anaerobic reduction of LF by NMNH exhibited saturation kinetics with a dissociation constant (K₁) of 0.11 M and a first order rate constant (k₂) of 7.1 sec^?1. Substitution of deuterium at the C-4 position of NMNH showed a 2.7 fold kinetic isotope effect on k₂ with no effect on K₁. During the reductive reaction, a long wavelength absorbing species was observed. The formation of this species was too fast to be measured by stopped flow techniques but its rate of disappearance was the same as the rate of reduction of the flavin. The turnover number of LF with O₂ and NMNH was significantly faster than the rate of anaerobic reduction of LF by NMNH. Therefore, the preequilibrium complex must react directly with O₂.     

Dinosterol side chain biosynthesis in a marine dinoflagellate, Crypthecodinium cohnii

The heterotrophic dinofiagellate, Crypthecodinium cohnii, cultured in a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly synthesized 4α-monomethyl compound dinosterol (4α,23,24-trimethylcholest-22-en-3β-ol). The MS fragmentation pattern indicated that the C-23 methyl group contained three deuterium atoms and was introduced intact by transmethylation from methionine. The C-24 methyl group contained only two deuterium atoms which is consistent with the production of a 24-methylenesterol intermediate which is subsequently reduced to give the 24-methyl side chain. Mechanisms are proposed to account for the production of the dinosterol side chain.     

Synthesis of 2′-Deuterio Tubercidin and Adenosine and the Corresponding Arabino Analogs

Abstract

The 2′-deuterio arabino analogs of tubercidin and adenosine have been prepared by Swern oxidation of the 3′,5′-TPDS derivatives of tubercidin and adenosine and reduction with NaBD_4. Subsequent inversion of stereochemistry at C-2′ yielded [2′-²H]tubercidin and [2′-²H]adenosine with 98% deuterium incorporation.     

Intramembrane Polarity by Electron Spin Echo Spectroscopy of Labeled Lipids

The association of water (D₂O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D₂O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12 atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the ¹⁴N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.     

Biosynthesis of acaterin: mechanism of the reaction catalyzed by dehydroacaterin reductase

Dehydroacaterin reductase is an enzyme which catalyzes the final step of acaterin biosynthesis, that is, the reduction of the C-4/C-5 double bond of dehydroacaterin. The mechanism of the reduction was investigated with a cell-free preparation obtained from the acaterin-producing microorganism, Pseudomonas sp. A 92. Incubation of dehydroacaterin in the presence of [4,4- 2H2]NADPH or D2O followed by 2H NMR analysis of the resulting acaterin revealed that the deuterium atom from NADPH was incorporated into the C-5 position of acaterin, while the deuterium atom from D2O was introduced into the C-4 position. It was further demonstrated that the pro-R hydrogen at C-4 of NADPH was stereospecifically utilized in this reduction.     

Deuterium transfer from [1,1-2-H] ethanol during metabolism of bile acids and cyclohexanone in the isolated perfused rat liver.

Deuterium transfer from [1,1-2-H]ethanol (95 atoms % excess) to reducible substrates was studied in the isolated perfused rat liver. The dueterium excess in cyclohexanol formed from cyclohexanone was somewhat lower (49 atoms%) than found under conditions in vivo, and this was also true of the deuterium excess in lithocholic acid formed from 3-oxo-5beta-cholanoic acid. These results may reflect a slower rate of ethanol oxidation in the isolated organ than in vivo. Cycloserine decreased the dueterium transfer to both substrates, whereas addition of lactate and malate resulted in an increased deuterium excess in cyclohexanol and a decreased deuterium excess in lithocholic acid. Addition of heavy water to the perfusion fluid resulted in labelling at C-3 of lithocholic acid formed from 3-oxo-5beta-cholanoic acid, and at C-3, C-4 and C-5 of 3alpha-hydroxy-5alpha-cholanoic acid formed from 3-oxo-4-cholenoic acid. The deuterium excess of hydrogens derived from NADPH (at C-3 and C-5) was approximately the same as that of hydrogen derived directly from water (at C-4). Thus, the hydrogen of NADPH is extensively exchanged with protons of water, which explains the dilution of deuterium with protium during the transfer from [1,1-2-H]ethanol via NADPH to the bile acids. The labelling at C-5 in the reduction of the 4,5-double bond indicates that different pools of NADPH are used for reduction of this double bond and the 3-oxo group, since in a previous study it was shown that deuterium is transferred from [1,1-2-H]ethanol only in the latter reaction.     

Selective Deuteration Labelling of 2′-Deoxyguanosine at the Carbon C-4’ Position

Abstract

Selective incorporation of deuterium within the sugar moiety of nucleosides and oligonucleotides can be used for different purposes including isotopic effect determination in mechanistic studies, massspectrometry fragmentation investigations, nuclear magnetic resonance analyses. We wish to report a simple method which allows the selective deuteration labelling of 2'-deoxyguanosine at the C-4'position through the intermediary of 9-(2-deoxy-B-D-erythropento-1,5-dialdo-114-furanosyllquanine. Heating of aqueous pyridine solution [1:11 of 2′-deoxyguanosine-5′-aldehyde for 1 hr at 60°C leads to a partial epimerisation of carbon C-4' with subsequent formation of 9-(2-deoxy-α-L-threopento-1,5-dialdo-1,4-furanosyl)guanine in 40% yield. A likely intermediate of this reaction appears to be a 5'-enol derivative. Similar treatment of 2′-deoxyguanosine-5′-aldehyde in D₂0-pyridine [1-1] gives after NaBH₄ reduction 60% of 2′-deoxyguanosine which is selectively deuterated at the C-4′ position. The extend of the isotopic labelling was up to 95% as determined by high resolution electron impact mass spectrometry and ¹H NMR analyses. Heating of the aqueous pyridine solution of 2′-deoxyguanosine-5′-aldehyde for a longer period (3–4 hrs) gave rise to two other nucleosides which where assigned as 9-(2-deoxy-α-D-threo-pentofuranosy1)guanine and 9-(2-deoxy-n-L-erythro-pentofuranosyl)guanine. A retro-aldol mechanism appears to be involved in the epimerization reaction which takes place at carbon C-3′.     

Reversible reaction via a carbanion intermediate in the elimination of ammonia from l-histidine catalysed by histidine ammonia-lyase

l-[5′-²H₂]Histidine was used as a substrate to investigate the enzymatic reaction mechanism with histidine ammonia-lyase from Pseudomonas fluorescens. The study was performed to determine the exchange rate of deuterium at C-5′ of the imidazole ring with solvent hydrogen relative to the net urocanic acid production. The extent of hydrogen exchange at C-5′ of histidine or urocanic acid was measured by gas chromatography—mass spectrometry—selected ion monitoring, monitoring the molecular ion intensities of the respective gas chromatographic derivatives, at m/z 380 and 379 for histidine and at m/z 267 and 266 for urocanic acid. The observed hydrogen exchange at C-5′ suggested a reversible mechanism via a carbanion intermediate in the reaction with histidine ammonia-lyase.     

Biosynthese des ramifications en C-24 des phytosterols de Physarum polycephalum et d'Ochromonas danica

The side chain C-24 alkylation mechanism of phytosterols of Physarum polycephalum and Ochromonas danica, cultured in a nutrient medium containing methionine-CD₃, has been studied. Depending on species, five or four deuterium atoms were incorporated respectively in C-24 ethyl phytosterols. No relation could be established between the stereochemistry of C-24 and the alkylation mechanism at this position.     

Biosynthetic intermediates in the conversion of fucosterol to oogoniol

When grown in the presence of [CD₃]-methionine Achlya heterosexual produces oogoniols containing two deuterium atoms which are located at C-28     

Stereochemistry of the β-cyanoalanine synthetase and S-alkylcysteine lyase reactions

The stereochemistry of the replacement of the SH-group of cysteine by CN catalyzed by β-cyanoalanine synthetase was studied using cysteine stereospecifically tritiated at C-3. Analysis of the resulting β-cyanoalanine by conversion into fumarate via aspartate and malate showed that the reaction had occurred with retention of configuration at C-3. Using cystine stereospecifically labeled at C-3 with tritium or with tritium and deuterium, it was found that the α,β-elimination reaction catalyzed by S-alkylcysteine lyase involves stereo-specific replacement of the β-substituent of the substrate by a hydrogen derived from the solvent, D₂O or H₂O, with retention of configuration to give pyruvate containing a chiral methyl group. The results are discussed, particularly in the light of mechanistic proposals by Braunstem and co-workers.     

Sensitive non-radioactive determination of aminotransferase stereospecificity for C-4' hydrogen transfer on the coenzyme

A sensitive non-radioactive method for determination of the stereospecificity of the C-4′ hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in ²H₂O with a substrate amino acid resulted in PMP labeled with deuterium at C-4′ in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4′-²H]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The ²H at C-4′ is retained with the PLP if the aminotransferase in question transfers C-4′ hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the ²H is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC–MS/MS for the presence or absence of ²H. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay.     

Identification of 4-O-methyl-D-xylose as a constituent of the cell wall of Chlorella vulgaris

From the cell wall of a strain of Chlorella vulgaris a sugar was isolated after acid hydrolysis and was identified as 4-O-methyl-D-xylose by the following criteria: (i) mass spectroscopy of its alditol acetate revealed characteristic primary fragments with m/e 117 and m/e 261, and, when one deuterium atom was substituted at C-1, with m/e 262 instead of m/e 261; (ii) after demethylation with BCl₃, xylose was identified as its parent sugar by chromatographic methods; (iii) L-iditol: NAD 5-oxidoreductase (sorbitol dehydrogenase) catalyzed the oxidation of its alditol, but not of 4-O-methyl-L-xylitol. 4-O-Methyl-D-xylose amounted to approx. 10% of the cell walls' dry weight or 1.6% of the cells' dry weight.     

Ergosterol biosynthesis in Mucor pusillus

Ergosterol, 22-dihydroergosterol, obtusifoliol and 24-methylene-24,25-dihydrolanosterol, isolated from Mucor pusillus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. Ergosterol and 22-dihydroergosterol, isolated from M. pusillus grown in the presence of mevalonic acid-[2-¹⁴C, (4R)-4-³H₁] had ¹⁴C:³H atomic ratios of 5:3. The significance of these results in terms of sterol biosynthesis in this organism in general and alkylation at C-24 in particular is discussed.     

9,16-dihydroxy-10-oxo-hexadecanoic acid,a novel component in citrus cutin

When grapefruit cutin was treated with [³H]NaBH₄ and subsequently depolymerized with LiA1H₄, a radioactive component which was more polar than 1,7,16-trihydroxyhexadecane was released. This component was identified by mass specttrometry as 1,7,8,16-tetrahydroxyhexadecane. Mass spectrometry of the tetraols derived from NaBD₄ reduction folllowed by LiAlD₄ depolymerization and from NaBH₄ reduction followed by LiA1D₄ depolymerization indicated that these tetraols were derived from a dihydroxy C₁₆ acid which contained a carbonyl group at C-10 or C-16. Periodate cleavage and permanganate oxidation of the labeled tetraol showed that the ³H was located at C-10. Thus the cutin monomer from which the tetraol was generated was identified as 9,16-dihydroxy-10-oxo-hexadecanoate. This identity was confirmed by NMR analysis of the C₁₆ tetraol obtained by LiA1H₄ reduction of Citrus cutin which had been treated with NaBD₄. This dihydroxyoxo-C₁₆ acid was found to be a minor component of the fruit peel cutin from grapefruit (4.2%), lime (0.1%), lemon (1.2%) and orange (0.3%). 9,10,16-Trihydroxyhexadecanoic acid was also identified as a minor component (0.1–1.9%) in these cutins.     

Sphingosine kinase 2 prevents the nuclear translocation of sphingosine 1-phosphate receptor-2 and tyrosine 416 phosphorylated c-Src and increases estrogen receptor negative MDA-MB-231 breast cancer cell growth: The role of sphingosine 1-phosphate receptor-4

We demonstrate that pre-treatment of estrogen receptor negative MDA-MB-231 breast cancer cells containing ectopically expressed HA-tagged sphingosine 1-phosphate receptor-2 (S1P₂) with the sphingosine kinase 1/2 inhibitor SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) or the sphingosine kinase 2 selective inhibitor (R)-FTY720 methyl ether (ROMe) or sphingosine kinase 2 siRNA induced the translocation of HA-tagged S1P₂ and Y416 phosphorylated c-Src to the nucleus of these cells. This is associated with reduced growth of HA-tagged S1P₂ over-expressing MDA-MB-231 cells. Treatment of HA-S1P₂ over-expressing MDA-MB-231 cells with the sphingosine 1-phosphate receptor-4 (S1P₄) antagonist CYM50367 or with S1P₄ siRNA also promoted nuclear translocation of HA-tagged S1P₂. These findings identify for the first time a signaling pathway in which sphingosine 1-phosphate formed by sphingosine kinase 2 binds to S1P₄ to prevent nuclear translocation of S1P₂ and thereby promote the growth of estrogen receptor negative breast cancer cells.     

Absolute stereochemistry of methanopterin and 5,6,7,8-tetrahydromethanopterin

The configuration at the C-9 of methanopterin (MPT) has been determined by comparing the circular dichroism (CD) spectra of MPT and its hydrolytic fragment, 1-[4-[[1-(2-amino-7-methyl-4-hydroxy-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-D -ribitol (HP-1), with the CD spectra of a series of model compounds of known stereochemistry. These compounds included (S)-6-[1-(4-carboxymethylanilino)ethyl]pterin, (S-6(1-hydroxyethyl)-7-methylpterin, (S-6-(1-hydroxyethyl)pterin, (R)-6-(1-phenoxyethyl)pterin, D (+)-neopterin, and L -biopterin. From this comparison it was concluded that MPT has the R configuration at C-9 and is thus configurationally related to D (+)-neopterin, which has the S configuration at C-1. From previous work establishing the relative stereochemistry at C-6, C-7, and C-9 of N⁵-N¹⁰-methenyl-5,6,7,8-tetrahydromethanopterin (N⁵-N¹⁰-methenyl-H₄MPT) as R, S, and R, respectively, it is clear that the remaining asymmetric carbons at C-6 and C-7 of H₄MPT have the S and S configuration, respectively. Comparison of these latter two positions to the equivalent carbons in 5,6,7,8-tetrahydrofolate (H₄folate) show that the steps involved in the biological reduction of MPT to H₄MPT occur with the same stereochemical outcome as those involved in the biological reduction of folate to H₄folate. © 1996 Wiley-Liss, Inc.     

Characterization of two enzyme proteins catalyzing NADP+/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 from swine kidney

Two proteins (pI 4.8 and 5.8) capable of catalyzing NADP⁺/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 but not at C-11 have been purified to homogeneity from swine kidney. Both proteins exhibited identical molecular weight and subunit size. Similar amino acid composition, antigenic determinants, and coenzyme and substrate specificity were also found. The molecular weight of the enzyme as determined by gel filtration was 29,000. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel gave a value of 29,500 indicating the presence of a single polypeptide chain. Either enzyme protein utilized a variety of prostaglandins (PGS) as substrates. PGA₁-glutathione conjugate and PGB₁ were found to be the best substrates for prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase activities, respectively. For prostaglandins having dual reactive groups in a single molecule, the rate of oxidation of PGF_2α at C-15 was comparable to that at C-9, whereas the rate of reduction of 15-keto-PGE₂ at C-15 was far greater than that at C-9.     

Superoxide- and 1,1-Diphenyl-2-picrylhydrazyl Radical-scavenging Activities of Soyasaponin β g Related to Gallic Acid

Soyasaponin β g at 1 mM had 8% scavenging activity for O₂ ^-, and 25 μM β scavenged 20.9% for the DPPH radical (IC₅₀: 63.8 μM). In the soyasaponin β g-gallic acid system, synerigistic effects were observed at a low level of gallic acid concentration. The spin density distribution calculated by the MNDO/AM1 method showed unpaired electron localization on the carbons at C-4 and C-6, and on the ketone group at C-4 of the DDMP moiety. Furthermore, for soyasaponin β g, the MNDO/AM1 method gave an ionization potential of 8.38 eV, electron affinity of 1.16 eV and Mulliken electronegativity of 4.77 eV. Based on this evidence, the synergistic antiradical effects of the soyasaponin β g-gallic acid system are assumed to involve two-electron reduction from gallic acid.