Synthesis and utility of 14C-labeled nicotinamide cofactors

A new method for the synthesis of the reduced form of beta-nicotinamide [U-14C]adenine dinucleotide 2(')-phosphate([Ad-14C]NADPH) is presented. The present synthesis results in a radioactive material with a specific activity that is greater than 220 mCi/mmol. This method could easily be adapted for syntheses of 14C-labeled NADH, NADP(+), or any nicotinamide cofactors with radiolabels in other positions. Since these cofactors are so ubiquitous, the use and applications of such labeled material has broad implications. The utility of the labeled cofactor for determination of substrates for nicotinamide-dependent enzymes in the nano- to femtomole scale, in alternative enzymatic assays, and in kinetic isotope effect studies is discussed.     

Microscale synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate as a cofactor for thymidylate synthase

A one-pot synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively. The current procedure offers high-yield, high-purity, and microscale-quantity synthesis. In this procedure, two enzymes were used simultaneously in the reaction mixture. The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-[4-xH]-labeled reduced nicotinamide adenine dinucleotide phosphate. The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-[6-xH]5,6,7,8-tetrahydrofolate (S-[6-xH]H4F). The enzymatic reactions were followed by chemical trapping of S-[6-xH]H4F with formaldehyde to form the final product. Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization. Two analytical methods were developed to follow the reaction progress. Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.     

Selective Transacylation of 1-O-Alkylglycerophosphoethanolamine by Docosahexaenoate and Arachidonate in Rat Brain Microsomes

The mechanism involved in the enzymic acylation of 1-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) in brain microsomes was investigated in comparison with the acylation of 1-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC). Both the alkyllsophospholipids were acylated without exogenously added cofactors to similar extents. The [14C]arachidonoyl moiety of exogenously added 1-stearoyl-2-[14C]arachidonoyl-GPC was transferred to the alkyllysophospholipids and the transfer was not inhibited by exogenously added free arachidonate. These results indicated that the transferase activity was due to a transacylase that catalyzes the transfer of fatty acids between intact phospholipids. The addition of CoA increased the acylation of 1-[3H]alkyl-GPC two or three times with a high acceptor concentration, and the highest rate of acylation of 1-[3H]alkyl-GPC was observed in the presence of CoA, ATP, and Mg2+. On the other hand, the addition of such cofactors only slightly increased the acylation of 1-[3H]alkyl-GPE. HPLC analysis revealed that docosahexaenoate and arachidonate were transferred to the second position of both [3H]alkyllysophospholipids without cofactors and that other fatty acids were transferred to much lower extents. With the addition of cofactors, the acylation of 1-[3H]alkyl-GPC by both docosahexaenoate and arachidonate increased 1.5-2 times, and high amounts of palmitate, oleate, and linoleate were newly transferred. High amounts of oleate were also transferred to 1-[3H]alkyl-GPE in the presence of cofactors but the acylation by both docosahexaenoate and arachidonate scarcely increased on the addition of these cofactors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of 5-chloroformycin A, 5-chloro-2''-deoxyformycin A and certain related 5,7-disubstituted 3-beta-D-ribofuranosylpyrazolo[4,3-d] pyrimidines from formycin A.

A facile synthesis of 7-amino-5-chloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5-chloroformycin A, 6), 7-amino-5-chloro-3-(2-deoxy-beta-D-erythro-pentofuranosyl) pyrazolo [4,3-d]-pyrimidine (5-chloro-2'-deoxyformycin A, 13) and certain related 5,7-disubstituted pyrazolo[4,3-d]pyrimidine ribonucleosides is described starting with formycin A. Thiation of tri-O-acetyloxoformycin B (4b) with phosphorus pentasulfide, followed 3-beta-D-ribofuranosyl-7-thioxopyrazolo[4,3-d] pyrimidin-5(1H,4H,6H)-one (3b) in excellent yield. Chlorination of 4b with either phosphorus oxychloride or phenyl phosphonicdichloride furnished the key intermediate 5,7-dichloro-3-(2,3, 5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (5a), which on deacetylation afforded 5,7-dichloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5b). Ammonolysis of 5a with liquid ammonia gave 6, whereas with MeOH/NH3, a mixture of 6 and 7-methoxy-5-chloro-3-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine (7) was obtained. Reaction of 6 with lithium azide and subsequent hydrogenation afforded 5-aminoformycin A (10). Treatment of 5a with thiourea gave 5-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) pyrazolo[4,3-d]pyrimidine-7(1H,6H)-thione (8a), which on further reaction with sodium hydrosulfide furnished 3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine-5,7(1H,4H,6H)-dithione (11). The four-step deoxygenation procedure using phenoxythiocarbonylation of the 2'-hydroxy group of the 3', 5'-protected 6 gave 5-chloro-2'-deoxyformycin A (13).     

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.     

Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

Since phospholipids are major components of all serum lipoproteins, the role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine is made both by the CDP-choline pathway and by the methylation of phosphatidylethanolamine, which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanolamine). Monolayer cultures of rat hepatocytes were incubated in the presence of [methyl-3H]choline, [1-3H] ethanolamine, or [3-3H]serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the cultured medium. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine derived from [1-3H]ethanolamine were markedly lower (approximately one-half and less than one-tenth, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of phosphatidylcholine made from [methyl-3H]choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with [3-3H]serine, the specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were significantly higher in the lipoproteins than in the cells. These data indicate that there is not a random and homogeneous labeling of the phospholipid pools from the radioactive precursors. Instead, specific pools of phospholipids are selected, on the basis of their routes of biosynthesis, for secretion into lipoproteins.     

Compartmentation of dCTP pools disappears after hydroxyurea or araC treatment in lymphocytes.

The calculated rate of DNA synthesis using [5-3H]TdR was about 4 times higher than in the case of [5-3H]CdR labeling, even after correction for the specific radioactivities of the intracellular pools. These data show a compartmentation of dCTP pools in lymphocytes. Hydroxyurea increased the specific activities of both dTTP and dCTP pools so that the calculated rate of DNA synthesis became equal. The same effect was found for araC treatment, but not for fluorodeoxyuridine. dCTP was supplied from CTP which is the lowest ribonucleotide pool in lymphocytes. Different functions of the two dCTP pools are proposed: one serving DNA replication; the other one supplies phospholipid precursors and DNA repair.     

Characterization of the pathways for phosphatidylethanolamine biosynthesis in Chinese hamster ovary mutant and parental cell lines

A tritium suicide procedure was devised to facilitate the isolation of Chinese hamster ovary cell mutants defective in phosphatidylethanolamine biosynthesis. One mutant with a 20-50% reduction in [3H]ethanolamine incorporation was chosen for further analysis and was shown to have reduced activity of CTP: phosphoethanolamine cytidylyltransferase. Levels of phosphatidylethanolamine and rates of its biosynthesis were compared in the mutant and parent cell lines. Despite the reduced activity of the CDP-ethanolamine pathway in the mutant, levels of phosphatidylethanolamine were the same in mutant and parent cells. Rates of phosphatidylethanolamine synthesis de novo, as measured by incorporation of 32PO4 into phosphatidylethanolamine, were also the same in mutant and parent cells, as was the rate of incorporation of [3H]serine into both phosphatidylserine and phosphatidylethanolamine. After a long term labeling with [3H]serine, the specific radioactivity of phosphatidylserine was the same as that of phosphatidylethanolamine, and there was no difference in the specific radioactivities of the two lipids between mutant and parent cells. These results implicate decarboxylation of phosphatidylserine as the sole route for synthesis of phosphatidylethanolamine under normal culture conditions.     

Synthesis of [¹¹C]uric acid, using [¹¹C]phosgene, as a possible biomarker in PET imaging for diagnosis of gout

The synthesis and in vivo evaluation of (11)C -labeled uric acid ([(11)C]1), a potential imaging agent for the diagnosis of urate-related life-style diseases, was performed using positron emission tomography (PET) image analysis. First, the synthesis of [(11)C]1 was achieved by reacting 5,6-diaminouracil (2) with (11)C-labeled phosgene ([(11)C]COCl(2)). The radiochemical yield of [(11)C]1 was 37±7% (decay-corrected based on [(11)C]COCl(2)) with specific radioactivities of 96-152GBq/μmol at the end of synthesis (n=6). The average time of radiosynthesis from the end of bombardment, including formulation, was about 30min with >98% radiochemical purity. Second, the synthetic approach to [(11)C]1 was optimized using 5,6-diaminouracil sulfate (3) with [(11)C]COCl(2) in the presence of 1,8-bis(dimethylamino)naphthalene. [(11)C]1 was synthesized in 36±6% radiochemical yield, 89-142GBq/μmol of specific radioactivities, and 98% radiochemical purity by this method (n=5). This allowed the synthesis of [(11)C]1 to be carried out repeatedly and the radiochemical yield, specific radioactivities, average time of synthesis, and radiochemical purity of [(11)C]1 were similar to those obtained using 2. PET studies in rats showed large differences in the accumulation of radioligand in the limbs under normal and hyperuricemic conditions. Thus, an efficient and convenient automated synthesis of [(11)C]1 has been developed, and preliminary PET evaluation of [(11)C]1 confirmed the increased accumulation of radioactivity in the limbs of a rat model of hyperuricemia.     

Striatal Dopamine Release In Vitro: A β-Adrenoceptor-Regulated Response Not Mediated Through Cyclic AMP

The effects of (-)isoproterenol (10(-6) M), dibutyryl cyclic AMP (10(-3) M), and the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) (10(-4) M) on in vitro [3H]dopamine ([3H]DA) efflux and synthesis were studied in rat striatal slices continuously superfused with [3H]tyrosine. The beta-adrenoceptor agonist (-)isoproterenol induced an immediate and significant facilitation of [3H]DA efflux but did not alter [3H]DA synthesis as measured by [3H]H2O formation. In contrast, both dibutyryl cyclic AMP and IBMX enhanced [3H]DA synthesis as well as efflux. The presence of IBMX in the superfusing medium did not potentiate the augmentation of [3H]DA efflux caused by (-)isoproterenol. Additionally, the blockade of [3H]DA synthesis by alpha-methyl-p-tyrosine (10(-4) M) completely prevented the action of dibutyryl cyclic AMP on [3H]DA efflux. However, under similar conditions, (-)isoproterenol was still able to increase [3H]DA efflux. The results suggest that (-)isoproterenol can modify striatal DA release through a mechanism not involving cyclic AMP.     

Efficient synthesis and biological evaluation of all A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 and its 20-epimer

An improved synthesis of the diastereomers of 1alpha,25-dihydroxyvitamin D3 (1) was accomplished utilizing our practical route to the A-ring synthon. We applied this procedure to synthesize for the first time all possible A-ring diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of the C-3 hydroxyl group to the olefin by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A-ring enynes (3). i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield. Palladium-catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethylene)cholestan-25-ol, followed by deprotection, afforded the requisite diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). The biological profiles of the synthesized stereoisomers were assessed in terms of affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP). HL-60 cell differentiation-inducing activity and in vivo calcium-regulating potency in comparison with the natural hormone.     

Tartrate dehydrogenase reductive decarboxylation: stereochemical generation of diastereotopically deuterated hydroxymethylenes

Tartrate dehydrogenase catalyzes the reductive decarboxylation of meso-tartrate to glycerate. Concomitant with the ketonization of the intermediate enolate the C3 hydroxymethylene of glycerate necessarily acquires a proton from solvent. In D2O, the proton is shown to be added stereospecifically to form (2R,3R)-[3-2H]glycerate. The 1H-NMR assignments of the diastereotopic C3 protons of glycerate were confirmed by the enzymatic conversion of [1R-2H]fructose-6-phosphate to (2R,3R)-[3-2H]glycerate. The decarboxylation-protonation occurs with retention of configuration, implying that the general acid is positioned on the same face of the intermediate as the departing carboxylate. The stereochemically pure (2R,3R)-[3-2H]glycerate is readily synthesized and serves as a chiral hydroxymethylene synthon as demonstrated by the synthesis of (2S,3R)-[3-2H]serine.     

High-yield synthesis of a [3H]ethylenediamine ditetrodotoxin derivative

[3H]Tetrodotoxin [( 3H]TTX) and a [3H]ethylenediamine derivative of TTX are the most widely used ligands for the study of the Na channel. The former ligand presents a low specific radioactivity (1 Ci/mmol) while the latter is highly labeled (30 Ci/mmol). However, its two-step synthesis, i.e., mild oxidation followed by coupling of [3H]ethylenediamine, has been described with a low overall yield of 1.7%. In this work, more favorable experimental conditions are defined for the limiting reaction, i.e., the oxidation step, using [14C]testosterone as a model molecule. Applied to the oxidation of tetrodotoxin, this procedure produces yield values of 30-50%, as determined by high-performance liquid chromatography. Moreover, two oxidized TTX molecules appear to be covalently linked to [3H]ethylenediamine, yielding a new labeled tetrodotoxin derivative with a specific radioactivity of 45 Ci/mmol and a dissociation constant of 0.6 nM for electroplax membranes.     

Plant peptide hormone phytosulfokine (PSK-alpha): synthesis of new analogues and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In our studies on structure/activity relationship in PSK-alpha the synthesis of a series of analogues was performed: [H-D-Tyr(SO3H)1]- (9), [H-Phe(4-SO3H)1]- (10), [H-D-Phe(4-SO3H)1]- (11), [H-Phg(4-SO3H)1]- (12), [H-D-Phg(4-SO3H)1]- (13), H-Phe(4-NHSO2CH3)1]- (14), [H-D-Phe(4-NHSO2CH3)1]- (15), [H-Phe(4-NO2)1]- (16), [H-D-Phe(4-NO2)1]- (17), [H-Phg(4-NO2)1]- (18), [H-D-Phg(4-NO2)1]- (19), [H-Hph(4-NO2)1]- (20), [H-Phg(4-OSO3H)1]- (21), [Phe(4-NO2)3]- (22), [Phg(4-NO2)3]- (23), [Hph(4-NO2)3]- (24), [H-Phe(4-SO3H)1, Phe(4-SO3H)3]- (25) [H-Phe(4-NO2)1, Phe(4-NO2)3]- (26), [H-Phg(4-NO2)1, Phg(4-NO2)3]- (27), [H-Hph(4-NO2)1, Hph(4-NO2)3]- (28) and [Val3]- PSK-alpha (29). For modification of the PSK-alpha peptide chain the novel amino acids and their derivatives were synthesized, such as: H-L-Phg(4-SO3H)-OH (1), H-D-Phg(4-SO3H)-OH (2), Fmoc-Phg(4-SO3H)-OH (3), Fmoc-D-Phg(4-SO3H)-OH (4), Boc-Phg(4-NHSO2CH3)-OH (5), Boc-D-Phg(4-NHSO2CH3)-OH (6) Boc-Phe(4-NHSO2CH3)-OH (7), and Boc-D-Phe(4-NHSO2CH3)-OH (8). Peptides were synthesized by a solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK according to the Matsubayashi et al. test.     

Heterogeneity in the metabolism of the arachidonoyl molecular species of glycerophospholipids of rabbit alveolar macrophages. The interrelationship between metabolic activities and chemical structures of the arachidonoyl molecular species

The relative incorporation of [3H]arachidonic acid (20:4) into individual molecular species containing 20:4 at the 2 position (18:1-20:4, 16:0-20:4 and 18:0-20:4 species) of diacyl and ether-linked glycerophosphocholine, glycerophosphoethanolamine and glycerophosphoinositol of rabbit alveolar macrophages has been measured by reversed-phase high-performance liquid chromatography (HPLC). The rate of incorporation of [3H]20:4 into the molecular species of glycerophospholipids was greatly influenced by their structures. The reversed-phase HPLC analysis allowed elucidation of the influence of structural differences, such as the nature of the polar head group, the fatty chain at the 1 position and the chemical form of the bond of the fatty chain attached at the 1 position on the uptake of [3H]20:4 by comparison of the specific radioactivities of arachidonoyl molecular species having the same structures, except that one of the three kinds of moiety was different. The specific radioactivities of the molecular species containing choline head groups were significantly higher than those containing ethanolamine and inositol moieties. The specific radioactivities of diacyl molecular species were considerably higher than those of ether-linked molecular species. The nature of the fatty chain attached at the 1 position also influenced the uptake of [3H]20:4 into glycerophospholipids. The arachidonoyl molecular species containing 18:1 at the 1 position were preferentially labelled with [3H]20:4 as compared to the corresponding 16:0-20:4 and 18:0-20:4 species either of diacyl or ether-linked glycerophospholipids. The present results suggest that the acyltransferase involved in the incorporation of 20:4 into glycerophospholipids has selectivity for the structures of glycerophospholipids and the order of selectivity of this enzyme for the arachidonoyl molecular species, deduced in the present experiments, was as follows: choline head group greater than ethanolamine and inositol groups, acyl bond greater than ether and vinyl ether bonds, 18:1 fatty chain greater than 16:0 and 18:0 fatty chains at the 1 position. Comparison of the metabolic activities of all major arachidonoyl molecular species of glycerophospholipids having a single structure is reported here for the first time.     

Metabolism of unique diarachidonoyl and linoleoylarachidonoyl species of ethanolamine and choline phosphoglycerides in rat testes

Selected molecular species of rat testicular 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines were quantitated as their diradylglycerobenzoate derivatives, using a recently developed high-performance liquid chromatographic method. Increased amounts of docosapentaenoic acid were found in glycerophospholipids containing ether moieties compared with the diacyl phospholipids (e.g., docosapentaenoate-containing species comprised more than 80% of the alkylacyl subclass of the ethanolamine phosholipids as opposed to 29.3% of the diacyl subclass). Within 2 h after intratesticular injections of [5,6,8,9,11,12,14,15-3H]arachidonic acid, the 20:4-20:4 and 18:2-20:4 molecular species of the diacyl subclass of both the choline and ethanolamine glycerophosphatides had the highest specific radioactivities. These unique molecular species (20:4-20:4 and 18:2-20:4) also exhibited the largest percentage decrease in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonic acid, which indicates these two species possess a high metabolic turnover. Two of the arachidonate-containing molecular species (18:1-20:4 and 18:0-20:4) in the ethanolamine plasmalogens showed only a small decrease in specific radioactivity, whereas a third species (16:0-20:4) actually had a 44% increase in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonate. These data indicate that the 20:4-20:4, 18:2-20:4 and 18:1-20:4 species of phosphatidylcholine and/or phosphatidylethanolamine are most rapidly labeled after administration of [3H]arachidonic acid and that they appear to serve as the source of the [3H]arachidonate that is ultimately transferred to ethanolamine plasmalogens.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

Specific tritium labeling of glucosyl- and galactosylceramides at the 6-position of the carbohydrate moiety using CrO3-graphite

A new procedure for introducing tritium into the carbohydrate portions of glucosyl- and galactosylceramides was developed using a new catalyst, CrO3-graphite, which specifically oxidizes the primary alcohol group to the aldehyde. About 10% of the glycolipid was converted to the aldehyde and the aldehyde produced was then reduced back to the original form with KB3H4. After methanolysis, more than 96.7% of the radioactivities of [3H]glucosyl- and [3H]galactosylceramides were found to be located in the carbohydrate portions, and the specific activities of the [3H]galactosyl- and [3H]glucosylceramides were 2.08 to 4.30 X 10(4) cpm/nmol, which could be increased greatly by purifying the aldehydes and reducing them with KB3H4. In addition, beta-galactosidase activity was successfully determined with [3H]galactosylceramide as the enzyme substrate; the Km was 18.73 mM and the Vmax was 11.63 nmol/mg/h, indicating that no significant structural modification occurs during the oxidation.     

2-Keto-3-fluoroglutarate: a useful mechanistic probe of 2-keto-glutarate-dependent enzyme systems

2-Keto-3-fluoroglutaric acid prepared by acid hydrolysis of its diethyl ester is stable, as the free acid in aqueous solution at pH 2, and can be stored at -20 degrees C for several years. Both enantiomers are reduced by NADH in the presence of glutamate dehydrogenase (EC 1.4.1.2) to the two diastereomers of 3-fluoro-L-glutamate, which are stable at neutral pH and at high pH unless heated. 2-Keto-3-fluoroglutarate exists in solution almost entirely as a hydrate both at low and neutral pH. Both enantiomers of ketofluoroglutarate react with the pyridoxamine forms of aspartate, alanine and 4-aminobutyrate transaminases to give fluoride release. 2 mol of cosubstrate amino acid react for each mol of ketofluoroglutarate (KFG) when starting from the pyridoxamine form of the enzyme: 2 RCHNH2COOH + KFG + H2O----F- + NH4+ + glutamate + 2 RCOCOOH. Both diastereomers of fluoroglutamate are decarboxylated by glutamate decarboxylase (EC 4.1.1.15) with fluoride release: KFG + H2O----CO2 + F- + HCOCH2CH2COOH. By contrast, only one isomer of fluoroglutamate will react with the pyridoxal form of glutamate-oxalacetate transaminase to give fluoride release: HOOCCHNH2CHFCH2COOH + H2O----4F- + NH4+ + HOOCCOCH2CH2COOH. The enzymatic decarboxylation of 3-fluoroisocitrate produces only one enantiomer of ketofluoroglutarate, which is reduced to threo (2R,3R)-3-fluoroglutamate by NADH and glutamate dehydrogenase: [2R,3S]-HOOCCH(OH)CF(COOH)CH2COOH + NADP+----[3R]-KFG + CO2 + NADPH + H+. The proton, 13C, and 19F-NMR parameters of ketofluoroglutarate and the two fluoroglutamate diastereomers are presented. These molecules are useful probes of enzymatic mechanisms thought to involve carbanion intermediates.     

Practical stereoselective synthesis of (2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl)acetic acid

Highly enantioselective asymmetric hydrogenation of readily accessible olefins, (E)- and (Z)-[1-(toluene-4-sulfonyl)-1,2,3, 4-tetrahydro-1H-benzo[b]azepin-5-ylidene]acetic acid (4a and 4b, respectively) and [1-(toluene-4-sulfonyl)-2, 3-dihydro-1H-benzo[b]azepin-5-yl]acetic acid (4c), is presented as an efficient and straightforward route to (R)-[1-(toluene-4-sulfonyl)-2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl]acetic acid [(R)-1] which is a key intermediate for the synthesis of non-peptide AVP V2-agonist. Hydrogenation of carboxylic acid 4c gave (R)-1 in quantitative yield and 85% ee using Ru(OAc)2[(S)-H8-BINAP], a Ru(II) complex of a partially hydrogenated BINAP (H8-BINAP), as a catalyst. When (R)-1 of 76% ee was transformed into the corresponding isopropylamide 6, pure enantiomer (R)-6 was obtained in 75% yield by recrystallization from MeOH.