Biosynthesis of the 7-methylated pterin of methanopterin.

The incorporation of [15N]glycine and [U-methyl-2H]methionine into methanopterin by growing cells of a methanogenic bacterium was measured to establish the biosynthetic route of the methylated pterin in the structure. The tetrahydromethanopterin produced by the cells was oxidatively cleaved to produce 7-methylpterin, and the amount of label incorporated into this pterin was measured by gas chromatography-mass spectrometry of the ditrimethylsilyl derivative of this compound. Approximately 27% of the 7-methylpterin and the guanine present in the cell was derived from the fed [15N]glycine. [U-methyl-2H]methionine was incorporated with the initial retention of all three deuteriums. These results are consistent with the biosynthesis of the pterin of methanopterin originating from GTP and its 7-methyl group arising from the methyl group of methionine.     

Tracer studies of the interconversion of R- and S-methylmalonic semialdehydes in man.

Two human subjects were given separate oral doses of sodium [2H6]isobutyrate and [methyl-2H3]thymine and the labelling patterns of urinary metabolites were determined. Ingestion of deuterated isobutyrate resulted in the excretion of 2H5-labelled S-3-hydroxyisobutyric acid, formed on the direct catabolic pathway, and of S- and R-[2H4]-3-hydroxyisobutyric acids, formed by the reduction of S- and R-methylmalonic semialdehydes respectively. Only the R-enantiomer of urinary 3-hydroxyisobutyric acid was labelled by thymine. This labelling pattern indicates a flow from S- to R-methylmalonic semialdehyde, suggesting that the R-enantiomer is the substrate of methylmalonic semialdehyde dehydrogenase.     

Biosynthesis and characterization of a series of deuterated cis,cis-octadeca-6,9-dienoic acids

[4,4-2H2]-, [5,5-2H2)-, [6-2H]-, [7-2H]-, [8,8-2H2)-, [11,11-2H2]-, [14,14-2H2]- and [18,18,18-2H3]-cis,cis-octadeca-6,9-dienoic (isolinoleic) acid were synthesized by supplementing cultures of the protozoan Tetrahymena with the corresponding deuterated cis-octadeca-9-enoic (oleic) acids. The cultures were harvested, the deuterated isolinoleic acids isolated and analyzed for purity by GC and TLC, and the structure and the level and position of deuteration of each fatty acid determined by 13C-NMR spectroscopy. The 13C resonances of all 18 carbons were also assigned based upon alpha-carbon deuterium isotope shifts and by comparison of the spectra to those of other polyunsaturated fatty acids. The results illustrate the utility of a biological approach for the synthesis of deuterated polyunsaturated fatty acids in yields suitable for 2H-NMR studies of membranes and possibly human metabolism.     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.     

Properties of 7-substituted deoxyguanosines formed by cis-diamminedichloroplatinum(II)

cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with deoxyguanosine and guanosine at pH 6 and the reaction products were purified by HPLC. The products were characterized by UV and 1H-NMR spectra and by incubating cis-Pt with 7-methylguanosine, N2-methylguanosine, [8-3H]guanosine and [U-14C]guanosine. The main product was shown to be a cross-link, in which cis-Pt was linked to the N-7 atoms of two guanines. In the other product cis-Pt was bound monofunctionally to the N-7 atom of guanine. The cis-Pt adducts had many unique properties compared to other N-7 alkylated guanosines. When cis-Pt was incubated with [8-3H]guanosine, the products still had their 8-3H-radioactivity. At pH 10 at room temperature the imidazole ring of the monoadduct was not opened in 20 days, while the half-life of imidazole ring-opening for 7-methyldeoxyguanosine was 21.5 min. The half-lives of acid-catalyzed depurination in 0.1 M HCl at 70 degrees C for deoxyguanosine, 7-methyldeoxyguanosine and the monoadduct were 30 s, 48 s and 35 min, respectively.     

Stereoselective synthesis of chirally deuterated (S)-D-(6-(2)H(1))glucose

Chirally deuterated (S)-D-(6-(2)H(1))glucose has been prepared in good overall yield from d-(6,6'-(2)H(2))glucose by a short, five-step synthesis from D-(6,6-(2)H(2))glucose utilizing (R)-(+)-Alpine-Borane [(R)-9-[(6,6-dimethylbicyclo[3.1.1]hept-2-yl)methyl]-9-borabicyclo[3.3.1]nonane]. Suitably protected methyl 2,3,4-tri-O-benzyl-D-(6,6-(2)H(2))glucopyranoside was prepared and the deuterated O-6 primary alcohol was oxidized to an aldehyde by Swern oxidation. Stereoselective reduction with nondeuterated (R)-(+)-Alpine-Borane gave methyl 2,3,4-tri-O-benzyl-(6S)-D-(6-(2)H(1))glucopyranoside, which was deprotected under standard conditions to afford the title compound. The key stereoselective reduction step was achieved in 90% yield. The preparation uses economical, commercially available starting materials and will be useful for elucidating biosynthetic mechanisms.     

Synthesis and rearrangements of D-glucosyl esters of aspartic acid linked through the 1- or 4-carboxyl group.

Catalytic hydrogenation of 2,3,4,6-tetra-O-benzyl-1-O-[1-benzyl N-(benzyloxycarbonyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (1alpha) in acetic acid-2-methoxyethanol gave 1-O-(L-beta-aspartyl)alpha-D-glucopyranose (2alpha) contaminated with 2-O-(L-alpha-aspartyl)-D-glucopyranose (8). Evidence that 8 was formed from the 1-oyl isomer of 1alpha, namely 2,3,4,6-tetra-O-benzyl-1-O-[4-benzyl N-(benzyloxycarbonyl)-L-aspart-1-oyl]-alpha-D-glucopyranose (7alpha), via 1 leads to 2 acyl migration, was obtained by submitting the deprotected D-glucosyl ester to successive N-acetylation, esterification, and O-acetylation; the final product was identified as a approximately 4:1 mixture of 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (4alpha) and 1,3,4,6-tetra-O-acetyl-2-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-D-glucopyranose (6) which were also prepared by definitive methods. On the other hand, deprotection of 1beta gave isomerically pure 2beta which was converted into the peracetylated ester derivative 4beta; an explanation for the differences in aglycon isomeric purity of 2alpha and 2beta is given. Hydrogenolysis of 7beta under the above conditions led to intermolecular transesterification with scission of the C-1 ester bond to give 1-(2-methoxyethyl) L-aspartic acid and D-glucose. Catalytic hydrogenation of 7alpha and 7beta, performed in the presence of trifluoroacetic acid, afforded 1-O-(L-alpha-aspartyl)-alpha- and -beta-D-glucopyranoside trifluoroacetate salts (11alpha and 11beta), respectively. The structure of 11beta was established by successive conversion into 2,3,4,6-tetra-O-acetyl-1-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-beta-D-glucopyranose (5beta) which was also prepared by definitive methods. Analogous treatment of 11alpha gave the N-acetyl derivative 12 which underwent 1 leads to 2 acyl migration during esterification with diazomethane to give the N-acetyl methyl ester derivative 10; acetylation of 10 afforded 6.     

Synthesis and chemical behaviour of D-glucosyl esters of glutamic acid having the side-chain carboxyl group involved in the glycosidic linkage.

Simultaneous and stepwise deprotection of the fully benzylated D-glucosyl esters of 1-benzyl N-benzyloxycarbonyl- and N-tert-butyloxycarbonyl-L-glutamic acid (1 and 5, respectively) have been examined. Catalytic hydrogenation of 1 led to intramolecular aminolysis to give pyroglutamic acid and D-glucose, but similar treatment in the presence of trifluoroacetic acid afforded both anomers of 1-O-(L-gamma-glutamyl)-D-glucopyranose, which were characterized as trifluoroacetates (2alpha and 2beta) and converted into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-glutam-5-oyl]-D-glucopyranose (4) which was also prepared by a definitive method. Hydrogenolysis of 5 gave both anomers of 1-O-[N-(tert-butyloxycarbonyl)-L-gamma-glutamyl]-D-glucopyranose (6), which, upon treatment with trifluoroacetic acid at - 10 degrees, afforded 2alpha and 2beta, respectively. The structure of 6beta was established by its conversion into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-beta-D-glucopyranose (7beta), whereas similar treatment of 6alpha gave a mixture of 1,3,4,6-tetra-O-acetyl-2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (9) and 7alpha. A 1 leads to 2 acyl migration occurred during esterification of the aglycon carboxyl group of 6alpha with diazomethane to give 2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (8).     

Synthesis and reactions of O-acetylated benzyl alpha-glycosides of 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-N-acetylmuramoyl-L- alanyl-D-isoglutamine esters: the base-catalysed isoglutamine in equilibrium glutamine rearrangement in peptidoglycan-related structures

Condensation of benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-3-O-[(R)-1-carboxyethyl]-alpha-D-glucopyranoside (2) and its 4-acetate (4) with L-alanyl-D-isoglutamine benzyl ester via the mixed anhydride method yielded N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-lacto yl)-L- alanyl-D-isoglutamine benzyl ester (5) and its 4-acetate (6), respectively. Condensation by the dicyclohexylcarbodi-imide-N-hydroxysuccinimide method converted 2 into benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside 1',4-lactone (7). In the presence of activating agents, 7 underwent aminolysis with the dipeptide ester to give 5. Zemplén O-deacetylation of 5 and 6 led to transesterification and alpha----gamma transamidation of the isoglutaminyl residue to give N-(2-O-[benzyl 2-acetamido-6-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyr anosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (8) and -glutamine methyl ester (9). Treatment of 6 with MgO-methanol caused deacetylation at the GlcNAc residue to give a mixture of N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-4-O-acetyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (11) and -glutamine methyl ester (12). Benzyl or methyl ester-protection of peptidoglycan-related structures is not compatible with any of the reactions requiring alkaline media. Condensation of 2 with L-alanyl-D-isoglutamine tert-butyl ester gave N-(2-O-[benzyl 2-acetamido- 6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2,3-d ideoxy- alpha-D-glucopyranosid-3-yl]-(R)-lactoyl-L-alanyl-D-isoglutamine tert-butyl ester (16), deacetylation of which, under Zemplén conditions, proceeded without side-reactions to afford N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-la cotyl)-L- alanyl-D-isoglutamine tert-butyl ester (17).     

Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells

The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta-adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.     

Biosynthesis of tetrahydrofolate. Stereochemistry of dihydroneopterin aldolase

7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, [1',2',3',6,7-13C5]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated [6alpha,6,7-13C3]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-[6,7,9,11-13C4]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with [13C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2H predominantly in the pro-S position.     

An efficient route to novel 4,5-di- and 2,4,5-tri substituted imidazoles from imidazo[1,5-a]-1,3,5-triazine (5,8-diaza-7,9-dideazapurine) derivatives

Two new types of imidazole derivatives: N-(2-R1-5-R2-1H-imidazol-4-yl) thioureas 7a-g and N-(2-R1-5-R2-1H-imidazol-4-yl) formamides 8b,c,g were obtained in high yields by the hydrolytic degradation of 6-R1-8-R2-2-thioxo-2,3-dihydroimidazo[1,5-a]-1,3,5-triazin-4(1H)-ones 5a-g and 6-R1-8-R2-imidazo[1,5-a]-1,3,5-triazin-4(3H)-ones 6b,c,d, respectively. The tautomeric preferences of the new imidazoles were determined.     

Biosynthesis of methanopterin

The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. [methylene-2H]-6-(Hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from [methyl-2H3]methionine, as confirmed by the incorporation of two C2H3 groups into 6-ethyl-7-methylpterin, a pterin-containing fragment derived from methanopterin. Cells grown in the presence of [methylene-2H]-6-(hydroxymethyl)pterin, [ethyl-2H4]-6-[1 (RS)-hydroxyethyl]pterin, [methyl-2H3]-6- (hydroxymethyl)-7-methylpterin, [ethyl-2H4, methyl-2H3]-6-[1 (RS)-hydroxyethyl]-7-methylpterin, and [1-ethyl-3H]-6-[1 (RS)-hydroxyethyl]-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic [methylene-3H]-7,8-H2-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H2-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.     

The developmental pattern of homologous and heterologous tRNA methylation in rat brain differential effect of spermidine

UsingS-adenosyl-L-[Me-¹⁴C] methionine, rat cerebral cortex methyltransferase activity was determined during the early postnatal period in the absence of addedEscherichia coli tRNA and in its presence. [Me-¹⁴C] tRNA was purified from both systems and its [Me-¹⁴C] base composition determined. The endogenous formation of [Me-¹⁴C] tRNA (homologous tRNA methylation) was totally abolished in the presence of 2.5 mM spermidine, whereasE. coli B tRNA methylation (heterologous methylation) was markedly stimulated. Only [Me-¹⁴C] 1-methyl guanine and [Me-¹⁴C]N ²-methyl guanine were formed by homologous methylation, there being an inverse shift in their relative proportions with age. Heterologous tRNA methylation led, additionally, to the formation of [Me-¹⁴C]N ₂ ² -dimethyl guanine, 5-methyl cytosine, 1-methyl adenine, 5-methyl uracil, 2-methyl adenine, and 1-methyl hypoxanthine. A comparison of heterologous tRNA methylation between the whole brain cortex (containing nerve and glial cells) and bulk-isolated nerve cell bodies revealed markedly lower proportions of [Me-¹⁴C]N ₂-methyl andN ₂ ² -dimethyl guanine and significantly higher proportions of [Me-¹⁴C] 1-methyl adenine in the neurons. The present findings suggest (1) that homologous tRNA methylation may provide developing brain cells with continuously changing populations of tRNA and (2) that neurons are enriched in adenine residue-specific tRNA methyltransferases that are highly sensitive to spermidine.This research was supported by grant NS-06294 of the United States Public Health Service.     

Isolation and identification of products from alkylation of nucleic acids: ethyl- and isopropyl-purines.

Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-, 7- and 9-alkylguanines; 1-, 3-, 7- and 9-alkyladenines. The products were identified from their characteristic u.v-absorption spectra, by comparison with either known ethyladenines or with the corresponding known methyladenines, and were also characterized by mass spectrometry. Their chromatographic properties on paper, t.l.c. and various columns were determined. DNA was alkylated in neutral solution with 14C-labelled alkyl methanesulphonates and the ratios of the alkylpurines formed were obtained, and compared for alkylation by methyl, ethyl and isopropyl methanesulphonates and by N-methyl-N-nitrosourea. The extents of alkylation at O-6 of guanine relative to those at N-7 of guanine varied with the reactivity of the methylating agents according to the predictions of Swain & Scott (1953) relating nucleophilicity of the groups alkylated with the substrate constants of the alkylating agents. The relative extents of alkylation at N-3 of adenine did not follow this correlation.     

Enantiospecific quantification of hexobarbital and its metabolites in biological fluids by gas chromatography/electron capture negative ion chemical ionization mass spectrometry.

A highly sensitive and specific assay based on gas chromatography/electron capture negative ion chemical ionization mass spectrometry has been developed for the analysis of the enantiomers of hexobarbital and its major metabolites in human urine and plasma. S-(+)-(5-2H3)hexobarbital and R-(-)-(5-2H3)hexobarbital were synthesized for clinical studies along with (+/-)-(1,5-2H6)hexobarbital and the deuterated major metabolites for use as internal and reference standards. Hexobarbital enantiomers and their metabolites were analyzed after pentafluorobenzyl and trimethylsilyl derivatization, following solid-phase extraction from plasma and urine. Intense negative ion spectra were observed for all of the derivatives. The base peak in the spectra corresponded to the M-pentafluorobenzyl anion [M-PFB]- except for 1,5-dimethylbarbituric acid, where M-. was the most abundant ion. The applicability of the method was demonstrated by following the plasma concentration-time profiles and urinary excretion in a male extensive metabolizer of mephenytoin who was given a pseudoracemic oral dose of hexobarbital containing equal 50 mg amounts of S-(+)-2(H0)hexobarbital and R-(-)-(2H3)hexobarbital. Marked stereoselective disposition was observed, with the R-(-)-enantiomer being more efficiently metabolized, primarily by alicyclic oxidation and ring cleavage.     

Substituent--directed aralkylation and alkylation reactions of the tricyclic analogues of acyclovir and guanosine

Aryl or tert-butyl substituent in the 6 position of 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-9-oxo-6-R-5H-imidazo[1,2-a]purine (6-R-TACV) 1 partly directs aralkylation reactions into unusual positions: N-4 to give 3 and C-7 to give N-5,7-disubstituted or N-4,7-disubstituted derivatives. In the case of alkylation the effect is limited to aryl substituent and position N-4. Replacement of acyclic moiety of 1 with a ribosyl one like in 7 prevents N-4 substitution. Cleavage of the third ring of 3b to give 3-benzylacyclovir 10 is an example of a new short route to 3-aralkyl-9-substituted guanines.     

Tetrahydrobiopterin analogues: solution conformations of 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyltetrahydropterins as determined by proton nuclear magnetic resonance

The conformation of tetrahydrobiopterin analogues in aqueous solution at 23 degrees C has been determined by analyzing the 200-MHz 1H NMR spectral parameters of the enzymatically active 6-methyltetrahydropterin, 7-methyltetrahydropterin, and cis- and trans-6,7-dimethyl-5,6,7,8-tetrahydropterins. Each of these cofactors, with the exception of the cis-6,7-dimethyl isomer, exhibited an unusually small trans 6H-7H spin-spin coupling (8.5-9.1 Hz). An empirical equation that accounts for the effects of substituent electronegativity and orientation on vicinal couplings [Haasnoot, C. A. G., deLeeuw, F. A. A. M., & Altona, C. (1980) Tetrahedron 36, 2783-2792] predicted this coupling to be 11.3-11.6 Hz. We attribute the discrepancy between the calculated and experimentally observed values of this coupling to hyperconjugation of the axially oriented C7-H bond with the pi orbital of the vinylogous amide protein of the pterin ring (N8-C8a = C4a-C4 = O) rather than conformational averaging. The trans 6H-7H interproton distance in the 6-methyl analogue is calculated to be 3.0 A from the measured decrease in the spin-lattice relaxation rate of the axially oriented C7 proton after specific deuteration at C6. This is consistent with the single-conformer interpretation. Chemical shift comparisons of the methyl resonances of these analogues, NOE measurements from selectively deuterated analogues, and the differential sensitivities of axially vs. equatorially disposed ring protons to protonation at N5 all indicate that (i) the methyl substituents at both the C6 and C7 positions markedly prefer equatorial-like orientations and (ii) the tetrahydropterin ring is, with the exception of a pronounced pucker at C6, nearly planar.(ABSTRACT TRUNCATED AT 250 WORDS)