Regulatory control of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase in Streptomyces antibioticus

Streptomyces antibioticus possesses a tryptophan-inhibitable 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthetase whose synthesis is also repressed by L-tryptophan. Studies of the DAHP synthetase obtained by ammonium sulfate fractionation of a crude extract derived from S. Antibioticus revealed that the enzymic activity was only partially inhibited by tryptophan. Inhibition of the DAHP synthetase activity was strongly pH dependent at values below 7.0. A number of tryptophan analogues was noted to inhibit the enzyme; by contrast, other aromatic amino acid end products failed to affect DAHP synthetase activity. Chorismic acid, a key intermediate in aromatic amino acid biosynthesis, was ineffective as an inhibitor when used alone; however, if supplied with L-tryptophan, a further reduction of DAHP synthetase activity (15--25%) was routinely observed.     

Fluorine-containing analogues of intermediates in the Shikimate pathway.

The phosphoenolpyruvate analogue (Z)-phosphoenol-3-fluoropyruvate is a substrate for phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase from Escherichia coli. In the presence of excess erythrose 4-phosphate, apparent KM values of 65 and 38 muM were observed for phosphoenol-3-fluoropyruvate and phosphoenolpyruvate, respectively. Because the apparent Vmax for phosphoenol-3-fluoropyruvate is only 1.17% of that for phosphoenolpyruvate, one can study the former as an inhibitor of 3-deoxy-arabino-heptulosonic acid-7-phosphate synthase. Kinetic experiments showed phosphoenol-3-fluoropyruvate to be competitive with respect to phosphoenolpyruvate. Two distinguishable Ki values of 8 and 48 muM were obtained. The product (3S)-3-deoxy--3-fluoro-arabino-heptulosonic acid 7-phosphate was purified, characterized, and shown to act as a substrate for 5-dehydroquinate synthase. 3-Deoxy-3-fluoro-arabino-heptulosonic acid 7-phosphate, in contrast to 3-deoxy-arabino-heptulosonic acid 7-phosphate reacts slowly or not at all with reagents specific for 2-keto-3-deoxy sugars and is relatively resistant to oxidative cleavage by sodium periodate. The expected product of periodate oxidation, 3-fluoro-3-formylpyruvate, cannot be detected. This observation was clarified by studies with model compounds.     

5-Dehydro-3-deoxy-D-arabino-heptulosonic acid 7-phosphate. An intermediate in the 3-dehydroquinate synthase reaction.

3-Dehydroquinate synthase was purified to homogeneity from Escherichia coli. It was found to be a single polypeptide chain of Mr = approximately 57,000. Reaction mixtures of pure enzyme and the substrate, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, were incubated for short times and treated with NaB3H4. The resulting 3-deoxyheptonic acid 7-phosphate was degraded with sodium periodate, and formic acid representing C-5 of the substrate was isolated. The presence of 3H in the formate corresponding to 15% of the enzyme was interpreted as indicating a 5-dehydro derivative of the substrate as an intermediate of the reaction. Quinic acid, resulting from reduction of 3-dehydroquinate with NaB3H4, was also isolated and degraded with periodate. The formate from C-4 of the quinate was unlabeled, indicating that 3,4-bisdehydroquinate is not an intermediate.     

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli.

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium.

The first committed step of aromatic amino acid biosynthesis in Salmonella typhimurium was shown to be catalyzed by three isoenzymes of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. Mutations in each of the genes specifying the isoenzymes were isolated and mapped. aroG, the structural gene for the phenylalanine-inhibitable isoenzyme, was linked to gal, and aroH, the structural gene for the tryptophan-inhibitable isoenzyme, was linked to aroE. aroF, the structural gene for the tyrosine-inhibitable isoenzyme, was linked to pheA and tyrA, which specify the phenylalanine- and tyrosine-specific branch-point enzymes, respectively. The phenylalanine-inhibitable isoenzyme was the predominant DAHP synthase in wild-type cells, and only the tryosine-inhibitable isoenzyme was completely repressed, as well as inhibited, by low levels of its allosteric effector. The DAHP synthase isoenzymes were separated by chromatography on diethylaminoethyl-cellulose with a phosphate gradient which contained enolpyruvate phosphate to protect the otherwise unstable phenylalanine-inhibitable isoenzyme. No cross-inhibition of either the tyrosine- or phenylalanine-inhibitable isoenzyme was observed at inhibitor concentrations up to 1 mM. The tryptophan-inhibitable isoenzyme was partially purified from extracts of a strain lacking the other two isoenzymes and shown to be inhibited about 30% by 1 mM tryptophan. A preliminary study of interference by tryptophan in the periodate-thiobarbiturate assay for DAHP suggested a combined effect of tryptophan and erythrose 4-phosphate, or an aldehydic compound resulting from degradation of erythrose 4-phosphate by periodate.     

The synthesis of 3-deoxyheptulosonic acid 7-phosphate.

3-Deoxyheptulosonic acid 7-phosphate was obtained by a one-step chemical synthesis through condensation of oxalacetate with erythrose 4-phosphate. This reaction occurs at measurable rates only in the presence of a metal ion; Co2+ and Ni2+ are the most effective catalysts. The Co2+ catalyzed condensation of oxalacetate and erythrose 4-phosphate proceeds at room temperature and neutral pH. Since erythrose 4-phosphate can be replaced by any free aldehyde tested thus far, this type of a homogeneous catalysis opens new synthetic routes to alpha-keto-gamma-hydroxy-fatty acids and their derivatives.     

Microbial synthesis of p-hydroxybenzoic acid from glucose.

A series of recombinant Escherichia coli strains have been constructed and evaluated for their ability to synthesize p-hydroxybenzoic acid from glucose under fed-batch fermentor conditions. The maximum concentration of p-hydroxybenzoic acid synthesized was 12 g/L and corresponded to a yield of 13% (mol/mol). Synthesis of p-hydroxybenzoic acid began with direction of increased carbon flow into the common pathway of aromatic amino acid biosynthesis. This was accomplished in all constructs with overexpression of a feedback-insensitive isozyme of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase. Expression levels of enzymes in the common pathway of aromatic amino acid biosynthesis were also increased in all constructs to deliver increased carbon flow from the beginning to the end of the common pathway. A previously unreported inhibition of 3-dehydroquinate synthase by L-tyrosine was discovered to be a significant impediment to the flow of carbon through the common pathway. Chorismic acid, the last metabolite of the common pathway, was converted into p-hydroxybenzoic acid by ubiC-encoded chorismate lyase. Constructs differed in the strategy used for overexpression of chorismate lyase and also differed as to whether mutations were present in the host E. coli to inactivate other chorismate-utilizing enzymes. Use of overexpressed chorismate lyase to increase the rate of chorismic acid aromatization was mitigated by attendant decreases in the specific activity of DAHP synthase and feedback inhibition caused by p-hydroxybenzoic acid. The toxicity of p-hydroxybenzoic acid towards E. coli metabolism and growth was also evaluated.     

Synthesis of 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate by phosphorylation of methyl (methyl 3-deoxy-d-arabino-heptulopyranosid)onate

3-Deoxy-d-arabino-2-heptulosonic acid 7-phosphate (5), the first committed intermediate in aromatic amino acid biosynthesis, has been synthesized in good yield by treatment of methyl (methyl 3-deoxy-d-arabino-2-heptulopyranosid)onate with diphenylphosphoric chloride under mild conditions to give the 7-diphenyl phosphate. Catalytic removal of the phenyl residues, followed by base-catalyzed hydrolysis resulted in formation of (methyl 3-deoxy-d-arabino-2-heptulopyranosid)onic acid dihydrogen 7-phosphate (4), which yielded a crystalline tris-(cyclohexylammonium) salt. Acid-catalyzed hydrolysis of 4 afforded 5, which was used to purify 3-dehydroquinate synthase.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Modulation of phosphoenolpyruvate synthase expression increases shikimate pathway product yields in E. coli

Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed-batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3-dehydroshikimic acid. Shikimate pathway byproducts 3-deoxy-D-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3-dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3-dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.     

Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase.

Dehydroquinate synthase, the enzyme which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to 5-dehydroquinate, has been purified from Bacillus subtilis in association with chorismate synthase and NADPH-dependent flavin reductase. The enzyme was only active when associated with chorismate synthase, whereas the flavin reductase could be separated from the complex with retention of dehydroquinate synthase activity. The enzyme requires NAD and either Co2+ or Mn2+ for maximal activity. The activity was completely inhibited by EDTA. The Km of the enzyme for DAHP, NAD, and Co2+ were estimated to be 1.3 X 10(-4), 5.5 X 10(-5), and 5.5 X 10(-5) M, respectively. Enzyme activity was completely inhibited by NADH and the inhibition was not reversed by the addition of NAD, NADPH and NADP were not inhibitory. The enzyme was unstable to heat and lost all activity at 55 degrees C. A protein fraction which did not adsorb to phosphocellulose was found to inhibit the enzyme.     

Fatty acid synthesis and the oxidative pentose phosphate pathway in developing embryos of oilseed rape (Brassica napus L.)

The potential role of the plastidial oxidative pentose phosphate pathway (OPPP) in providing the NADPH for fatty acid synthesis in plastids from developing embryos of Brassica napus (L.) has been investigated. Measurements of distributions of enzyme activities in fractions obtained from homogenates of isolated embryos have revealed that the glucose 6-phosphate and 6-phosphogluconate dehydrogenases are present in both cytosol and plastid, as is ribose 5-phosphate isomerase. However, transketolase and transaldolase are most probably confined to the plastid, while ribulose 5-phosphate epimerase is essentially cytosolic, although a very small proportion of plastid-localized activity cannot be ruled out. The activity of the OPPP in intact plastids was measured by the release of (14)CO(2) from [1-(14)C]glucose 6-phosphate. Activity was detectable in the absence of electron sinks created by the addition of metabolites to the incubation media and was stimulated 1.3-, 3.2-, and 7.9-fold by the respective additions of glutamine plus 2-oxoglutarate, cofactors and substrates for fatty acid synthesis, or methyl viologen. An increase in OPPP activity in response to additions that are absolutely required for fatty acid synthesis in these isolated plastids provides direct evidence that these two processes are connected, most probably by NADP/NADPH metabolism. The OPPP activity with methyl viologen was more than twice that during fatty acid synthesis, suggesting that the latter is not limited by OPPP capacity. Light energy may also contribute to reductant provision and, consistent with the possibility of maintenance of a balance of NADPH from light and the OPPP, glucose 6-phosphate dehydrogenase activity in the isolated plastids was decreased by light or by DTT.     

Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes.

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.     

Synthesis of δ-aminovaleramide

In Pseudomonas, δ-aminovaleramide³ is both a product of the l-lysine oxygenase reaction and a substrate for δ-aminovaleramide deamidase. We report the synthesis and certain physical, chemical, and biological properties of δ-aminovaleramide. δ-Aminovaleric acid (I) is converted to δ-carbobenzoxy-δ-aminovaleric acid (II) and then to δ-carbobenzoxy-δ-aminovaleric acid methyl ester (III). Ammonolysis of (III) forms δ-carbobenzoxy-δ-aminovaleramide (IV), which on removal of the carbobenzoxy group yields δ-aminovaleramide.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

Stereospecific synthesis and two-dimensional 1H-NMR investigation of isoursocholic acid.

This report describes the chemical synthesis of isoursocholic acid (3 beta, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) from its corresponding 3 alpha-analog. The method consists of refluxing a mixture of ursocholic acid, triphenylphosphine, and diethyl azodicarboxylate in benzene solution with an acid such as formic acid. The sterically pure ester (3 beta-formate) so formed after saponification with LiOH-aqueous methanol then allowed an easy access to the epimer of the starting acid. Large scale preparative separation and purification of the final product and synthetic intermediates were accomplished by flash column chromatography of their methyl ester derivatives. Structural assignment of the isourscholic acid molecule was confirmed by complete analysis of proton NMR spectra using 2-D NMR correlation experiments which rigorously established the (3 beta/3 alpha) and (7 beta/7 alpha) hydroxyl configurations in the isoursocholic and ursocholic acids. It is suggested that the isoursocholic acid will be useful as a reference compound and as a substrate in studies dealing with the hepatic inversion of the 3 beta-hydroxy group.     

Coupling of ethanol metabolism to lipid biosynthesis: labelling of the glycerol moieties of sn-glycerol-3-phosphate,a phosphatidic acid and a phosphatidylcholine in liver of rats given [1,1-2H2]ethanol

The mechanism behind ethanol-induced fatty liver was investigated by administration of [1,1-²H₂]ethanol to rats and analysis of intermediates in lipid biosynthesis. Phosphatidic acid and phosphatidylcholine were isolated by chromatography on a lipophilic anion exchanger and molecular species were isolated by high-performance liquid chromatography in a non-aqueous system. The glycerol moieties of palmitoyl-linoleoylphosphatidic acid, the corresponding phosphatidylcholine and free sn-glycerol-3-phosphate were analysed by GC/MS of methyl ester t-butyldimethylsilyl derivatives. The deuterium labelling in the glycerol moiety of the phosphatidic acid was 2–3-times higher than in free sn-glycerol-3-phosphate, indicating that a specific pool of sn-glycerol-3-phosphate was used for the synthesis of phosphatidic acid in liver. The results indicate that NADH formed during ethanol oxidation is used in the formation of a pool of sn-glycerol-3-phosphate that gives rise to triacylglycerol and possibly fatty liver.     

Nucleic acids and alkylating agents: carbon-13 magnetic resonance studies of the methylation of ribonucleic acid with methyl methanesulfonate.

Direct examination of the interaction of ribonucleic acid with the alkylating agent, methyl methanesulfonate, by carbon-13 magnetic resonance spectroscopy has demonstrated the potential usefulness of this new spectroscopic method in studying the interaction between nucleic acids and biologically active agents. By comparison with the model compounds isolated from the reactions of mononucleotides with methyl methanesulfonate, five methylated products, 7-methylguanosine (49.7%), 1-methyladenosine (21.2%), 3-methyl cytosine (12.7%), 1-methylguanosine (7.5%), and methyl phosphate (8.9%, tentative) have been identified in the alkylated RNA from the reaction without buffer, whose pH is maintained using a pH stat; three methylated products, 7-methylguanosine (61.1%), 1-methyladenosine (20.5%), and 3-methylcytosine (18.4%) have been identified from the reaction in phosphate buffer. These quantitative differences may account for the failure of previous structure-activity correlations. The limits of the biological interpretation of the present and previous chemical results are discussed.