Sites of alkylation of poly(U) by agents of varying carcinogenicity and stability of products.

Several alkylating agents of widely varying reported carcinogenicity (dimethylsulfate, diethylsulfate, ethylmethanesulfonate, methylnitrosourea, ethylnitrosourea and ethylnitrosoguanidine) were reacted with poly(U) at pH values ranging from 4.5 to 7.5. All nucleophilic centers (internal phosphate groups, ribose hydroxyls, and O2, N-3 and O4 sites of the uracil base) were found reactive, though to different extents, at neutrality and in slightly acid solution. The distribution of products is a function of the alkylating agent and pH. The nitroso compounds are more reactive toward oxygens than are dialkylsulfates and alkylalkanesulfonates. The ratio of N : O alkyl products is strongly pH dependent, primarily due to the N-3 being most reactive at the higher pH values, while the diester is most reactive at the lower pH values. The extent of reaction of the O2, O4 or 2'-O or ribose is not greatly affected over the pH range tested. At pH 5.0 alkyl ribophosphotriesters mainly lose alchol to re-form a stable phosphodiester. With increasing OH- concentration, the favored reaction is chain scission at the 3'-O-P bond.     

The alkylation of 2′-deoxyguanosine and of thymidine with diazoalkanes. Some observations on O-alkylation

The reaction in ether-methanol between 2'-deoxyguanosine and diazomethane or its ethyl or n-butyl homologue gives 1-, O(6)- and 7-alkyl-2'-deoxyguanosine. N(2),O(6)-Dimethyl-2'-deoxyguanosine was also detected. The hydrolysis of the methyl and the ethyl derivatives gives the corresponding alkylguanines: the O(6)-alkyl-2'-deoxyguanosines were sequentially hydrolysed, first to 2-amino-6-alkoxypurines, subsequently to guanine. The mass spectra of O(6)-alkyl-2'-deoxyguanosines (methyl and ethyl) and of the corresponding 2-amino-6-alkoxypurines were determined. The reaction of diazomethane with thymidine afforded O(4)-methylthymidine, in addition to the previously detected 3-methylthymidine.     

Methylation and ethylation of uridylic acid and thymidylic acid. Reactivity of the ring and phosphate as a function of pH and alkyl group.

At pH 6.8 in aqueous solution (4 hr, 22 degrees), all methylating agents tested, i.e., dimethyl sulfate, methyl methanesulfonate, and methylnitrosourea, react with both the N-3 of the ring and the phosphate of UMP and dTMP. Although the extent of reaction varies from 17 to 76%, the ratio of phosphate/ring methylation is approximately 4. Both the 3-methyl nucleotides and methyl ester of 3-methyl nucleotides are identified, as well as the methyl esters of unmodified UMP and dTMP. At pH 8.2 the extent of total methylation is similar but reactivity of the N-3 is increased and that of the phosphate decreased so that the phosphate/ring ratio is approximately 1. At pH 6 almost all reaction is with the phosphate group. Uridine, under the same conditions, is methylated at pH 6.8 to form 15% 3-methyluridine and, at pH 8.2, the N-3 of uridine and thymidine is methylated to about 50%. Neither uridine nor UMP forms detectable ribose methyl products at any of these pH's. The comparable ethylating agents (diethyl sulfate, ethyl methanesulfonate, and ethylnitrosourea) are less reactive and the total ethylation of UMP or dTMP is about 1/5 that of methylation. There is little ethylation of the N-3 but the phosphate is alkylated to a relatively high extent so that the phosphate/base ratio at pH 6.8 is 10-23, and at pH 8.2 the ratio is 5-8. The fact that ethylating agents have a greater affinity than methylating agents for alkylating phosphates is proposed as the basis for the previously reported analytical data in which ethylating agents, acting on DNA or RNA at neutrality, form more phosphotriesters than the analogous methylating agents.     

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.     

Microwave-Assisted Synthesis of O′-Adamantylated Uracil-Derived Nucleosides

Abstract

Microwave-induced synthesis of O′-adamantyl derivatives of AZT, thymidine, 2′-deoxyuridine and uridine was investigated. Contrary to heterocyclus adamantylation of uracil and uridine in trifluoroacetic acid, the microwave-induced reaction provided sugar-substituted compounds.     

Reaction products from N-methyl-N-nitrosourea and deoxyribonucleic acid containing thymidine residues. Synthesis and identification of a new methylation product, O4-methyl-thymidine

1. DNA was treated with N-methyl-N-nitrosourea at pH7-8, 37 degrees C, degraded to yield 3- and 7-methylpurines and deoxyribonucleosides and the reaction products were separated by chromatography on ion-exchange resins. The following methods for identification and determination of products were used: with unlabelled N-methyl-N-nitrosourea, u.v. absorption; use of methyl-(14)C-labelled N-methyl-N-nitrosourea and use of [(14)C]thymine-labelled DNA. 2. The synthesis of O(4)-methylthymidine and its identification by u.v. and mass spectroscopy are reported. 3. 3-Methylthymidine and O(4)-methylthymidine were found as methylation products from N-methyl-N-nitrosourea with thymidine and with DNA, in relatively small yields. Unidentified products containing thymine were found in enzymic digests of N-methyl-N-nitrosourea-treated DNA, which may be phosphotriesters. 4. The possible role of formation of methylthymines in mutagenesis by N-methyl-N-nitrosourea is discussed.     

Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.     

32P-postlabeling of N-7, N2 and O6 2'-deoxyguanosine 3'-monophosphate adducts of styrene oxide

Adducts were prepared by reacting styrene oxide with 2-deoxyguanosine 3'-monophosphate (dGMP). Four isomeric N-7-, two diastereomeric N2- and three isomeric O6-adduct were isolated and characterized. The adducts were used as substrates in the 32P-postlabeling reaction. No phosphorylation products were seen with the N-7-alkylation products. One diastereomeric N2-adduct was labeled with 20% efficiency and the second with a markedly lower efficiency. Two of the three O6-adducts were labeled with 5% and the third with 10% labeling efficiency. The results suggest that large N-7-dGMP adducts are very poor substrates of T4 polynucleotide kinase. The diastereomeric products are labeled at different efficiencies indicating stereoselectivity in the kinase reaction.     

Dealkylation rates of O6-alkyldeoxyguanosine, O4-alkylthymidine and related compounds in an alkyl-transfer system

Bacterial O6-alkylguanine-DNA alkyltransferase (AGT) removes alkyl group from O6-alkylguanine and O4-alkylthymine residues in DNA, both of which are considered to be DNA damages most related to the induction of cancer and/or mutation. The repair process involves alkyl-transfer of an O-alkyl group to the active site of the enzyme, where an SH-group of cysteine residue plays the role of alkyl acceptor. In order to elucidate the chemical characteristics of substrates for this enzyme, dealkylation rates of O6-alkyldeoxyguanosine, O4-alkylthymidine and related compounds were measured using an alkyl-transfer system. Thiophenol-triethylamine system was employed as an alkyl acceptor and twenty-one O-alkyl compounds were tested. Dealkylation proceeded with pseudo first order kinetics. The half-life of O6-methyldeoxyguanosine (MedG) was 122 h and no remarkable dependence on N-9 substituents (H, CH3 and deoxyribose) was observed. A compound lacking 2-NH2 group underwent demethylation about three times faster than O6-methylguanines did, while, a compound lacking imidazole moiety underwent demethylation about 2.5 times more slowly. The half-life of O4-methylthymidine (MedT) was 38 h and no remarkable dependence on N-1 (H, CH3 and deoxyribose) and C-5 (H and CH3) substituents was observed. Deethylation proceeded much more slowly than demethylation. Substitution of selenophenol for thiophenol resulted in a 4.5 times faster MedG demethylation rate. Demethylation rates were moderately correlated with values for NMR chemical shift of CH3 group, an indicator of electron density, although the correlation curves of a series of MedG and MedT derivatives were quite different. This result suggests that some different rate-determining factors other than electron density are playing a role. These findings may be of help in resolving the details of the mechanisms of enzymic repair by bacterial and mammalian AGT.     

New UDP-GlcNAc C4 epimerase involved in the biosynthesis of 2-acetamino-2-deoxy-L-altruronic acid in the O-antigen repeating units of Plesiomonas shigelloides O17

Plesiomonas shigelloides is a ubiquitous waterborne pathogen responsible for diseases such as diarrhea and bacillary dysentery, commonly afflicting infants and children. This bacterium is endowed with an O-antigen gene cluster consisting of 10 consecutive reading frames. One of these, designated wbgU (orf3), has been overexpressed and biochemically characterized to show that it encodes a uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) C4 epimerase, only the second microbial enzyme characterized to have this activity. Epimerization is an equilibrium reaction resulting in a 70:30 ratio of UDP-GlcNAc to uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc), irrespective of the initial substrate. The K(m) values for UDP-GalNAc and UDP-GlcNAc are 131 microM and 137 microM, respectively. WbgU is also capable of converting nonacetylated derivatives but with much lower efficiency. It contains a tightly bound nicotinamide adenine dinucleotide [NAD(H)] molecule and requires no other cofactors for activity. We propose here that this enzyme catalyzes the first of the three transformations in the biosynthetic pathway of 2-acetamino-2-deoxy-L-altruronic acid, an unusual sugar present in the O-specific side chains of lipopolysaccharide of P. shigelloides O17 and its close relative Escherichia coli Sonnei.     

Reaction of N3-benzoyl-3',5'-O-(di-tert-butylsilanediyl)uridine with hindered electrophiles: intermolecular N3 to 2'-O protecting group transfer

The compound N3-benzoyl-3',5'-O-(di-tert-butylsilanediyl)uridine 2 was alkylated with various alkyl iodides in CH3CN in the presence of base. Normal 2'-O-alkylated products were obtained with methyl or benzyl iodide. If hindered alkyl iodides with beta-branching such as 2-ethylbutyl iodide were used as electrophiles under the same conditions, N3-alkyl-2'-O-benzoyl uridine derivatives were produced. This unexpected transformation is usually dormant with reactive alkylating agents, but expressed with sterically hindered, less reactive electrophiles. This unwanted reaction gives isomeric products whose spectra differ in only subtle ways from target compounds.     

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

1. The following methods for hydrolysis of methyl-(14)C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH(4) (+) form) at pH6 or 8.9, or on Dowex 50 (H(+) form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O(6)-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose-phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson-Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson-Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly through phosphotriester formation, but this mechanism is not proven.     

Studies on the metabolic activation of beta-keto nitrosamines: mechanisms of DNA methylation by N-(2-oxopropyl)-N-nitrosourea and N-nitroso-N-acetoxymethyl-N-2-oxopropylamine

At pH 7.35, N-(2-oxopropyl)-N-nitrosourea (OPNU) reacted with calf thymus DNA to yield O6-methylguanine, 7-methylguanine and 3-methyladenine. Kinetic measurements of the base catalyzed decomposition of OPNU and the extent of methylation of DNA by OPNU suggested that methylnitrosourea is not formed as an intermediate product. Diazoacetone, acetic acid and methanol were identified as products of decomposition of OPNU at pH 7.35. Reaction of OPNU with N-methylmaleimide yielded the product resulting from 1,3-dipolar cycloaddition of diazomethane. Hydrolysis of N-nitroso-N-acetoxymethyl-N-2-oxopropylamine (NAMOPA) in the presence of hog liver esterase also produced diazoacetone, acetic acid and methanol. Enzymatic hydrolysis of NAMOPA in the presence of DNA produced O6-methylguanine, 7-methylguanine and 3-methyladenine. These results suggest that OPNU undergoes base-catalyzed decomposition and NAMOPA undergoes enzymatic hydrolysis to yield the same intermediate, 2-oxopropyldiazotate. This diazotate then reacts either by protonation followed by loss of water to form diazoacetone, or by internal nucleophilic attack by the diazotate oxygen on the carbonyl carbon to form an oxadiazoline intermediate which then collapses to form acetate and the methylating agent diazomethane. These reaction schemes are used to suggest the mechanism by which N-nitroso-2-oxopropylpropylamine methylates hepatic DNA in vivo.     

Metabolism of pyrimidine deoxyribonucleosides in Neurospora crassa.

The experiments in this report involve the following series of reactions which were previously demonstrated with purified enzyme preparations from Neurospora crassa: thymidine a yields thymine ribonucleoside b yields thymine c yields 5-hydroxymethyluracil d yields 5-formyluracil e yields uracil-5-carboxylic acid f yields uracil. The evidence for some of the reactions occurring in vivo has been incomplete and for others totally lacking. In this paper intact cells of Neurospora are shown to be capable of converting the substrates of each of the reactions to the corresponding products. Studies are described which were carried out in vivo and in vitro with the pyrimidineless strains pyr-4,uc-1,uc-2 and pyr-4,uc-1,uc-3, developed by Williams and Mitchell. The results reported in the present paper indicate that (reaction a) and the uc-3 mutation affects thymine 7-hydroxylase (reactions c,d, and e). Evidence is presented for the 2'-hydroxylase reaction being the major, if not only, way by which Neurospora can initiate the conversion of thymidine to the pyrimidines of nucleic acids and for the 2'-hydroxylation of thymidine and deoxyuridine being catalyzed by the same enzyme. Deoxycytidine was shown not to be hydroxylated in intact cells but instead deaminated to deoxyuridine, which in turn was converted to uridine. Further studies with the uc-3-carrying strain showed that an enzyme other than thymine 7-hydroxylase can also convert 5-formyluracil to uracil-5-carboxylic acid.     

Interaction of adenylic acid with alkaline earth metal ions in the crystalline solid and aqueous solution. Evidence for the sugar C'2-endo/anti, C'3-endo/anti and C'4-exon/anti conformational changes

The reaction of adenosine 5'-monophosphoric acid (H2-AMP) with the alkaline earth metal ions has been investigated in aqueous solution at neutral pH. The solid salts of Mg-AMP.5H2O, Ca-AMP.6H2O, Sr-AMP.7H2O and Ba-AMP.7H2O were isolated and characterized by Fourier transform infrared, 1H-NMR spectroscopy and X-ray powder diffraction measurements. Spectroscopic and other evidence showed that the Sr-AMP.7H2O and Ba-AMP.7H2O are isomorphous, whereas the Mg-AMP.5H2O and Ca-AMP.6H2O are not similar. The Mg2+ binding is through the N-7 (inner-sphere) and the phosphate group (outer-sphere via H2O), while the Ca2+ binds to the phosphate group (inner-sphere) and to the base N-7 site (outer-sphere through H2O). The Sr2+ and Ba2+ bind to H2O molecules, H-bonding to the N-7, N-1 and the phosphate group (outer-sphere). In aqueous solution, an equilibrium between the inner- and outer-sphere metal ion bindings can be established. The sugar moiety exhibited C'2-endo/anti conformation, in the free H2-AMP acid and the magnesium salt, C'3-endo/anti in the calcium salt and unusual C'4-exo/anti, in the strontium and barium salts.     

Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3''-5'')thymidine.

1. The ethyl phosphotriester of thymidylyl(3'-5')thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates.     

Alkylation by propylene oxide of deoxyribonucleic acid, adenine, guanosine and deoxyguanylic acid

1. Propylene oxide reacts with DNA in aqueous buffer solution at about neutral pH to yield two principal products, identified as 7-(2-hydroxypropyl)guanine and 3-(2-hydroxypropyl)adenine, which hydrolyse out of the alkylated DNA at neutral pH values at 37 degrees C. 2. These products were obtained in quantity by reactions between propylene oxide and guanosine or adenine respectively. 3. The reactions between propylene oxide and adenine in acetic acid were parallel to those between dimethyl sulphate and adenine in neutral aqueous solution; the alkylated positions in adenine in order of decreasing reactivity were N-3, N-1 and N-9. A method for separating these alkyladenines is described. 4. Deoxyguanylic acid sodium salt was alkylated at N-7 by propylene oxide in neutral aqueous solution. 5. The nature of the side chain in the principal alkylation products was established by mass spectrometry, and the nature of the products is consistent with their formation by the bimolecular reaction mechanism.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 2. Lipid A, its structural variability, the linkage to the core oligosaccharide, and the biological activity of the lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1-->6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS.     

The effect of trans-[Rh(4-ethylpyridine)4Cl2]Cl x 2H2O on nucleic acid and protein syntheses in Escherichia coli

The effect of the transition metal compound trans-[Rh(4-ethylpyridine)4Cl2]Cl x 2H2O on the syntheses of DNA, RNA, and protein has been investigated for an auxotrophic bacterial strain, Escherichia coli JS-1, incapable of thymidine, uridine, and histidine syntheses. At low concentration (7.4 x 10(-6) M), this rhodium complex interferes with normal cell division and induces the formation of filaments comparable to those observed in the presence of the cis-(NH3)2PtClx antitumour agents. Once the suppressed growth rate of the filamenting cells has been taken into account, the rhodium compound is found not to alter macromolecular synthesis. Again this is consistent with similar observations made for the platinum compounds.     

Introduction of antigenic determining 2,4-dinitrophenyl residues into 4-thiouridine, N3-(3-L-amino-3-carboxypropyl) uridine and tRNA-Phe from E. coli.

The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product.