Evidence for DNA adducts in rat liver after adminstration of N-nitrosopyrrolidine

Liver DNA isolated from rats given N-nitrosopyrrolidine contained amounts of an unidentified fluorescent adduct which were dose dependent. The adduct formed gradually over the first 12 hr after administration and was slowly removed from the DNA $\text{in vivo}$. Fluorescence and chromatographic properties of the adduct suggested the compound was a substituted guanine; also, the putative adduct was readily removed from DNA by neutral thermal hydrolysis, as are 3-alkyladenines and 7-alkylguanines. Evidence was also obtained for the formation of 7-methylguanine in liver DNA of N-nitrosopyrrolidine-treated rats; however, N-nitrosopyrrolidine was not the methyl source for this alkylated guanine.     

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.     

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III

The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.     

Synthesis of non-glycosidic 4,6'-thioether-linked disaccharides as hydrolytically stable glycomimetics

Michael addition of 1,2:3,4-di-O-isopropylidene-6-thio-alpha-D-galactose (2) to 2-propyl 6-O-acetyl-3,4-dideoxy-alpha-D-glycero-hex-3-enopyranosid-2-ulose (1) afforded, as the major diastereoisomer, 2-propyl 6-O-acetyl-3-deoxy-4-S-(6-deoxy-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranos-6-yl)-4-thio-alpha-D-threo-hexopyranosid-2-ulose (3, 91% yield). Reduction of the carbonyl group of 3, followed by O-deacetylation gave the two epimers 7 (alpha-D-lyxo) and 8 (alpha-D-xylo) in a 1:2 ratio. On removal of the protecting groups of 8 by acid hydrolysis, formation of an 1,6-anhydro bridge was observed in the 3-deoxy-4-thiohexopyranose unit (10). The free non-glycosidic thioether-linked disaccharide 3-deoxy-4-S-(6-deoxy-alpha,beta-D-galactopyranos-6-yl)-4-thio-alpha,beta-D-xylo-hexopyranose (11) was obtained by acetolysis of 10 followed by O-deacetylation. A similar sequence starting from the enone 1 and methyl 2,3,4-tri-O-benzoyl-6-thio-alpha-D-glucopyranoside (12) led successfully to 2-propyl 3-deoxy-4-S-(methyl 6-deoxy-alpha-D-glucopyranos-6-yl)-4-thio-alpha-D-lyxo-hexopyranoside (17) and its alpha-D-xylo analog (19, major product). In this synthetic route, orthogonal sets of protecting groups were employed to preserve the configuration of both reducing ends and to avoid the formation of the 1,6-anhydro ring.     

Synthesis of modified tetrasaccharide sequences of N-glycoproteins

The tetrasaccharides O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D- mannopyranosyl-(1----6)]-O-(4-deoxy-beta-D-lyxo-hexopyranosyl)-(1- ---4)-2- acetamido-2-deoxy-alpha, beta-D-glycopyranose (22) and O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl-(1----6)]-O- beta-D-talopyranosyl-(1----4)-2-acetamido-2-deoxy-alpha, beta-D- glucopyranose (37), closely related to the tetrasaccharide core structure of N-glycoproteins, were synthesized. Starting with 1,6-anhydro-2,3-di-O-isopropylidene-beta-D-mannopyranose, the glycosyl donors 3,6-di-O-acetyl-2-O-benzyl-2,4-dideoxy-alpha-D-lyxo- hexopyranosyl bromide (10) and 3,6-di-O-acetyl-2,4-di-O-benzyl-alpha-D-talopyranosyl bromide (30), were obtained in good yield. Coupling of 10 or 30 with 1,6-anhydro-2-azido-3-O-benzyl-beta-D-glucopyranose to give, respectively, the disaccharides 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2-O-benzyl-4 -deoxy- beta-D-lyxo-hexopyranosyl)-beta-D-glucopyranose and 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(3,6-di-O-acetyl-2,4-di-O-ben zyl- beta-D-talopyranosyl)-beta-D-glucopyranose was achieved with good selectivity by catalysis with silver silicate. Simultaneous glycosylation of OH-3' and OH-6' of the respective disaccharides with 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl chloride yielded tetrasaccharide derivatives, which were deblocked into the desired tetrasaccharides 22 and 37.     

Alkylation by 1,2:5,6-dianhydrogalactitol of deoxyribonucleic acid and guanosine.

DNA was alkylated in neutral solution at 37 degrees C with 1,2:5,6-dianhydrogalactitol and hydrolysed to yield two principal products, identified as 7-galactitylguanine and 1,6-dideoxy-1,6-di(guanin-7-yl)galactitol. The reaction products were separated by chromatography on Sephadex G-10 and Dowex 50 (H+ form). The two compounds were also obtained by reaction between dianhydrogalactitol and guanosine in acetic acid. The products were characterized from their u.v.-spectral data by comparison with those of the 7-alkylguanines and were also identified by mass spectrometry.     

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.     

In vivo study on alkylation site in DNA by the bifunctional dianhydrogalactitol

In vivo alkylation of Yoshida sarcoma cell DNA by ³H-labelled 1,2:5,6-dianhydrogalactitol (DAG) yielded N-7 monogalactitylguanines and 1,6-di-(guanin-7-yl)-galactitol, similar to the alkylated products obtained by in vitro reaction of DNA with dianhydrogalactitol in neutral solution. The ratio between monoalkylguanines and diguaninyl product was 2–2.5, slightly increasing with doses. Persistence of alkylated products in DNA was followed in function of time. There was no significant loss of either monoalkylated bases or diguaninyl derivative during the observation period i.e. 7–24 h after treatment. In contrast, the physical measurements of the amount of renaturable DNA showed a rapid opening of cross-links in the same period. Taking the presence of diguaninyl moiety as an indicator of cross-links in DNA, these two latter findings show an apparent contradiction which could be reconciled however by the mechanism proposed by Reid and Walker (Biochim. Biophys. Acta, 179 (1969) 179) for the removal of cross-linkage induced by HN₂. Accordingly, one arm of the cross-links is removed, probably enzymically, leaving the DNA non-renaturable, while the other arm of cross-link is still covalently attached to the DNA molecule rendering possible the detection of diguaninyl moiety in DNA at some later time. This concept for the removal of cross-links from DNA seems to be supported by our results too.     

Alkylation of DNA by melphalan in relation to immunoassay of melphalan-DNA adducts: characterization of mono-alkylated and cross-linked products from reaction of melphalan with dGMP and GMP

A product expected to result from cross-linking of guanine bases in DNA by melphalan (4-(2-(di-guanin-7-yl))ethylamino-L-phenylalanine) was obtained from hydrolysis of melphalan-treated sodium deoxyguanylate at pH7 and characterized by U.V. and mass spectra. When tested in a competitive immunoassay using an antibody specific for melphalan-alkylated DNA it showed an affinity intermediate between that of melphalan-alkylated DNA and melphalan. From this and other assays it seemed possible that the cross-linked moiety in DNA was recognised by the antibody, but that its conformation differed from that of the free base tested, sufficiently to account for the discrepancy. It seemed possible that cross-linked guanine nucleotides would provide a better model, and these were therefore isolated, characterised and tested. Products derived from cross-linking of guanylic acid moieties through N-7 and N-7, and through N-7 and phosphate, had higher affinity than the cross-linked base, approximately the same as for alkylated native DNA, but less than for alkylated denatured DNA or RNA.     

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. III. Biosynthetic pathways

The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2,3-13C]succinate or [1,4-13C]succinate was investigated by using gas chromatography-mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo. The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C4, C5 and C6 sugars without the use of fructose-1,6-bisphosphate aldolase (which has not been detected in P. denitrificans) is proposed. The 13C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo.     

Synthesis of an appropriately protected core glycotetraoside, a key intermediate for the synthesis of "bisected" complex-type glycans of a glycoprotein

A stereocontrolled synthetic route to a glycotetraoside, allyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-3, 6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O- benzyl- 2-deoxy-6-O-p-methoxy-phenyl-2-phthalimido-beta-D-glucopyranoside, an important intermediate for the synthesis of "bisected" complex type glycans of glycoproteins has been established by employing two glycosyl donors, 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate and 4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-alpha-D-mannopyranosyl bromide, and a glycosyl acceptor, allyl O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4) -3-O- benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranoside.     

Compounds similar to acyclovir. II. Synthesis of acyclic analogs of 2'-deoxynucleosides

Acyclic analogues of nucleosides, viz.9- and 3-(1,6-dihydroxy-4-oxahex-3-yl)adenine, 9-(1,6-dihydroxy-4-oxahex-3-yl)guanine, 1-(1,6-dihydroxy-4-oxahex-3-yl)cytosine and thymine, with C3'-C4' bond of the furanose ring cleaved, have been prepared by condensation of trimethylsilyl derivatives of nucleic acid bases with 1,4,6-triacetoxy-3-oxahexane in the presence of Lewis acids followed by treatment with metanolic ammonia.     

The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O(2) adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.     

Synthesis of chitotetraose and chitohexaose based on dimethylmaleoyl protection

tert-Butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside was readily transformed into the disaccharide glycosyl donor, 3,4,6-tri-O-acetyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-alpha/beta-D-glucopyranosyl trichloroacetimidate, and the disaccharide glycosyl acceptor, tert-butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside. A TMSOTf-catalysed coupling of the acceptor with the donor afforded the respective tetrasaccharide derivative, which can be transformed to chitotetraose. tert-Butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-4-O-phenoxyacetyl-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside was converted into donor 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-4-O-phenoxyacetyl-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl trichloroacetimidate. Its coupling with benzyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside, followed by dephenoxyacetylation, gave benzyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside, whose glycosylation furnished, after replacement of the DMM-group by the acetyl moiety and subsequent deprotection, chitohexaose.     

Synthesis of lacto-N-neotetraose and lacto-N-tetraose using the dimethylmaleoyl group as amino protective group.

The disaccharide donor O-[2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido - alpha,beta-D-glucopyranosyl] trichloroacetimidate (7) was prepared by reacting O-(2,3,4,6-tetra-O-acetyl- alpha-D-galactopyranosyl) trichloroacetimidate with tert-butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2- dimethylmaleoylamido-glucopyranoside to give the corresponding disaccharide 5. Deprotection of the anomeric center and then reaction with trichloroacetonitrile afforded 7. Reaction of 7 with 3'-O-unprotected benzyl (2,4,6-tri-O-benzyl-beta-D-galactopyranosyl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside (8) as acceptor afforded the desired tetrasaccharide benzyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-(3,6-di-O- benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl)-(1-->3)- (2,4,6- tri-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D- glucopyranoside. Replacement of the N-dimethylmaleoyl group by the acetyl group, O-debenzylation and finally O-deacetylation gave lacto-N-neotetraose. Similarly, reaction of O-[(2,3,4,6-tetra-O-acetyl-beta- D-galactopyranosyl)-(1-->3)-4,6-O-benzylidene-2-deoxy-2-dimethylmalei mido- alpha,beta-D-glycopyranosyl] trichloroacetimidate as donor with 8 as acceptor afforded the desired tetrasaccharide benzyl (2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-(1-->3)-(4,6-benzylidene-2-deoxy-2-dimethylmaleimid o- beta-D-glucopyranosyl)-(1-->3)-(2,4,6-tri-O-benzyl-beta-D-galactopyranos yl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside. Removal of the benzylidene group, replacement of the N-dimethylmaleoyl group by the acetyl group and then O-acetylation afforded tetrasaccharide intermediate 15, which carries only O-benzyl and O-acetyl protective groups. O-Debenzylation and O-deacetylation gave lacto-N-tetraose (1). Additionally, known tertbutyldimethylsilyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->3)-4,6-O-benzylide ne- 2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside was transformed into O-[2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)- (1-->3)-4,6-di-O-acetyl-2-deoxy-2-dimethylmaleimido-alpha,beta-D- glucopyranosyl] trichloroacetimidate as glycosyl donor, to afford with 8 as acceptor the corresponding tetrasaccharide 22, which is transformed into 15, thus giving an alternative approach to 1.     

The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo.

The sites and extent of ethyl products of neutral ethylation of HeLa cell DNA by [14-C]diethyl sulfate, [14-C]ethyl methanesulfonate, and [14-C]ethylnitrosourea have been determined in vitro and in vivo, and found to differ significantly depending on the ethylating agents. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of HeLa cell DNA in the following order: 7-ethylguanine greater than 3-ethyladenine greater than 1-ethyladenine, 7-ethyladenine greater than 3-ethylguanine, 3-ethylcytosine, O-6-ethylguanine. Ethyl bases accounted for 84-87% of the total ethyl groups associated with HeLa cell DNA. Ethylnitrosourea, in contrast, has particular affinity for the O-6 position of guanine. It ethylates the bases of HeLa cell DNA in the following order: O-6-ethylguanine, 7-ethylguanine greater than 3-ethyladenine greater than 3-ethylguanine, 3-ethylthymine greater than 1-ethyladenine, 7-ethyladenine, 3-ethylcytosine. Ethylation of the bases only accounts for 30% of the total ethylation in the case of ethylnitrosourea. The remaining 70% of the [14-C]ethyl groups, introduced in vivo and in vitro, are in the form of phosphotriesters which after perchloric acid hydrolysis are found as [14-CA1ethanol and [14-C]ethyl phosphate. In contrast, phosphotriesters amounted to only 8-20% of total ethylation in in vivo or in vitro diethyl sulfate and ethyl methanesulfonate treated HeLa cell DNA, and 25% of the total methylation in in vitro methylnitrosourea treated HeLa cell DNA. Alkylation at the N-7 and N-3 positions of purines in DNA destabilizes the glycosidic linkages. Part of 7-ethylguanine and 3-ethyladenine are found to be spontaneously released during the ethylation reaction. Incorporation of the 14-C of the alkylating agents into normal DNA bases of HeLa cells can be eliminated by performing the alkylations, in the presence of cytosine arabinoside, for 1 hr.     

Synthesis and in vivo diuretic activity of some novel pyrimidine derivatives

A series of 1,6-dihydropyrimidine-2-amine derivatives and 1,6-dihydropyrimidine-2-thiol derivatives were synthesized by the reaction of substituted 1,3-diphenylprop-2-en-1-one (chalcones) with guanidine hydrochloride and thiourea, respectively. All the synthesized compounds were in good agreement with elemental and spectral data. The synthesized compounds were screened in vivo for diuretic activity. The four compounds 2d, 2e, 3d and 3e, which showed moderate to good diuretic activity, were evaluated for their toxicity studies and none of the compounds showed any toxicity of the liver as compared with control. However, compounds 3e and 3d showed diuretic properties more than that of standard (acetazolamide) and were long acting. Overall, compound 3e, 6-(2,6-dichlorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidine-2-thiol, was found to be the most promising candidate of the series.     

Streptococcus pneumoniae type XIV polysaccharide: synthesis of a repeating branched tetrasaccharide with dioxa-type spacer-arms

β-Glycosides of 2-acetamido-2-deoxy- -glucopyranose were synthesized, using either 7-methoxycarbonyl-3,6-dioxa-1-heptanol or 8-azido-3,6-dioxa-1-octanol. Selective β-lactosylation of 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-3-O-benzyl-2-deoxy-β- -glucopyranoside with hepta-O-acetyl-lactosyl-trichloroacetimidate, followed by β-galactosylation of the secondary hydroxyl group with O-(2,3,4,6-tetra-O-acetyl-- -galactopyranosyl)trichloroacetimidate, catalytic hydrogenolysis, and O-deacetylation, gave 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-2-deoxy-4-O-β- -galactopyranosyl-6-O-(4-O-β- -galactopyranosyl-β- -glucopyranosyl)β- -glucopyranoside. Selective β-lactosylation of 8-azido-3,6-dioxaocytl 2-acetamido-3-O-benzyl-2-deoxy-β- -glucopyranoside with hepta-O-acetyl-lactosyl bromide in the presence of silver triflate, followed by condensation with 2,3,4,6-tetra-O-acetyl-- -galactopyranosyl bromide in the presence of silver triflate, catalytic hdyrogenolysis, and O-deacetylation, gave 8-azido-3,6-dioxaoctyl 2-acetamido-2-deoxy-4-O-β- -galactopyranosyl-6-O-(4-O-β- -galactopyranosyl-β- -glucopyranosyl)-β- glucopyranoside.     

Alkylation of nucleic acid components with ethylenimine and its derivatives. IV. Alkylation of homopolynucleotides and DNA]

Alkylation of homopolynucleotides and DNA by thio TEPA and monoaziridine diethyl phosphate was studied. The modification affected nucleic bases and terminal phosphate groups but not internucleotide phosphate groups. It was shown that the main center of modification in poly(A) was the N1 atom, whereas the products of N6- and N3-alkylations were formed in smaller amounts. In poly(G), the alkylation proceeded predominantly at the N7 and, insignificantly, at the N1 atom of guanine; the pyrimidine N3 atom is alkylated poorly in poly(C) and even worse in poly(U). In the case of DNA, the major alkylated sites are the guanine N7 and the adenine N3; this results in DNA denaturation and the subsequent formation of products modified at N1 and N6 of adenine, N1 of guanine, and N3 of cytosine. An increase in the pH and ionic strength of the solution as well as the DNA denaturation decrease the reaction rate, whereas ultrasonic fragmentation enhances it. Upon alkylation, melting temperatures decrease, CD and UV spectra change, and DNA luminescence appears. To separate the reaction mixtures and identify the DNA alkylation products, chemical hydrolysis, ion-exchange and reverse-phase HPLC, and UV spectroscopy were used.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.