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1,N2-Etheno-2'-deoxyguanosine (1,N2-epsilon dGuo), not previously reported as a product of chloroacetaldehyde (CAA) reaction, has been synthesized and characterized. Reaction of deoxyguanosine with CAA in dimethylformamide in the presence of K2CO3 led to preparation of pure 1,N2-epsilon dGuo with 55% yield. pKa values are 2.2 and 9.2. The anionic form of the compound exhibits weak but defined fluorescence; the intensity is similar to that of N2,3-etheno-2'-deoxyguanosine (N2,3-epsilon dGuo) at neutrality. The stability of the glycosyl bond of 1,N2-epsilon dGuo (t1/2 = 2.3 h at 37 degrees C, pH 1) is 10-fold greater than of unmodified deoxyguanosine and at least one thousand-fold greater than of isomeric N2,3-epsilon dGuo. Reaction of CAA with model polynucleotides indicates that hydrogen bonding of guanine residues in the double-stranded structures is, as expected, an important factor in the formation of 1,N2-ethenoguanine. In contrast, the formation of isomeric N2,3-ethenoguanine is relatively independent of whether the DNA is single- or double-stranded. In salmon sperm DNA, reacted with CAA at neutrality, the formation of 1,N2-ethenoguanine could be demonstrated. However, we find the efficiency of formation of this adduct in double-stranded DNA to be lower than that of all other etheno derivatives.

Reliable methods have been developed for the synthesis of the 3'-O-[(diisopropylamino) (2-cyanoethoxy)phosphino]-5'-O-(4,4'- dimethoxytrityl) derivatives of 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxo-dGuo, 1) and 2'-deoxy-7,8-dihydro-8-oxoadenosine (8-oxo-dAdo, 2), and for the efficient incorporation of the latter into oligomeric DNA. Both methods rely on the conversion of the 2'-deoxy-8-bromopurine nucleosides 3 and 10 to their corresponding 2'-deoxy-8-(benzyloxy) nucleosides 4 and 12 followed by catalytic hydrogenation to generate the 8-oxo function at the C-8 position. The preparation of the phosphoramidites 8 and 19 required for the synthesis of a series of DNA oligomers was carried out under strictly anhydrous conditions. Failure to keep the systems dry resulted in great difficulties during the purification procedures, and erratic results when DNA synthesis was attempted. In the preparation of the DNA itself, it was found to be extremely important during the ammonia deprotection step to add an antioxidant. Otherwise aerial oxidation resulted in almost complete loss of the oligomer. However, when these special conditions were followed, oligomeric DNA containing 8-oxo-dGuo and 8-oxo-dAdo residues could be prepared in excellent yield. Analysis of selected DNA oligomers by enzymatic degradation and mass spectroscopic analysis confirmed the designated sequences and compositions.

Beginning with 3-cyclopenten-1-ylamine hydrochloride, the 5'-nor derivatives of carbocyclic 2'-deoxyguanosine (2), 2'-deoxyadenosine (3), and 2,6-diaminopurine 2'-deoxyribofuranoside (4) have been prepared. These compounds were evaluated for antiviral potential versus herpes simplex virus, varicella-zoster virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, and human immunodeficiency virus and found to lack activity. Also, compounds 2-4 were virtually nontoxic toward the host (human diploid fibroblast ESM and HEL) cells. These biological properties may be due to the inability of 2-4 to be phosphorylated to the requisite nucleotide level that is likely to be necessary for biological activity by correlation to carbocyclic 2'-deoxyguanosine (1), which possesses significant antiviral properties as a result of conversion to its 5'-triphosphate derivative.

Treatment of 2-amino-6-chloro-9-(beta-D-ribofuranosyl)purine (21) with TBDMS chloride/imidazole/DMF gave a separable mixture of 5'-O, 2',5'-bis-O (22), 3',5'-bis-O (23), and 2',3',5'-tris-O-TBDMS derivatives. Oxidation of 22 and 23 with CrO3/pyridine/Ac2O, treatment of the respective ketonucleosides with methylenetriphenylphosphorane, and deprotection gave 2-amino-6-chloro-9-[3(and 2)-deoxy-3(and 2)-methylene- beta-D-erythro-pentofuranosyl]purines (28 and 37) that were converted into other 2-amino-6-substituted-purine analogues. Tubercidin was converted into 2'-deoxy-2'-methylenetubercidin (49) by an analogous route. Inactivation of S-adenosyl-L-homocysteine hydrolase by 2'- and 3'-methyleneadenosine analogues was investigated. Mechanism-based inhibition of S-adenosyl-L-homocysteine hydrolase and anticancer and antiviral activities of 2'(and 3')-deoxy-2'(and 3')-methylenenucleoside analogues are discussed.

Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh8Gua). Because of differences in experimental design, the base pairing specificity of oh8G in vivo is not completely resolved. Here, oh8dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh8Gua in vivo. The first is a reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh8Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G----T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh8dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh8Gua with A. Seventy-six of 78 mutations were A----C substitutions, and two were G----T substitutions. These assays thus illustrate mutagenic replication of oh8Gua as template causing G----T substitutions and misincorporation of oh8Gua as substrate causing A----C substitutions, both caused by oh8Gua.A mispairs.

A series of four isomeric 2'- and 6'-fluorocarbocyclic guanosine analogues have been prepared and evaluated as potential anti-herpes agents. The racemic 2' beta-fluoro isomer 2-amino-1,9-dihydro- 9-[(1 alpha, 2 alpha, 3 beta, 4 alpha)-2-fluoro-3-hydroxy-4- (hydroxymethyl)cyclopentyl]-6H-purin-6-one (11a, C-AFG) and its 2' alpha-fluoro epimer 11b plus the chiral 6' beta-fluoro isomer 2-amino-1,9-dihydro-9-[[1S-(1 alpha, 2 alpha, 3 alpha, 4 beta)]- 2-fluoro-4-hydroxy-3-(hydroxymethyl)cyclopentyl]-6H-purine-6-one (11c) and its 6' alpha-fluoro epimer 11d were prepared from their respective fluoro amino diol hydrochlorides (6a,d). For comparison, the furanosyl compound 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)guanine (17, AFG) was prepared by coupling 2-amino-6-chloropurine with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha- D-arabinofuranosyl bromide followed by base hydrolysis. The 6' alpha-fluoro derivative 11d exhibited comparable activity to that of acyclovir (ACV) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro but was greater than 30-fold more active than ACV against HSV-1 and HSV-2 in vivo in the mouse systemic model. The 2' beta-fluoro derivative (11a, C-AFG) was extremely potent in vitro against HSV-1 and HSV-2 (ID50 0.006 and 0.05 micrograms/mL) and in vivo it was greater than 2 orders of magnitude more potent than ACV against HSV-1 and 70-fold more potent against HSV-2. The 2' alpha-fluoro 11b and 6' beta-fluoro 11c isomers were much less active.

The reaction of 2'-deoxyguanosine (1), substituted hydrazines (Hy) and oxygen, in the presence of Cu(II) as catalyst, yields 8-hydroxy-2'-deoxyguanosine (2). The rate of formation of 2 was found to be dependent upon the oxidation potential of the Hy and on structural factors of the Hy. The conversion of 1 to 2 under these conditions suggested that a Haber-Weiss/Fenton type of process was involved with Hy acting as reductant. However, the dependence of the rate of the conversion of 1 to 2 upon the structure of Hy suggested that the Hy substrates studied may be more directly involved in the process than that of a reducing agent. Additional studies of this reaction system, including the oxygen consumption, radical trapping studies and direct ESR measurements suggest that the conversion of 1 to 2, under the conditions used, involves the intermediacy of complex composed of the metal catalyst, 1, Hy and oxygen. The rate data for the conversion of 1 to 2 appear to be correlated with the carcinogenicity of Hy and therefore 2 may be an important DNA adduct in the carcinogenesis of hydrazines.

Juvenile English sole were exposed intramuscularly to nitrofurantoin (NF) and the levels of 8-hydroxy-2' deoxyguanosine (8-OH-dG) in liver, kidney and blood were determined using reversed-phase HPLC with electrochemical detection. Identification and quantitation of the 8-OH-dG in the samples was accomplished by comparison with standard 8-OH-dG, which was characterized by UV spectroscopy and fast-atom bombardment mass spectrometry. The levels of hepatic 8-OH-dG increased (r2 = 0.59, P = 0.015) with the dose of NF (0.10-10 mg NF/kg fish). In kidney and blood, however, the levels of 8-OH-dG were significantly higher than controls only at the highest dose tested. The level of binding in liver ranged from 0.37 to 0.76 fmol 8-OH-dG/micrograms DNA. The levels of hepatic 8-OH-dG reached a maximum (approx. 1 fmol 8-OH-dG/micrograms DNA) between 1 and 3 days after exposure, followed by a decrease to control levels (approx. 0.25 fmol 8-OH-dG/micrograms DNA) at 5 days post-exposure. These data demonstrate the first direct evidence for the formation of oxidized DNA bases resulting from the metabolism of a nitroaromatic compound by fish.

3-methyl-2'-deoxyguanosine (1a) is obtained from 2'-deoxyguanosine by a reaction sequence involving conversion into tricyclic 1,N-2-isopropeno derivative (4-desmethyl-2'-deoxywyosine, 3a) followed by methylation which results in 2'-deoxywyosine (2a) and final removal of the 1,N-2 blocking system. Compounds 1a and 2a undergo spontaneous hydrolytic cleavage of their glycosidic bonds at pH 7, 37 degrees C, which makes them the most labile of all known nucleosides composed of structural units occurring in nature.

2'-Deoxyguanosine (3) and native DNA both give rise to exocyclic 1,N2-(1,3-propano)-2'-deoxyguanosine adducts 6 and 7 upon treatment with acrolein (1), a known mutagen, in vitro under physiological conditions. The use of synthetic deoxyoligonucleotides containing adduct 6 or 7 could shed light on the mechanism of the mutagenicity of 1 and on the nature of the structural perturbations present in DNA duplexes where they are present. Unfortunately, this is precluded by the instability of 6 and 7 to the conditions of automated DNA synthesis. We have prepared 1,N2-(1,3-propano)-2'-deoxyguanosine (PdG) (8) as a stable model for 6/7. The structure of 8 has been verified by magnetic resonance, ultraviolet spectroscopy, and mass spectrometry. This moiety has been incorporated into oligodeoxynucleotides via solid-state synthesis technology. Negative ion fast atom bombardment (FAB) mass spectrometry of the pentaoligodeoxynucleotide 5'-GT(PdG)CG-3' verified the identity and position of the modified base. The validity of 8 as a model system for the adduct pair 6/7 in structural and biological studies of DNA duplexes is discussed.

HPLC with electrochemical detection is a highly sensitive and selective method for detecting the oxidatively modified DNA residue oh8dG. By this method, the detection of oh8dG from DNA and urine offers a powerful approach for assessing in vivo oxidative damage. Application of this technique to the detection of oh8dG from DNA permits the quantitation of the steady-state levels of this oxidatively modified deoxynucleoside and overcomes the detection problems associated with the extremely low levels present in DNA. In addition, the selectivity gained by this detection method eliminates the problem of separating the signal for oh8dG from normal deoxynucleosides. The quantitation of oh8dG in urine complements the measurement of oh8dG in DNA by estimating cumulative oxidative DNA damage in the body. In addition, the urinary assay provides a noninvasive means of measuring this type of damage in laboratory animals and human populations. Thus, an individual animal or human subject may be monitored over time, possibly under various prooxidant conditions, using oh8dG as a sensitive marker for oxidative DNA damage. This analytical approach may allow one to estimate the exposure of an individual to prooxidant conditions associated with lifestyle, genetic predisposition, degenerative diseases, and environmental toxins.

To study the nature and repair of the promutagenic DNA lesion O6-methylguanine, we have synthesized 8-14C-labeled O6-methyldeoxyguanosine triphosphate and investigated the kinetics of its incorporation into the synthetic copolymers poly(dC'm6dG) and poly(dT,m6dG). Deoxy[8-14C]guanosine was methylated with ethereal diazomethane and the products were separated by high-pressure liquid chromatography. O6-Methyldeoxy[14C]guanosine was converted to the 5'-monophosphate with carrot phosphotransferase and then to the 5'-triphosphate via the phosphorimidazolidate formed by the action of N,N'-carbonyldiimidazole. Although m6dGTP was a poor substrate for Escherichia coli DNA polymerase I, copolymers could be synthesized from dCTP or dTTP and m6dGTP with terminal deoxynucleotidyl transferase. The percent of m6dG in the polymer increased linearly as the percentage of m6dGTP in the polymerization mixture was increased to 20% of the total. The percent incorporation of m6dGTP into poly(dT,m6dG) was, however, higher than into poly(dC,m6dG). Good yields of both polymers were readily obtained. The stability of O6-methyldeoxyguanosine in poly(dT,m6dG) was found to be pH dependent, and the half-life has been measured at four different pH values.

Hypoxanthine, guanine, and 6-thiopurine nucleosides of 6-deoxy-D-allofuranose have been prepared as potential antitumor agents. Thus, reaction of 6-deoxy-beta-D-allofuranosyl bromide (1) with the trimethylsilyl derivatives of hypoxanthine and guanine afforded mixtures of the 9- and the 7-substituted bases, which were separated and deblocked with ammonia to give 9-(6'-deoxy-beta-D-allofuranosyl)hypoxanthine (6), 7-(6'-deoxy-beta-D-allofuranosyl)hypoxanthine (7), 9-(6'-deoxy-beta-D-allofuranosyl)guanine (8), and 7-(6'-deoxy-beta-D-allofuranosyl)guanine (9). The two nucleosides with the purine joined at the N-9 position, namely, 6 and 8, are easily distinguished from the other two nucleosides (7 and 9), having N-7 junctions, by their NMR spectra. Reaction of 1 with the trimethylsilyl derivative of 6-chloropurine afforded 10, which upon treatment with thiourea and deblocking gave 9-(6'-deoxy-beta-D-allofuranosyl)-6-thiopurine (12). The hypoxanthine and guanine nucleosides showed no inhibition of mouse leukemia L1210 when tested in vivo, but the thiopurine nucleoside 12 showed strong inhibition of growth of L1210 both in vivo and in vitro. Compound 7 strongly inhibited purine nucleoside phosphorylase (KI = 8.8 X 10(-5) M), while compounds 8, 9, 6, and 12 were inactive.

DNA is subject to constant oxidative damage from endogenous oxidants. The oxidized DNA is continuously repaired and the oxidized bases are excreted in the urine. A simple routine analytical procedure is described for urinary 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage adduct, as an indicator of oxidative damage in humans and rodents. This adduct was purified from human urine and characterized. The described assay employs a series of solid-phase extraction steps that separate 8-hydroxy-2'-deoxyguanosine from other urinary constituents, followed by analysis by gradient reversed-phase HPLC coupled to a dual-electrode high-efficiency electrochemical detection system. Analysis of urine from three species by this method indicates that mice excrete approximately 3.3-fold more 8-hydroxy-2'-deoxyguanosine than humans (582 vs. 178 residues per cell per day), a result that supports the proposal that oxidative damage to DNA increases in proportion to species-specific basal metabolic rates.

The synthesis of a new modified etheno-2'-deoxyguanosine is reported. N2,3-Ethenodeoxy-guanosine (epsilon dGuo) is a product in double-stranded DNA treated with the carcinogen vinyl chloride in vivo or its metabolite chloroacetaldehyde in vitro. The lability of its glycosyl bond has, however, interfered with its isolation from DNA. The synthesis, starting with O6-benzyl-2'-deoxyguanosine 5'-phosphate, reacted with bromoacetaldehyde, could only be accomplished in slightly alkaline media, which prevented significant loss of the sugar. The 5'-phosphate also decreased the lability of the glycosyl bond. The resulting compound, when deprotected, was converted to N2,3-ethenodeoxyguanosine 5'-triphosphate, as well as the corresponding nucleoside. Fluorescence, UV, and 1H NMR data were consistent with the assigned structures and almost identical with those of the previously synthesized much more stable ribo analogues [Kuśmierek et al. (1987) J. Org. Chem. 52, 2374-2378]. A systematic study of the pH-dependent glycosyl bond cleavage gave a t1/2, 37 degrees C, pH 6, of approximately 3.5 h for the nucleoside and 7-10 h for the nucleotides. Comparison, under the same conditions, of stability of the glycosyl bond in poly(dG,epsilon dG-dC) showed an increased stability of 2 orders of magnitude, t1/2 = approximately 600 h. The rate of sugar loss was, in all cases, greatly decreased at higher pH's, over the range of pH 5-9. These stability data indicate that when slightly alkaline conditions can be used, studies on incorporation of epsilon dGuo into polymers for in vitro mutagenesis studies are possible.

Deoxyguanosine was reacted with trichloropropylene oxide (TCPO), epichlorohydrin and glycidol in aqueous solution and the products were isolated by high pressure liquid chromatography (HPLC). For each compound 7-alkyldeoxyguanosine was the main adduct; TPCO reacted about 3 times faster than epichlorohydrin and about 10 times faster than glycidol. The pKa's of the 7-alkyldeoxyguanosines were 6.7, 6.8 and 6.9 for the TCPO-, epichlorohydrin-, and glycidol-adducts, respectively. The respective half-lives of imidazole ring-opening were 6.3, 15 and 27 hrs (pH 8.5, 28 degrees) and those of depurination 1.9, 3.4 and 4.4 hrs (pH 5.5, 28 degrees).

A method was developed to synthesize 2'-deoxy-8,2'-methylene-cycloadenosine (1) and -cycloguanosine (2) which were new carbon-bridged cyclopurine nucleosides fixed in a high-anti torsional angle region. 3',5'-Di-O-acetyl-8-methanesulfonyl-2'-O-p-toluene-sulfonyladenosine+ ++ (3) or 2-acetamido-9- (3,5-di-O-acetyl-2-O-p-toluenesulfonyl-beta-D-ribofuranosyl)-6- ethoxy-8-methanesulfonyl-purine (9) was treated with sodium salt of ethyl malonate to give the cyclized nucleosides (4 and 10) in good yields, respectively. Subsequent decarboxylation and deblocking of 4 and 10 afforded 1 and 2 in crystalline form, respectively.

The C-8 position of deoxyguanosine (dGuo) was hydroxylated by ascorbic acid in the presence of oxygen (O2) in 0.1 M phosphate buffer (pH 6.8) at 37 degrees C. Addition of hydrogen peroxide (H2O2) remarkably enhanced this hydroxylation. The Udenfriend system [ascorbic acid, FeII, ethylenediaminetetraacetic acid (EDTA) and O2] was also effective for hydroxylation of dGuo in high yield. Guanine residues in DNA were also hydroxylated by ascorbic acid. Other reducing agents, such as hydroxylamine, hydrazine, dihydroxymaleic acid, sodium bisulfite and acetol, were also effective for the hydroxylation reaction, as were metal-EDTA complexes (FeII-, SnII-, TiIII-, CuI-EDTA). An OH radical seemed to be involved in this hydroxylation reaction in most of the above hydroxylating systems, but another reaction mechanism may also be involved, particularly when dGuo is hydroxylated by ascorbic acid alone or ascorbic acid plus H2O2. The possible biological significance of the hydroxylation of guanine residues in DNA in relation to mutagenesis and carcinogenesis is discussed.

The interaction of the mutagen 2-bromoacrolein (2BA) with DNA and thymidine was studied in vitro by reaction of [3-3H]2BA with thymidine, RNA, single-stranded DNA, and double-stranded DNA in phosphate buffer (pH 7.4). After purification of the nucleic acids, they were incubated at alkaline pH to convert any (hydroxybromo)propano(deoxy)-guanosine adducts to their dihydroxy analogues. After acid or enzymatic hydrolysis, the hydrolysates were analyzed by reversed-phase high-performance liquid chromatography. At a concentration of 1.6 mM, the fraction of 2BA that became covalently bound to DNA was 2.3% of the amount added. Only 3% of the radioactivity bound to DNA after extensive purification could be accounted for as cyclic 1,N2-(6,7-dihydroxy)-propanoguanine adducts. More 2BA became covalently bound to single-stranded DNA and RNA as compared with double-stranded DNA. However, high-performance liquid chromatographic analyses showed that formation of cyclic 1,N2-(6,7-dihydroxy)propanoguanine adducts was also a minor reaction with these macromolecules. Because these data showed that other type(s) of reaction(s) are more important in the reaction of 2BA with nucleic acids, we have investigated the reaction of 2BA with other nucleosides. It was found that 2BA reacted well with thymidine in vitro, and the major product was identified by 500 MHz 1H and 75.43 MHz 13C nuclear magnetic resonance and thermospray mass spectrometry as 3-(2"-bromo-3"-oxopropyl)thymidine. This adduct was unstable and decomposed upon storage. After enzymatic hydrolysis of [3H]2BA-modified double-stranded DNA and subsequent analysis of the hydrolysate by high-performance liquid chromatography, 22% of the covalently bound radioactivity to DNA coeluted with decomposition products of the 3-(bromooxypropyl)thymidine adduct. This indicates that reaction of 2BA with this nucleotide in DNA is a major reaction.

The N-hydroxylamine of the carcinogen 2-amino-3-methylimidazolo[4,5-f]quinoline (IQ) covalently bound to calf thymus DNA at pH 7, and the binding was 11% higher under acidic conditions (pH 5). The extent of N-hydroxy-IQ binding to single-stranded polynucleotides at neutral pH was in the following order: polyguanylic acid much greater than polyadenylic acid greater than polycytidylic acid = polyuridylic acid. The binding of the carcinogen to DNA, polyguanylic acid and polyadenylic acid at neutral pH was enhanced 6-, 4- and 2-fold respectively by the presumed in situ formation of N-acetoxy-IQ from N-hydroxy-IQ and acetic anhydride. N-(Deoxyguanosin-8-yl)-IQ was synthesized by reaction at pH 7 of N-acetoxy-IQ (formed in situ with N-hydroxy-IQ and acetic anhydride) with deoxyguanosine and the structure characterized by NMR, mass spectral and UV absorption spectral analyses. Reverse phase HPLC of enzymatically hydrolyzed DNA which had been reacted with N-hydroxy-IQ in vitro showed a major adduct which was chromatographically identical to synthetic N-(deoxyguanosin-8-yl)-IQ. In addition, N-acetoxy-IQ, generated chemically by acetic anhydride or enzymatically with mammalian acetyltransferase, formed one major adduct with DNA which was chromatographically identical to the synthetic N-(deoxyguanosin-8-yl)-IQ. The results indicate that N-hydroxy-IQ and N-acetoxy-IQ react with DNA forming primarily the N-(deoxyguanosin-8-yl)-IQ adduct.

O6-Ethylthioethyldeoxyguanosine has been synthesized from 6-chloro-3',5'-di-O-acetyldeoxyguanosine and characterized by UV, fluorescence, and mass spectrometry. High-pressure liquid chromatography studies have shown that this modified nucleoside is formed when the one-armed sulfur mustard, chloroethyl ethyl sulfide, reacts with deoxyguanosine. This result supports the hypothesis that the mutagenic effects of the sulfur mustards are caused in part by substitution of the O6-position of deoxyguanosine.

A facile synthesis of 7-beta-D-ribofuranosyl-3-deazaguanine (1) and certain 8-substituted derivatives of 1 via the sodium salt glycosylation method has been developed. Glycosylation of the sodium salt of methyl 2-chloro(or methylthio)-4(5)-cyanomethylimidazole-5(4)-carboxylate (5 and 13b) with 2,3,5-tri-O-benzoyl-D-ribofuranosyl bromide (6) gave exclusively methyl 2-chloro(or methylthio)-4-cyanomethyl-1-(2,3, 5-tri-O-benzoyl-beta-D-ribofuranosyl)imidazole-5-carboxylate (7 and 14a), respectively. Ammonolysis of 7 and 14a provided 6-amino-2-chloro(or methylthio)-3-beta-D-ribofuranosylimidazo-[4,5-c]pyridin-4(5H)-one (11 and 17), which on subsequent dehalogenation (or dethiation) gave 1. Similarly, reaction of the sodium salt of 5 and 13b with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose (8), and ammonolysis of the glycosylated imidazole precursors (9 and 16) gave 6-amino-2-chloro(or methylthio)-3-(2-deoxy-beta-D-erythro-pentofuranosyl) imidazo[4,5-c]-pyridin-4(5H)-one (10a and 15), respectively. Dehalogenation of 10a or dethiation of 15 gave 2'-deoxy-7-beta-D-ribofuranosyl-3-deazaguanine (10b). This procedure provided a direct method of obtaining 10b without the contaminating 9-glycosyl isomer 4.

The 3-(hydroxymethyl) branched homologue of 2-deoxyribofuranose was synthesized from the corresponding branched ribofuranose 2-O-(S-methyl dithiocarbonate) with tributyltin hydride in the first direct, one-step deoxygenation at C-2 of a ribofuranose. Nucleoside coupling afforded the corresponding 3'-branched 2'-deoxyribonucleosides of thioguanine. The alpha- and beta-nucleosides were equally inhibitory to growth of WI-L2 human lymphoblastoid cells, were phosphorylated and incorporated into the DNA of Mecca lymphosarcoma in mice to the same degree, and were more effective in these tests than the parent analogue alpha-2'-deoxythioguanosine. These results indicate that the hydroxy functions at the 3' and 5' positions of 2'-deoxynucleosides are interchangeable and that acceptance by the that the furanose ring oxygen and 2'-methylene are corresponding interchangeable, and that acceptance by the enzymes is improved if primary hydroxyls are provided at both the 3' and 5' positions.

A series of N2-substituted guanine derivatives was screened against mammalian thymidine kinase and the thymidine kinase encoded by type I herpes simplex virus to examine their capacity to selectivity inhibit the viral enzyme. Several bases, nucleosides, and nucleotides displayed selective activity. The mechanism of action of the most potent derivative, N2-phenyl-2'-deoxyguanosine (PhdG) was studied in detail. PhdG (a) inhibited the viral enzyme competitively with respect to the substrates thymidine and deoxycytidine, (b) was completely resistant to phosphorylation, (c) displayed limited toxicity for the HeLa cell lines employed as hosts for viral infection, and (d) selectively inhibited viral thymidine kinase function in intact cultured cells. The results indicate that the PhdG drug prototype has potential as a selective anti-herpes agent and as a novel molecular probe of the structure and function of herpes simplex thymidine kinase.

Several "sugar" modified acyclic nucleoside analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 2) were synthesized and evaluated for antiviral activity. The preparation generally involved the condensation of the acetoxymethyl ether of alcohols 6c-g and 10-12a with diacetylguanine to give adducts 7c-g and 14-16, which were then deprotected to afford analogues 9c-g and 17-19. Alternatively, alcohols 12a and 13a were converted to iodides via their tosylates 12b and 13b and then reacted with the sodium salt of guanine to afford, after deprotection, analogues 22 and 23. A crossed aldol-Cannizzaro reaction on aldehyde 27 readily afforded 28, which was deprotected to give analogue 29. An in vitro assay against HSV-1 showed that all compounds tested were less active than DHPG, though several were good substrates for the viral thymidine kinase. The more promising acyclic nucleosides 9c, 19, and 29 were evaluated in a mouse encephalitis model and proved ineffective at preventing death at a dose of 20 mg/kg.

The rodent carcinogens dimethylcarbamyl chloride (DMCC) and diethylcarbamyl chloride (DECC) react with dGuo (pH 7.0-7.5, 37 degrees C, 4 h) to form the O6-acyl derivatives 6-dimethylcarbamyloxy-2'-deoxyguanosine (6-DMC-dGuo) and 6-diethylcarbamyloxy-2'-deoxyguanosine (6-DEC-dGuo), respectively. Reaction of DMCC with dThd under identical conditions yielded 4-dimethylamino-thymidine (4-DMA-dThd). Compounds 6-DMC-dGuo and 6-DEC-dGuo undergo a nucleophilic aromatic substitution reaction with dimethylamine (DMA) to form 6-dimethylamino-2'-deoxyguanosine (6-DMA-dGuo) via displacement of the C-6 dialkylcarbamyloxy moiety. The substitution reaction did not take place when diethylamine or NH3 were substituted for DMA. The structures of the new compounds 6-DMC-dGuo, 6-DEC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were deduced from chemical analyses and syntheses, UV and nuclear magnetic resonance (NMR) spectra and electron impact, isobutane chemical ionization and source insertion isobutane chemical ionization mass spectra. It was postulated that 4-DMA-dThd was formed following reaction of the transient intermediate 4-DMC-dThd with DMA formed by hydrolysis of DMCC. Calf thymus DNA was reacted in vitro with DMCC (pH 7.0-7.5, 37 degrees C, 4 h) and the modified DNA hydrolyzed enzymatically to 2'-deoxynucleosides. Compounds 6-DMC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were identified in the hydrolysate by high-pressure liquid chromatography (HPLC). In an identical manner 6-DEC-dGuo was identified following in vitro reaction of DECC with calf thymus DNA. Compounds 6-DEC-dGuo and 6-DMC-dGuo possess novel structures with respect to the types of adducts known to be formed between carcinogens and bases in DNA. The implications of these findings with respect to chemical mutagenesis and carcinogenesis is discussed. The structural relationship between N4-dimethyl-5-methylcytosine (4-dimethylamino-Thy) formed in DNA following in vitro reaction with DMCC and 5-methylcytosine, the only modified base found in vertebrate DNA is noted.

N2-(p-n-Butylphenyl)-2'-deoxyguanosine (BuPdG) and its 5'-triphosphate (BuPdGTP), expected to be inhibitors of eukaryotic DNA polymerase alpha, have been synthesized. BuPdG was synthesized by two methods and characterized by 1H NMR and by chemical relation to guanosine. Direct synthesis involving silylated N2-(p-n-butylphenyl)guanine (BuPG) and 1-chloro-3,5-di-p-toluoyl-2-deoxyribofuranose in the presence of trimethylsilyl trifluoromethanesulfonate gave one alpha and two beta isomers of deoxyribonucleoside as determined by 1H NMR. However, NMR and UV spectra were equivocal in distinguishing between 7 and 9 isomers. The identity of the desired 9-beta-BuPdG was ultimately proved by its independent synthesis from the corresponding ribonucleoside. 1H NMR spectra of the O'-acetylated ribonucleosides of BuPG showed characteristic patterns of O'-acetylated guanosines, and their identity was proved by relating the products of the reaction of isomeric O'-acetylated 2-bromoinosines with p-n-butylaniline and with ammonia: the 2-bromoinosine which gave guanosine also gave the suspected 9-beta-ribonucleoside, BuPGr, and that which gave N7-beta-ribofuranosylguanine also gave the 7-beta isomer of BuPGr. BuPGr was transformed in a multistep procedure to give BuPdG, identical with the major beta isomer obtained by direct deoxynucleoside synthesis. The 5'-monophosphate of BuPdG was obtained by treatment of the nucleoside with phosphoryl chloride in trimethyl phosphate; the monophosphate reacted as the phosphoimidazolyl derivative with pyrophosphate to yield the 5'-triphosphate, BuPdGTP.(ABSTRACT TRUNCATED AT 250 WORDS)