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A simple method for the etherification at the O(6)-position of silyl-protected inosine, guanosine, and 2'-deoxyguanosine is described. Typically, a THF solution of the silylated nucleoside is treated with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and Cs(2)CO(3) under a nitrogen atmosphere. Conversion to the O(6)-(benzotriazol-1-yl) ethers occurs within about 10 min for inosine, and within about 60 min for guanosine and 2'-deoxyguanosine. Then, for reaction with alcohols, the reaction mixture is evaporated and the O(6)-(benzotriazol-1-yl) ether is treated with Cs(2)CO(3) and an appropriate alcohol, at room temperature. On the other hand, for reaction with phenols, Cs(2)CO(3) and the appropriate phenol are added to the reaction mixture without evaporation, and the reaction is carried out at 70°C. Subsequently, workup, isolation, and purification lead to the requisite O(6)-alkyl or O(6)-aryl ethers in good to excellent yields.

The radical-based alkylation of 8-bromoguanosine (1a) and 8-bromo-2'-deoxyguanosine (1b) at the C8 position has been investigated in aqueous solutions. Alkyl radicals were generated by scavenging of the primary species of γ-radiolysis by the alcohol substrate. These reactions result in the efficient formation of intermolecular C-C bonds in aqueous media, by using the reactivity of α-hydroxyalkyl radicals derived from alcohols with 1a and 1b. A mechanism for the formation of C8 guanine alkylated adducts has been proposed, based on the quantification of radiation chemical yields for the disappearance of starting material and the formation of all products. Two α-hydroxyalkyl radicals are needed to form an alkylated guanine, the first one adding to C8 followed by ejection of Br(-) with formation of guanyl adduct and the second one acting as reducing agent of the guanyl adduct.

Selenium modification of nucleic acids is of great importance in X-ray crystal structure determination and functional study of nucleic acids. Herein, we describe a convenient synthesis of a new building block, the 2'-SeMe-modified guanosine (G(Se)) phosphoramidite, and report the first incorporation of the 2'-Se-G moiety into DNA. The X-ray crystal structure of the 2'-Se-modified octamer DNA (5'-GTG(Se)TACAC-3') was determined at a resolution of 1.20 A. We also found that the 2'-Se modification points to the minor groove and that the modified and native structures are virtually identical. Furthermore, we observed that the 2'-Se-G modification can significantly facilitate the crystal growth with respect to the corresponding native DNA.

We describe the synthesis of protected phosphoramidites of deoxyriboguanosine and guanosine derivatives containing a thiopropyl tether at the guanine N2 (7a,b) for site-specific crosslinking from the minor groove of either DNA or RNA to a thiol of a protein or another nucleic acid. The thiol is initially protected as a tert-butyl disulfide that is stable during oligonucleotide synthesis. While the completed oligonucleotide is still attached to the support, or after purification, the tert-butyl thiol can readily be removed or replaced by thioethylamine or 5-thio-2-nitrobenzoic acid, which have more favorable crosslinking rates.

6-S-[2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxy-6-thiogua nosine (2) was synthesized in high yield from the corresponding 6-O-mesitylenesulfonyl derivative by the reaction with 2-ethylhexyl 3-mercapto-propionate. The phosphoramidite precursor derived from 2 was successfully applied to an automated DNA synthesizer to produce 2'-deoxy-6-thioguanosine containing ODN. The results showed that 2-ethylhexyl 3-mercaptopropionate is useful as an odor less reagent and also as an S-protecting group of 2'-deoxy-6-thioguanosine.

We describe two approaches for the synthesis of 2'-C-beta-methyl-2'-deoxyguanosine (3) via 2'-radical deoxygenation. One approach starts from 1,3,5-tri-O-benzoyl-alpha-d-ribofuranose (6) and gives 3 in 11 steps with 9.7% overall yield. The second approach starts from guanosine and gives 3 in 8 steps with 23% overall yield.

5'-Amino-5'-deoxyguanosine-5'-N-phosphoramidate (GNHP) was synthesised in four steps from guanosine. Its identity was confirmed using spectroscopic and kinetic studies. A chromatographic method for the purification of this unstable compound was developed. GNHP was found to initiate T7 RNAP promoted transcriptions to afford 5'-O3P-NH-RNA, which hydrolyses readily to yield 5'-H2N-RNA that can be conjugated to activated esters.

Nucleophilic aromatic substitution of 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-6-chloro-2-fluoro-9H-purine with N-(tert-butyldimethylsilyl) [15N]phthalimide in the presence of a catalytic amount of CsF at room temperature in DMF efficiently afforded the 6-chloro-2-[15N]phthalimidopurine derivative, which was subsequently converted to the [2-15N]guanosine derivative. The 2'-deoxy[2'-15N]guanosine derivative was also efficiently synthesized through a similar procedure.

Rates of alkylation of guanosine and 2'-deoxyguanosine with o-quinone alpha-(p-anisyl)methide were measured by flash photolysis in a series of aqueous sodium hydroxide solutions and bicarbonate ion, t-butylhydrogenphosphonate ion, and biphosphate ion buffers. The data so obtained provide rate profiles for these nucleoside plus quinone methide reactions over the range pH = 7-14, which furnish guanosine and deoxyguanosine acidity constants consistent with literature information. These profiles also provide rate constants that show the reaction of o-quinone alpha-(p-anisyl)methide with guanosine and deoxyguanosine to be fairly fast processes, considerably faster than the biologically wasteful reaction of the quinone methide with water, which is the ubiquitous medium in biological systems; that makes the quinone methide a potent guonosine and deoxyguanosine alkylator.

We have previously reported the use of a 13C tag at the C2 of 15N-multilabeled purine nucleosides to distinguish the adjacent-labeled 15N atoms from those in an untagged nucleoside. We now introduce the use of an indirect tag at the C8 of 15N7-labeled purine nucleosides. This tag allows unambiguous differentiation between a pair of 15N7-labeled purines in which only one is 13C8 labeled. Although the very small C8-N7 coupling (<1 Hz) precludes its direct detection in 1D 15N spectra, 2D 1H-15N NMR experiments display the large C8-H8 coupling (>200 Hz) because H8 is coupled to both N7 and C8. The 13C8 atom is introduced by means of a ring closure of the exocyclic amino groups of a pyrimidinone using [13C]sodium ethyl xanthate. Here, we present methods for the syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues.

Recently, we found a small Ca(2+)-dependent deoxyribozyme (unmodified), d(GCCTGGCAG(1)G(2)C(3)T(4)A(5)C(6)A(7)A(8)C(9)G(10)A(11)GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA downward arrow UGCCAGGC) ( downward arrow denotes the RNA cleavage site), in the presence of Ca(2+) and developed a functional SPR sensor chip with this deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769-2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified deoxyribozyme, RNA cleavage reactions were carried out using 24 mutant deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2'-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm(5)C (5-methyl-2'-deoxycytosine), or d(P)C (5-propynyl-2'-deoxycytosine). The K(m) values (Michaelis constants) with the mutants that lacked N7 and O6 of G(1) and O6 of G(2) were 4.5 and 6.6 times that of the unmodified one, respectively. The k(cat) value (cleavage rate constant) with the mutants that lacked O6 of G(10) was 0.025 times that of the unmodified one. The results of UV melting curves, SPR kinetics, and CD spectra supported the quantitative idea that the catalytic activity of the unmodified form was achieved using Ca(2+). On the basis of these results, a preliminary model for two G(1) x A(8) and G(2) x A(7) mismatched base pairs such as G(anti) x A(anti) formed in the catalytic loop is proposed. The factor of 10 increase in the k(cat)/K(m) value of the mutant deoxyribozyme, which has C(9) substituted with d(P)C, suggests that the base stacking interaction between the substituted propynyl group in dC and the nearest-neighbor base grew stronger. Thus, substituting d(P)C for dC in the catalytic loop would be one of the best ways to increase the catalytic activity of the deoxyribozyme.

The nucleophilic addition-elimination reaction of 2',3',5'-tri-O-acetyl-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]inosine (8) with [15N]benzylamine in the presence of triethylamine afforded the N2-benzyl[2-15N]guanosine derivative (13) in a high yield, which was further converted into the N2-benzoyl[2-15N] guanosine derivative by treatment with ruthenium trichloride and tetrabutylammonium periodate. A similar sequence of reactions of 2',3',5'-tri-O-acetyl-2-fluoro-06-[2-(methylthio)ethyl]inosine (9) and the 6-chloro-2-fluoro-9-(beta-D-ribofuranosyl)-9H-purine derivative (11), which were respectively prepared from guanosine, with potassium [15N]phthalimide afforded the N2-phthaloyl [2-15N]guanosine derivative (15; 62%) and 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-6-chloro-2-[15N]phthalimido-9H-purine (17; 64%), respectively. Compounds 15 and 17 were then efficiently converted into 2',3',5'-tri-O-acetyl [2-15N]guanosine. The corresponding 2'-deoxy derivatives (16 and 18) were also synthesized through similar procedures.

Nucleophilic substitution reaction of 6-chloro-2-fluoro-9-beta-D-ribofuranosyl-9H-purine derivative, prepared from guanosine, with potassium [15N]phthalimide at 40 degrees C for 9 h in DMF, followed by hydrolysis, afforded [2-(15)N]guanosine derivative efficiently. The corresponding 2'-deoxy derivative was also synthesized through a similar procedure.

A series of O6-(alkyl/aralkyl)guanosines and 2'-deoxyguanosine analogs extended to peracetyl and N2-acetyl derivatives, potentially water soluble, was synthesized. Each was associated with N'-(2-chloroethyl)-N-[2-(methylsulfonyl)ethyl]-N'-nitrosourea for in vitro evaluation on M4Beu melanoma cells of their ability to enhance the cytotoxic effect of this chloroethylnitrosourea, which is frequently reduced by repairs performed by O6-alkylguanine-DNA-alkyltransferase. Structure-activity analysis revealed that (i) benzyl and 4-halobenzyl are the O6-substituents required to afford a significant activity, (ii) 2'-deoxyguanosine derivatives demonstrate greater potency than guanosine analogs, (iii) acetylation, especially at the N2 position, generally results in compounds with moderate ability but may prevent incorporation of such nucleosides into DNA. Accordingly, O6-(4-iodobenzyl)-N2-acetylguanosine (3b) and O6-benzylperacetyl-2'-deoxyguanosine (2a), as well as O6-benzyl-N2-acetylguanosine (1b) and O6-benzyl-N2-acetyl-2'-deoxyguanosine (2b), by far the most water soluble, exhibit a good profile for further in vivo trials by the intravenous route.

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavage site in a double-stranded oligonucleotide identical to the U5 terminal sequence of the HIV genome. Integrase cleaves the 3'-terminal dinucleotide containing the fluorophore, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit integrase activity. This assay is specific for the 3'-processing reaction. The change in fluorescence intensity is linear over time and proportional to the rate of the reaction. This assay demonstrates the potential utility of this new class of fluorophore for continuous monitoring of protein/DNA interactions.

A series of over 70 difluoropurine analogs was synthesized by varying the C-2, 6 and 8 substituents about the purine ring system. After initial in vitro and in vivo screening, testing concentrated on the 2,6-diaminopurine analog (dFdAP) and the guanosine analog (dFdG). dFDAP appears to be a prodrug for dFdG. Both compounds significantly inhibited mammary tumor growth in mice, caused a moderate inhibition in ovarian and lymphosarcoma models, and demonstrated no activity in lung and melanoma models. This is a narrower spectrum of activity than that of gemcitabine (dFdC). The antitumor activity of dFdAP in human xenografts that are refractory to standard clinical agents was comparable or superior to that of gemcitabine. However, during the preliminary toxicology testing, dFdG was associated with several deaths caused by cardiac toxicity. Therefore, although dFdG is a potentially useful oncolytic, further investigation is required.

A "one-pot reaction" has been used for simultaneous protection of amino and O6-function of deoxyguanosine. N-(4-bromobutyl) phthalimide used for O6-protection generates an alkylamino function after mild base hydrolysis. Two sequences d(GGA) and d(GGATCC) have been synthesised with all protected guanosine units. Similarly, 2-(vinyl)pyridine has also been used for O6-protection following the same strategy.

Facile aerial oxidation is a general feature of guanine ribo- and 2'-deoxyribonucleosides that are substituted at the 8-position by an aminoaryl group. In previous work, it had been suggested that two of the major oxidation products are a pair of diastereomers having a spiro structure. These were presumed to be related by a chiral difference at the spiro carbon atom. The pattern of the oxidative process involves a contraction of the pyrimidine ring. It was thought to be analogous to that suggested by other investigators for the oxidation of uric acid, but for which no really definitive evidence had been presented. We have been able now to isolate in a crystalline state one of the diastereomers produced by the aerial oxidation of 8-phenylaminoguanosine under alkaline conditions. Analysis by X-ray diffraction has now confirmed the type of spiro structure promulgated previously. These findings also imply that spiro compounds are likely to be produced during the aerial oxidation of any 8-arylaminoguanine nucleoside or 2'-deoxynucleoside. In addition, this work adds considerable weight to the results of Poje and Sokolic-Maravic who proposed that a spiro intermediate is produced during the aerial oxidation of uric acid (12,13). However, they found this compound to be unstable to base, in contrast to the arylaminoguanine oxidation products. In the course of the above work we showed that the 8-arylamino derivatives of guanosine can be converted by the Barton deoxygenation method to the corresponding 2'-deoxyribonucleosides. This makes available a number of the latter compounds, which are not easily prepared by other methods.

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.