N-Substitution of Carbon 8 in Guanosine and Deoxyguanosine by the Carcinogen N-Benzoyloxy-N-Methyl-4-Aminoazobenzene in Vitro'

[CANCER RESEARCH 35, 832@843,March 1975) N-Substitution of Carbon 8 in Guanosine and Deoxyguanosine by the Carcinogen N-Benzoyloxy-N-methyl-4-aminoazobenzene in Vitro'

Jen-Kun Lin,2 Bernard Schmall,3 Ian D. Sharpe, Iwao Miura, James A. Miller,4 and Elizabeth C. Miller

McArdle Laboratory for Cancer Research. University of Wisconsin Medical Center. Madison, Wisconsin 53706 EJ-K. L., I. D. S.. J. A . M.. E. C. M.J, and institute of Cancer Research [B. SI. College of Pht'sicians and Surgeons, and Department of Chemistrt' [i. M.J, Columbia Universits'. New York, New York 10032

SUMMARY aminoazobenzene was synthesized. Attempts to devise an unambiguous synthesis of N-(guanosin-8-yl)-N-methyl-4- The major reaction products of the carcinogenic electro aminoazobenzene were not successful. phile N-benzoyloxy-N-methyl-4-aminoazobenzene with guanosine or deoxyguanosine were characterized as N- INTRODUCTION (guanosin-8-yl)- and N-(deoxyguanosin-8-yl)-N-methyl-4- aminoazobenzene from the following chemical. radiochemi N-Hydroxylation has been shown to be a key metabolic cal, and spectroscopic studies: (a) the presence of equimolar step in the activation of AAFâ€•and a number of related amounts of both N-methyl-4-aminoazobenzene (MAB) and aromatic amide and amine carcinogens for carcinogenic guanosine or deoxyguanosine residues was shown by the activity (29, 3 1). Esterification of these N-hydroxy carcino 3H:14C ratios of the products from the reaction of [prime gens converts them to electrophilic reactants, and there is ring-3H]-N-benzoyloxy-N-methyl-4-am inoazobenzene with strong evidence that this form of activation occurs in vivo. [8-'4C]guanosine or [8-'4Cjdeoxyguanosine and by the Thus, the carcinogen-macromolecule derivatives from the molecular weights of the trimethylsilyl derivatives of both tissues of animals given AAF, 4-acetylaminobiphenyl, 4- products: (b) substitution of the dye residue on its amino acetylaminostilbene, and their N-hydroxy derivatives that nitrogen was indicated by the retention in the products of have been characterized can also be formed nonenzymati the 3H:'4C ratio of [CH231-I + â€˜4CH3J-N-benzoyloxy-N- cally by reactions of esters of the hydroxamic acids or methyl-4-aminoazobenzene and by the release of MAB on hydroxylamines (1, 6, 17-19, 24, 28, 29, 31, 32). Further treatment of the nucleoside-dye derivatives with strong more, sulfotransferase, acetyltransferase, and phospho alkali in air: (c) substitution of the guanine residues in transferase activities for these and other related N-hydroxy position 7 or 8 was demonstrated by loss of 3H from compounds occur in various animal tissues (2, 3, 6, 14-16, [8-3H]guanosine or [8-3H]deoxyguanosine in the formation 37, 38), and correlative studies have suggested that the of the nucleoside-dye derivatives; (d) the stability of the sulfuric acid ester of N-hydroxy-2-acetylaminofluorene is a products to mild alkali (as contrasted to the lability of major ultimate carcinogenic metabolite of this hydroxamic 7-alkylguanosines) provided strong evidence that the substi acid in rat liver (6, 7, 10, 44, 48). tution was in position 8 of the guanine residue; (e) direct While the extension of these studies to the hepatic evidence of 8-substitution came from the acid hydrolysis of carcinogen MAB was hindered by an inability to synthesize guanosinyl- and deoxyguanosinyl-N-methyl-4-aminoazo N-hydroxy-MAB, Poirier et a!. (33) partially circumvented benzene to N-(guan-8-yl)-N-methyl-4-aminoazobenzene in the problem by synthesis of its benzoic acid ester, N-ben up to 50% yield; (f) comparisons of the proton or â€˜3Cnu zoyloxy-MAB. This ester, which has been used as a model clear magnetic resonance spectra or both of N-(guan-8-yl)- for the reactive esters of N-hydroxy-MAB that might be N-methyl-4-aminoazobenzene, M A B, N-(guanosin-8-yl)- formed in vivo, was strongly hepatotoxic in preweanling rats 2-acetylam inofluorene, 2-acetylam inofluorene, guanosine, and induced sarcomas at the s.c. injection site in adult rats and 7-methylguanosine with the spectra of the guanosine (33). Further, the products of the reactions of N-ben MAB product further confirmed that substitution had oc zoyloxy-MAB with methionine and tyrosine were identical curred at position 8 ofthe guanosine residue.

The new com pou nd N-(guan-8-yl)-N-methyl-4- 5 The abbreviations used are: AAF, 2-acetylaminofluorene: MAB, N-methyl-4-aminoazobenzene; N-hydroxy-MAB, N-hydroxy-N-methyl-4- 1 This work was supported by Grants CA-07l75, CA-15785, CRTY aminoazobenzene; N -benzoyloxy- M A B. N-benzoyloxy-N-methyl-4- 5002, and CA-02332 (to Dr. I. Bernard Weinstein) of the National Cancer aminoazobenzene; N-(guanosin-8-yl)-MAB, N-guanosin-8-yl-N-methyl-4- Institute. USPHS. aminoazobenzene; N-(deoxyguanosin-8-yI)-M A B. N-(deoxyguanosin-8- 2 Present address: Department of Biochemistry. College of Medicine, yl)-N-methyl-4-aminoazobenzene: N M R, nuclear magnetic resonance: National Taiwan University. Taipei, Taiwan. AB, 4-aminoazobenzene; N-(guan-8-yI)-MAB, N-(guan-8-yl)-N-methyl 3 Present address: Department of Chemistry, Queens' College. City 4-aminoazobenzene; guanosinyl-M A B, guanosinyl-N-methyl-4-aminoazo University of New York, Flushing. N. Y. I 1367. benzene; deoxyguanosinyl-M A B. deoxyguanosinyl-N-methyl-4-aminoazo

4 To whom requests for reprints should be sent. benzene: guan-8-yl-M A B, guan-8-yl-N-methyl-4-aminoazobenzene; gua Received September 24. 1974: accepted November 22. 1974. nosin-8-yl-AA F, guanosin-8-yI-2-acetylaminofluorene.

832 CANCER RESEARCH VOL. 35

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1975 American Association for Cancer Research. N-(Guan-8-yl)-MA B Nucleosidesfrom N-Benzoyloxy-MA B to products released on degradation of the liver protein from Perkin-Elmer R-26 C-I3FT spectrometer (courtesy of Dr. rats given MAB (21, 22, 33, 36). Nucleic acid-bound John Fleming. Perkin-Elmer Corp., Norwalk, Conn.). derivatives of N,N-dimethyl-4-aminoazobenzene and MAB Chemical shifts are in ppm relative to tetramethylsilane. also occur in the livers of rats fed these carcinogens, and these macromolecule-dye products have been degraded to Thin-Layer Chromatography yield nucleoside- and nucleotide-dye products (27, 34, 35. 41 -43). In view ofthe above findings it appeared likely that Cellulose plates (0.25 mm) were prepared from MN 300 the in vivo nucleic acid-bound dye derivatives might include GF-254 cellulose (Brinkman Instruments, Inc., Westbury. the same products formed by reaction of N-benzoyloxy N. Y.). Silica gel plates on plastic were purchased from MAB with guanosine and deoxyguanosine(33). The data Eastman Kodak Co., Rochester, N. Y. (Chromagram sheet presented in the accompanying paper (23) show that this 6060); 0.25-mm silica gel plates were prepared on glass from deduction was correct. Accordingly, the characterization of Brinkman I-IF-254 silica gel. The following solvent systems the reaction products ofguanosine and deoxyguanosine with were used: Solvent A, methanol:benzene (3:7); Solvent B, N-benzoyloxy-MAB became an important step in the aqueous phase of l-butanol:l-propanol:water (4: 1:5); Sol elucidation of the nature of the interactions of metabolite(s) vent C, Solvent B:glacial acetic acid (25: 1): Solvent D, of MAB with rat liver nucleic acids in vivo. The data Solvent B:concentrated NH4OH (100:1): Solvent E, presented in this paper identif@vthese in vitro reaction methanol:n-hexane ( I :9); Solvent F, glacial acetic acid:ben products as N-(guanosin-8-yl)- and N-(deoxyguanosin-8- zene (1:99). All measurements were by volume. Because of yl)-M A B. small variations in RF from plate to plate, known samples were chromatographed at the same time as samples to be identified. Where feasible, the standard was added to the MATERIALS AND METHODS unknown prior to chromatography; in other cases the known and unknown were chromatographed in adjacent Instrumentation spots on a single chromatogram. The samples to be used for determination of spectral properties were prepared by Corrected melting points were determined from inflec chromatography on glass plates: these plates were pre tions in rapid time-temperature melting curves obtained washed by ascension of the solvent to be used for the with the Accumelt apparatus (American Instrument Co., chromatography and then air dried prior to application of Silver Spring, Md.). All pH determinations were made at the sample. room temperature with a glass electrode. The electronic spectra were determined in a Beckman DB Determination of 3H and â€˜4C spectrophotometer equipped with a Sargent SR recorder. The infrared spectra were determined in KBr (infrared â€œCand 3H were determined in Packard Tri-Carb liquid grade) in a Beckman IR-lO instrument with 0.5-inch-diame scintillation counters: the measurements were corrected for ter discs in an evacuated Beckman No. 5020 die or with background and quenching. For analysis the samples were 5-mm discs prepared in a No. 616 microdie (Carle Instru dissolved in methanol or water and mixed with the scintilla ments, Inc., Anaheim, Calif.). The latter discs were tion fluid (25). mounted in a Beckman No. 46286 beam condenser. Thin-layer chromatograms were scanned with a Packard Gas-liquid chromatography of nonpolar dyes was carried Model 7201 radiochromatogram scanner set with a 2-mm out with a Barber-Colman Model 10 chromatograph slit width, a chart rate of0.l cm/mm, and linear ranges of 0 equipped with an argon-905r detector and a 0.7- x 60-cm to 100, 1000, or 3000 cpm. glasscolumn packedwith I 1%SE-30silicone rubber on Gas Chrom S (Applied Science Laboratories, State College, Chemicals Pa.). Flash heater, detector, and column temperatures of 240Â°,250Â°,and 200â€”220Â°,respectively, and a pressure of 8 Guanosine and deoxyguanosine were products of Sigma psiof argon were used. Chemical Co. (St. Louis. Mo.), while the 8-'4C and Mass spectral analyses were carried out on a Varian 8-3H-labeled compounds were obtained from Schwarz/ CH-7 mass spectrometer equipped with a mass marker. A Mann (Orangeburg, N. Y.). direct insertion probe (Variset Corp., Madison, Wis.) was MAB was synthesized by methylation of N-formyl-4- generally used. aminoazobenzene, and hydrolysis ofthe methylated product The proton NMR spectra recorded in this paper were was performed according to the procedure of Ishikawa et a!. obtained with Varian HA-tOO, Varian HR-220, and JEOL ( I I ); the product was identical in melting point (88â€”89Â°) and P5-100 instruments. The latter was equipped with PFT-lOO infrared spectrum with MAB formed by rearrangement of for proton Fourier transform analysis. The proton NMR N-methyldiazoaminobenzene (30). spectrum of 7-methylguanosine was obtained with the â€œC-and â€œH-LabeledMAB's. [prime ring-3H]MAB was Varian A-60A instrument (courtesy of William Simon, prepared in 33% yield by adapting the procedure of Sadtler Research Laboratories, Inc., Philadelphia, Pa.). All Ishikawa et a!. ( I I). [G-ring-3H}Aniline hydrochloride (0.5 but one ofthe â€˜3CNMR spectra were recorded on the JEOL mmole, 190 mCi/mmole; New England Nuclear, Boston, P5-100 instrument with Fourier transform capability. The Mass.) and I .53 mmoles of unlabeled aniline hydrochloride â€˜3CNMR spectrum of 7-methylguanosine was taken with a were diazotized with 2. 1 mmoles of sodium nitrite in

MARCH 1975 833

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1975 American Association for Cancer Research. f-K. Li,, et a!. aqueous solution containing 2 mmoles of HCI. The diazo brown oily residue was dissolved in 2 ml of acetone and 12 nium solution, buffered with sodium acetate, was coupled ml of ethanol, 60 ml of water were added, and the yellow with 2.2 mmoles of sodium aniline-w-methyl sulfonate and suspension was left undisturbed at 4Â° for 20 hr. The allowed to stand at 3Â°for 24 hr. after which the dye was N@benzoyloxy-MAB was collected by filtration on paper hydrolyzed at 95Â°for I hr with NaOH. The resulting AB with gentle suction, and the paper and precipitate were dried was extracted with benzene and formylated in 88% formic over Drierite in a vacuum for 2 days at 4Â°.The yellow acid at 110Â°for 4 hr. After complete removal of the formic orange microcrystalline precipitate (0.l7 g. I 1% of theory) acid (including 5 chases with ethanol), the N-formyl-4- melted at 89@9lO with decomposition as noted previously aminoazobenzene was methylated with methyl iodide in (33). The ratio of the absorbance at 1750 cm â€˜(ester alkaline ethanol. After hydrolysis of the product the dyes carbonyl) to that at 1600 cm â€˜(azolinkage) was used as an were extracted into benzene and chromatographed on index of purity. N-Benzoyloxy-MAB prepared by this alumina to yield 153 mg of [prime ring-3H]MAB (m.p. procedure gave ratios of 2.1 to 2.3. More recent prepara 86-87Â°), 28 mg of [prime ring-31-I]N,N-dimethyl-4- tions in which additional filtrations and decantations were aminoazobenzene and 42 mg of [prime ring-3H]AB. used to remove tarry materials before crystallization from [methy!-14C]MAB was prepared by reaction of benzene:n-hexane (1:10) have yielded preparations with a [â€˜4CJCI-131(1.0mmole, 1.0 mCi: New England Nuclear) melting point of 95-96Â° and an absorbance ratio (1750 with 10 mmoles ofAB according to the general procedure of cm':l600 cm') of 2.7. These preparations of N-ben Terayama et a!. (39). After chromatography on alumina the zoyloxy-MAB have shown no differences in their electro [methvl-'4C]MAB (66% yield, 140 mg. m.p. 88-89Â°) had a philic reactivity or carcinogenicity from those reported in specific radioactivity of 4.5 MCi/mg. [CH231-I + 14Cl-13]- this paper or previously (33, 45). MAB, prepared in the same manner in 80% yield from a The radioactive N-benzoyloxy-MAB's were synthesized mixture of [3H]CH3I (1.3 mmoles, 100 mCi; New England from the appropriately labeled MAB's diluted with nonradi Nuclear) and [â€˜4C]CH3I (1.3 mmoles, 1.95 mCi: New oactive MAB. [prime ring-3H]-N-Benzoyloxy-MAB had a England Nuclear) and 20 mmoles of AB had specific radio specific activity of3.5 MCi/mg: [methv!-'4C]-N-benzoyloxy activities of 2.7 x 102 @zCi/mgfor 3H and 5.4 .tCi/mg for MAB had a specific activity ofO.2 MCi/mg: and [CH23H + 14C. â€˜4CH31-N-benzoyloxy-MAB had specific activities of 8.9 â€˜4C-and 3H-Labeled N-Benzoyloxy-MAB's. Scaling zCi/mg for SI-I and 0.18 @zCi/mgfor â€˜4C. down the synthesis ofN-benzoyloxy-MAB (33) from 20-g to l-g samples of MAB resulted in only very low yields of N-benzoyloxy-M A B. Yields of approximately 10%(equiva Synthesis of N-(Guan-8-yl)-MAB lent to those obtained with the original synthesis) were obtained from I g of MAB by the following procedure. All This synthesis was based on the work of WÃ¶lcke el a!. operations were performed at 0-4Â° and all solutions were (47). Acetic anhydride (0.075 ml) was added to a solution of precooled to this temperature range. The chloroform was 137 mg of 3-hydroxyguanine (8, 46) in 30 ml of spectral predried with Drierite (anhydrous CaSO4). grade dimethyl sulfoxide. The mixture was stirred for 30 Benzoyl peroxide ( I . 14 g: Eastman), sodium carbonate mm, and 168 mg of MAB in 10 ml of dimethyl sulfoxide (0.72 g), Drierite (1.16 g). and chloroform (5 ml) were were then added. The stoppered flask was stirred for 22 hr. combined in a 300-mi round-bottom flask and cooled to and the reaction mixture was then poured into 300 ml of 2-3Â°. A cold solution of MAB (I g) in 3.4 ml of chloroform water. The flask was rinsed with 10 ml of â€¢dimethyl was added all at once, and the mixture was stirred sulfoxide, and the aqueous dimethyl sulfoxide solution was magnetically for 45 mm. Sixty ml of hexane were added. washed 5 times with 100 ml of ethyl ether (to remove and the contents were left at 3-4Â°without stirring for 16 hr. nonpolar products) and extracted 4 times with 150 ml of The supernatant was decanted from the tarry residuethat l-butanol. The combined butanol extracts were washed 3 precipitated and adhered to the sides of the flask; this times with 1000 ml of water and then evaporated to dryness supernatant and 20 ml of chloroform:hexane (1:9). which at 35Â°under reduced pressure. was used to wash the tar, were filtered through paper into a NaOH (400 ml of 0.5 N solution) was added to the separatory funnel. This solution was extracted as fast as residue, and the solution was extracted 4 times with 150 ml possible with 12 ml of 2 N NC1, twice with 12 ml of l N of ethyl ether: the final extract was colorless. The aqueous NaOH, and twice with 12 ml of2 N HCI. At this point tarry phase was cooled in an ice bath and neutralized to pH 7 with material formed and adhered to the sides of the funnel. The I N HC1, and the orange precipitate that formed was organic layer was transferred to a clean separatory funnel collected, washed with water, and dried under reduced and extracted rapidly twice with 12-mi portions of saturated pressure (yield. 87 mg). sodium bicarbonate solution and twice with 12-ml portions C ,8H ,8N8O2 (guanyl-N-methyl-4-aminoazobenzene mo of water. The organic layer was dried with 5 g of anhydrous nohydrate) sodium sulfate for 10 mm, and the clear brownish-orange solution was decanted and taken to dryness in a vacuum in a Calculated: C 57.14. H 4.79, N 29.61, 0 8.46 rotary flash evaporator. For this purpose the distilling flask Found: C 56.66. H 4.72, N 29.43, 0 7.92 was rotated in an ice:water mixture, and the condensing flask was cooled in a Dry Ice:acetone bath. The reddish The values for carbon and oxygen were somewhat outside

834 CANCER RESEARCH VOL. 35

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1975 American Association for Cancer Research. N-(Guan-8-y!)-MA B Nucleosides from N-Benzoyloxy-MA B the usual tolerance limit of 0.40% (0.48 and 0.54%, respec Hydrolyses of Guanosinyl- and Deoxyguanosinyl-MAB in tively, less than theory); no suitable conditions for recrys Weak Acid tallization of the product were found. The mass spectra of the product and its trimethylsilyl derivatives [prepared in For the absorption spectra approximately 300 sg of the 0.03 ml of N,O-bis(trimethylsilyl)acetamide and 0. 1 ml major product from the reaction of guanosine with N-ben acetonitrile at 99Â°for 5 mm] gave the correct molecular zoyloxy-MAB were heated in 3 ml of 10% methanolic 0.5 N weight. The spectrum of the product showed prominent HCI at 100Â° for I hr. The major reaction product of peaks at m/e = 360 (Mt). 255 (M@ â€”C6H5N2) and 21 1 deoxyguanosine was incubated in 0.05 N HCI at 37Â°for 18 (MAB). A small peak at m/e = 402 indicated the possible hr. After neutralization the reaction mixtures were ex presence of some acetylated guanyl-N-methyl-4- tracted once with n-hexane:benzene ( 1:7) and then twice aminoazobenzene. The mass spectrum of the trimethylsilyl with 1-butanol. After being washed with water, the butanol derivatives showed ions at m/e = 432, 504, and 576, the extracts were evaporated to dryness under reduced pressure molecular weights of the expected mono-, di-, and tri(tri at approximately 40Â°, and the residues were chromato methylsilyl) derivatives. No peaks of higher mass were ob graphed with Solvent A on silica gel thin layers on glass served. plates that had been prewashed with the same solvent. In The mode of synthesis [from the work of WÃ¶lckeet a!. each case a broad major dye-containing spot was seen over (47)], the formation of a tri(trimethylsilyl) derivative, and the RF range of 0.5 to 0.7. and smaller dye-containing spots the NMR spectrum (â€œResultsand Discussionâ€•)demon were obtained at the origin, at approximately RF 0.4, and strated that the product is an 8-substituted guanine deriva near the solvent front. The dye products were protected tive. The proton NMR spectrum showed the correct from light during the above procedures. integration and there were no absorption bands indicative of For the mass spectrum analyses 200-big quantities of an acetyl group in the â€˜3CNMR spectrum. nucleoside-dye were hydrolyzed in I N HC1 at 100Â°for 15 mm. After evaporation to dryness the hydrolysate was chromatographed on a silica gel thin layer with benzene: Reaction of N-Benzoyloxy-MAB with Nucleosides methanol:concentrated N H 4OH (70:30:0.75). The narrow dye spot at [email protected] was eluted with methanol, dried, and For the reaction of N-benzoyloxy-MAB with guanosine silylated as described above for N-(guan-8-yl)-MAB. or deoxyguanosine, the dye (5 to 10 mg, 15 to 30 smoles) was dissolved in 3 to 10 ml of methanol or absolute ethanol and mixed with 10 to 25 ml of 0.05 M sodium phosphate RESULTS AND DISCUSSION buffer, pH 7.0, containing 2.5 to 10 mg ofnucleoside. After incubation at room temperature for 16 to 20 hr. most of the Products Formed on Reaction of N-Benzoyloxy-MAB with alcohol was removed under reduced pressure at 40Â°,and the Guanosine or Deoxyguanosine major share of the nonpolar dye was extracted into 20 ml of hexane. The aqueous layer was then extracted twice with As shown earlier (33), N-benzoyloxy-MAB reacts with l5-ml volumes of 1-butanol, and the combined butanol guanosine and deoxyguanosine at neutrality to yield polar extracts were taken to dryness under reduced pressure. The dye derivatives. These derivatives were extractable from residue was dissolved in 20 ml of acetone, the solution was aqueous solution with 1-butanol but not with hexane, tightly passed through a column of silica ( I .2 x 7 cm, 50 to 200 adsorbed to columns of silica from acetone solutions, and mesh; G. Frederick Smith Chemical Co., Columbus, Ohio) readily eluted with methanol. Thin-layer chromatography previously equilibrated with acetone, and the column was of the methanol eluates on silica gel thin-layer plates washed with acetone (approximately 100 ml) until the revealed 4 yellow fractions (Chart I). As estimated from the washings contained no yellow color. The dye adsorbed at absorption at 370 nm of methanol solutions of the dye the top of the column was then eluted with 60 ml of fractions, the dyes with the lowest RF's accounted for about methanol, which was concentrated under reduced pressure 85% of the butanol-soluble dyes. These fractions also and streaked on thin-layer plates of Silica HF-254 for contained â€˜4Cwhen the reaction mixtures contained [8- development with Solvent A. The major nucleoside-dye â€˜4C]guanosineor [8-'4C]deoxyguanosine. On the basis of product was eluted from the silica with methanol and their â€˜4Ccontent the yields of these guanosine- and deox rechromatographed under the same conditions 1 to 3 yguanosine-dye derivatives from the reactions of N-ben additional times. The 2nd chromatography, especially with zoyloxy-MAB and the nucleosides were 3 to 5%: these large preparations, sometimes revealed a minor yellow band values are in agreement with earlier data (33). When these which moved just ahead of the major nucleoside-dye dye- and â€œC-containing fractions were rechromatographed product: this band, which had little absorption at or below on silica thin layers with Solvents C or D, the radioactivity 260 nm, was separated from the desired product. and dye moved together. The characterization of these For isolation of sufficient nucleoside-dye product for guanosine- and deoxyguanosine-dye derivatives (hereafter NMR analyses, 12 replicate incubations, each containing 23 called guanosinyl- and deoxyguanosinyl-MAB) form the mg of N-benzoyloxy-MAB and 25 mg of guanosine, were subject of this paper. carried out in a similar manner. The product was isolated by The 2 minor dye fractions at RF's of 0.5 to 0.65 from chromatography on 0.5-mm silica gel plates. reaction mixtures of N-benzoyloxy-MAB and [8-

MARCH 1975 835

â€˜4C]guanosineor [8-'4C]deoxyguanosine also contained Solvents E and F and by gas-liquid chromatography â€˜4C.They have not been investigated further. indicated that about one-fourth of the dye was MAB. The Little or no radioactivity was present in the dye-contain rest of the dye in this fraction was not characterized. ing fraction near the top of the silica gel thin layers from reaction mixtures of N-benzoyloxy-MAB and [8- Chemical and Radiochemical Studies on Guanosinyl- and â€˜4Cjguanosine or [8- â€˜4Cjdeoxyguanosine. Analysis of these Deoxyguanosinyl-MAB dye fractions by silica gel thin-layer chromatography with

â€”500 Gross Composition and Molecular Weight. The major nucleoside-dye products from the reaction of N-benzoyloxy [8-@'C}GLt@NOSINEâ€”G MAB with guanosine or deoxyguanosine each contained equimolar amounts of dye residues and nucleoside residues, as evidenced by their content of 3H from [prime ring-3H]-N-

C.@ benzoyloxy-MAB and of â€˜4Cfrom the [8-'4C]guanine @ G-MAB nucleosides (Table 1). â€” ,:,@4, Confirmation of these results and the masses of the 2 F,000 5 10 Sâ€” w 0N-BENZOYLOXY-MAB I- residues were obtained by mass spectroscopy of the tn z methylsilylation products of the nucleoside-dyes. Silylation of approximately 30 @gof guanosinyl-MAB gave a pro duct with a molecular ion at m/e = 852 [the theoretical U) value for a penta(tnimethylsilyl)guanosinyl-N-methyl-4- aminoazobenzene]. A larger peak occurred at m/e = 780 [the theoretical value for a tetra(trimethylsilyl)guanosinyl N-methyl-4-am inoazobenzene]. Sim ilar treatment of deoxyguanosinyl-MAB yielded peaks at m/e = 764 and 692 [the theoretical values for a tetra- and a tni(tnimethyl silyl)deoxyguanosinyl-MAB]. No peaks of higher mass C-. @ @V///4V : dG :dGp,iA@ were observed in either spectrum. These results are con @ â€”N-BENZOYLOXY-MAB , â€” sistent with the presence of 1 MAB residue (mass = 210) @ b sr and 1 guanosine residue (mass = 282) or deoxyguanosine N-BENZOYLOXY-MAB CONTROL residue (mass = 266) in these nucleoside-dyes. F /1/ â€˜//, Evidenee for a MAB Residue. The guanosinyl-MAB @ ///,â€”@â€˜@ â€˜,@,â€˜â€˜â€˜ tb F obtained on reaction of guanosine with [prime ring-3H + ORIGIN CM SOLVENT â€˜4CH3]-N-benzoyloxy-MAB had the same 3H:'4C ratio as FRONT the N-benzoyloxy-MAB used for the reaction (Table 2). Chart I. Products obtained by reaction of [8-'4Cjguanosine or of Additionally, reaction of guanosine or deoxyguanosine with [8-'4C]deoxyguanosine with N-benzoyloxy-MAB at pH 7. The butanol [CH23H + â€˜4CH3]N-benzoyloxy-MAByieldedproducts in soluble material from the reaction mixtures was adsorbed to and eluted which the 3H:'4C ratio was very similar to that of the from silica columns and then chromatographed on silica gel thin layers N-benzoyloxy-MAB (Table 2). Thus, each of the nucleo with Solvent A. The graphs show the radioactivity scans of the thin layer plates depicted below each graph. The yellow dye-containing spots are side-dye derivatives contained a MAB residue with an intact indicated by hatching on the chromatograms. The positions occupied by N-methyl group. guanosine (G), deoxyguanosine (dG), guanosinyl-MAB (G-MAB), and Furthermore, hydrolysis at 100Â°for 12hr in 6 N NaOH in deoxyguanosinyl-MAB (dG-MAB) are indicated by dotted and solid lines. air liberated MAB in approximately 34% yield from the respectively. guanosine derivative and in approximately 10% yield from

Table I The ratios of MA B residues to nucleoside residues in the major products formed by reaction of N-benzoyloxy-MA B with guanosine or deoxyguanosine [prime ring-3H]-N-Benzoyloxy-MAB (16.6 @moIes;specific activity, 1.49 @Ci/@mole)in 5 ml of methanol was allowed to react with a solution of [8-'4C]guanosine (19. 1Mmoles; 0. 15 MCi/zmole) or of [8-'4C]deoxyguanosine (17.6 Mmoles; 0.22 gzCi/zmole) in 10 ml of phosphate buffer, pH 7.0, at room temperature for 20 hr. The reaction mixtures were worked up as described in â€œMaterials and Methodsâ€• and guanosinyl- and deoxyguanosinyl-MAB were isolated first by thin-layer chromatog raphy on Silica Gel HF-254 with Solvent A and then by chromatography on cellulose thin layers with Solvent D. The moles of MAB and of nucleoside residues were calculated from the specific activities of each of the reactants. residues: MABresidues residues nucleoside residuesGuanosinyl-MAB1.60 (Mmole)Nucleoside (@mole)MAB

l021.02Deoxyguanosinyl-MAB5.01 x 1O21.56 x x [email protected] x [email protected]

836 CANCER RESEARCH VOL. 35

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1975 American Association for Cancer Research. N-(Guan-8-yT)-MA B Nuc!eosides from N-Benzoyloxy-MA B the deoxyguanosine derivative (Table 3). The alkaline indicative of either N-7 or C-8 substitution of the guanine degradation to MAB was apparently oxidative in nature, residue (40). since much less MAB (only up to 5% oftheory) was formed These results are at variance with analogous data from an when the hydrolyses were carried out in evacuated sealed earlier study in this laboratory (33). Repetition and reex tubes. Guanosinyl-M A B, deoxyguanosinyl-M A B, and amination of the latter experiments with the labeled guano MAB also yielded some AB with this aerobic alkaline sines indicated that the erroneous conclusion that 3H in treatment. position 8 of guanosine was.not removed on reaction with Unlike the report by Warwick (42) that 50% of the dye N-benzoyloxy-MAB was due to contamination of the polar from N-benzoyloxy-MAB-treated RNA was released as dye product with non-dye nucleoside products (benzoylated MAB by acid hydrolysis, hydrolysis of guanosinyl-MAB at nucleosides?6) in the thin-layer chromatography on cellulose 100Â°for I hr in I N I-ICl yielded only 5% of the theoretical with l-butanol:acetic acid:water (50:11:25) and to differ amount of MAB (Table 3). Only a very small amount of AB ences in the relative efficiencies of determining 3H and â€˜4C was formed in this acid hydrolysis. in guanosine when it is in solution or adsorbed on cellulose. The dyes from the acid and alkaline degradations were When the major nucleoside-dye derivative was adequately shown to be identical with authentic MAB and AB by separated from other products and when the products were coincidence of the retention times at 3 column temperatures assayed for radioactivity in the absence of adsorbant, there upon gas-liquid chromatography and by thin-layer chroma was no significant content of 3H in the nucleoside-dye tography on silica gel sheets (Eastman 6060) with Solvent E products. (RF's of 0.44 and 0.23 for MAB and AB, respectively) and Stability of the Purine Rings to Mild Alkali. Guanine with Solvent F (RF's of 0.58 and 0.23 for MAB and AB, nucleosides that contain a substituent in postion 7 are respectively). M A B, isolated by thin-layer chromatography. readily hydrolyzed by mild alkali at the 8,9-bond to yield the was also identified by mass spectral analysis. corresponding 5-formylaminopyrimidine derivatives (20). These data thus exclude the methyl group of the MAB Thus, at pH 8.7 and 30Â°the absorbance at 272 nm of a residue as a site of attachment of the dye residues to the 7-methylguanosine solution nearly doubled within 2.5 hr. nucleoside residues. The release of MAB from the nucleo Under the same conditions no change was observed in the side-dyes by hydrolysis shows that the MAB residue is spectrum of the guanosinyl- or deoxyguanosinyl-MAB attached to the nucleosides through its amino nitrogen. solutions at 2.5 or 20 hr (Table 5). These data thus provide Absence of 3H in Products Derived from [8-3H]Guanosine strong evidence that the guanine rings in these derivatives or [8-3H]Deoxyguanosine. On reaction of N-benzoyloxy were not substituted in position 7. MAB with a mixture of [8-3H}guanosine and [8-14C]guano Formation of N-(Guan-8-yl)-MAB by Acid Hydrolysis of sine or with the corresponding deoxyguanosine derivatives, Guanosinyl- and Deoxyguanosinyl-MAB. Hydrolysis of the 3H:'4C ratios of the major polar dyes were only 3 to 6% either guanosinyl- or deoxyguanosinyl-MAB in hot acid those of the nucleosides in the reaction mixtures (Table 4). under conditions that would be expected to cleave the The reaction thus led to removal of tritium from position 8 glycosidic bond liberated a dye-containing product that had ofguanosinyl- and deoxyguanosinyl-MAB, a result strongly the same RF on silica gel thin layers in methanol:benzene or benzene:methanol:NH4OH as did N-(guan-8-yl)-MAB (see â€œMaterialsandMethodsâ€•).Yields of up to 50% of product Table 2 were obtained. The electronic absorption spectrum in Ratios of3H to â€˜4Cinthe guanosinvl- and deoxvguanosinvl-MA B obtained methanol of this product was very similar to that of by reaction ofnucleosides with [CH23H + â€˜4CH3]-N-benzovloxs'-MA B or with [prime ring-3H + â€˜4CH3]-N-ben:ovloxv-MA B N-(guan-8-yl)-MAB (Chart 2). The infrared spectrum of [CH23H+ â€˜4CH3JN-Benzoyloxy-MAB(ISj.@moles:specificactivities, this product in KBr also closely matched that of synthetic 2.93 @tCi/.tmoIe for 3H and 0.058 @Ci/@zmo1eforâ€˜4C)in6 ml of methanol N-(guan-8-yl)-MAB chromatographed on silica gel in was incubated with a solution of 35 @moIesofguanosine or deoxyguanosine methanol:benzene. In each spectrum absorption maxima in 20 ml of 0.05 @iphosphate buffer, pH 7.0. [prime ring-3H + were noted at 2900(w), 2840(w), 1740(w), 1600(w), 1560(s), â€˜4CH3)-N-Benzoyloxy-MAB ( 18 @zmoIes;specific activities. 5.6 MCi/Mmole for 3H and 0.20 @zCi/@moIefor L4C)in 5 ml ofmethanol was incubated with 1380(s), 1260(w), 1120(m), 1020(w), 920(w), 820(w), a solution of 21 pmoles ofguanosine in 10 ml of0.05 M phosphate buffer, 750(m), and 610(w) cm â€˜.In the mass spectra of the pH 7.0. After 20-hr incubations, the nucleoside-dye derivatives were tnimethylsilyl derivatives of this product ions were noted at chromatographed first on thin layers ofSilica Gel HF-254 with Solvent A m/e = 432 [molecular weight of the mono(tnimethylsilyl) and then on cellulose thin layers with Solvent D. derivative of N-(guan-8-yl)-M A B], 504 [di(tnimethylsilyl) 3H:CH23H derivative], and 576 [tri(tnimethylsily) derivative]. No peaks of higher mass were observed. Thus the properties of the +prime ring â€˜4CH3N-Benzoyloxy-MABCompound assayed for radioactivityâ€˜4CH33H + 6 The suggestion of benzoylated nucleosides as reaction products of reaction5028Guanosinyl-MAused in N-benzoyloxy-MAB with guanosine is by analogy to the observations that BFrom N-acetoxy-4-acetylaminobiphenyl. N-acetoxy-2-acetylam inophenan layer4928Fromsilica thin threne. and N-acetoxy-4-acetylam inoazobenzene acetylate guanosine (Ref. layer49Deoxyguanosinyl-MABFromcellulosethin 31: E. C. Miller and J. A. Miller. unpublished data). These findings are based on the formation of a product that is less polar than guanosine. layer49Fromsilica thin retains tritium from [8-3Hjguanosine and 14C from the N-14C-acetoxy cellulose thin layer48 labeled amides. and has an electronic absorption spectrum very similar to that of guanosine.

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Table 3 Release of MA B and A B on alkaline or acid degradation ofguanosinvl- and deoxvguanosinyl-MA B The major nucleoside-dye derivative from the reaction of guanosine or deoxyguanosine with N-benzoyloxy MAB was isolatedfrom silicagel thin layersdevelopedwith Solvent A. For the alkalinedegradation the dye @ (60 to 80 in 0.05 ml of methanol) was added to 4 ml of 6 N NaOH, and the solution was heated under reflux in air for 12 hr at 1000. The reaction mixture was extracted with benzene:hexane (1:1). For the acid degrada tion the dye (70 @zg)in4 ml of I N HCI was heated under reflux at l000 for I hr. The solution was then made alkaline and extracted with benzene:hexane (1:1). The extracts were analyzed for MAB and AB by gas chro matography.

degradationMAB(%)AB(%)MAB(%)AB(%)Guanosinyl-MAB34Â±degradationAcid

CompounddegradedAlkaline

2.0(3)0.5(3)Deoxyguanosinyl-MAB9.6 8.4Â° (IO)b16 Â±3.4(10)5.3 Â± (6)MAB94Â± Â± 0.7 (6)5.8 Â±1.1 10 (4)7.0 Â±2.5 (4)88, 95 (2)0.5 (2)

, Average Â±S.D. h Numbers in parentheses, number of samples.

(- Results for individual samples.

Table4 Ratio of @Hto â€œCin the major the derivatives obtained by reaction of I.0 N-IGUAN-8-YL)-MAB @ [8-3H. 4Cjguanosine. or 3 4C)deoxvguanosine with

iV-ben:ovloxv-MA B L*J 0 GUANOSINYL-MAB N-Benzoyloxy-MAB (30 @zmoles)in 10 ml of methanol was incubated z 4 @0 for 20 hr at room temperature with 20 ml of phosphate buffer. pH 7.0, ... .â€˜, .__â€¢\ HYDROLYSIS which contained 15 Mmoles of [8-3H,'4C]guanosine or [8-3H,14C)deox go 5 I â€˜..PRODUCTOF .... \, /@ .â€˜ GUANOSINYL-MAB yguanosine. The reaction mixtures were worked up as described in 4 â€œMaterials and Methods.â€• and the nucleoside-dyes were isolated first by @ chromatography on Silica Gel HF-254 thin layers with Solvent A and then HYDROLYSISPRO CT OF DEOXYGUANOSINYL-MAB@' @ by chromatography on cellulose thin layers with Solvent D. I I I _I_@_I _1@_ I I _L I zbo 5W 340 @() â€˜I@U â€˜3W @JU Deoxy WAVELENGTH,NM Guanosinyl-MABguanosinyl Chart 2. UV absorption of dark-equilibrated solutions of N-(guan-8- MAB yl)-MAB and guanosinyl-MAB(each 0.036 M)in methanol. The lower 2 Experi Experi (Experi curves are for acid hydrolysis products obtained from guanosinyl- and 3)Nucleoside ment Iment 2ment deoxyguanosinyl-M AB, respectively.

reactant7.63935Eluate Comparative Analysis of the Proton NMR Spectra of productFromof nucleoside-dye I5.03.5Fromsilica thin layer2. Guanosinyl-MAB and Related Compounds cellulose thin layer0.21 .22.1 Substitution ofthe MAB residue at C-8 ofguanosine was further indicated by a comparative analysis of the proton Table 5 NMR (100 and 220 MHz) spectra of MAB, guanosine, The relative stabilities of 7-methvlguanosine and guanosinyl- and guanosinyl-MA B, guan-8-yl-M A B, and N-(guanosin-8- deoxvguanosinvl-MA B to mild alkali yl)-2-acetylaminofluorene (Chart 3). Solutions of 7-methylguanosine (0.034 mg/mI) and of the guanosinyl In the spectrum of MAB the 3 methyl protons appeared MAB (0.14 mg/mI) or deoxyguanosinyl-MAB(0.18mg/ml) were incu as a doublet at 2.79 Ã´and the aromatic protons were seen as bated in 30% methanol in 0. 1 M potassium phosphate buffer, pH 8.7, at 300. Spectrawere determinedat0,2.5,and 20 hr. multiplets centered at 7.73, 7.48, and 6.67 t5, which inte grated for 4, 3, and 3 protons, respectively. On exchange Relativeindicatedwavelength7-Methyl absorptionat the with D2O the multiplet at 6.67 Ã´was reduced to a 2-proton doublet. This multiplet thus contains the amino proton and Guano Deoxygua 2 aromatic protons. guanosinesinyl-MABnosinyl-MABTime nm)0((hr)(272 nm)(262 nm)(262 The N- I proton of guanosine appeared as a broad absorption band at 10.71 @,the C-8 proton appeared as a .00)2.51.920.961.14202.220.971.07I.00)( I.00)( I sharp singlet at 7.91 Ã´,and the 2-amino protons appeared as a broad absorption band at 6.42 tI. The ribosyl C- Iâ€˜proton is seen as a doublet centered at 5.70 @5.Theassignments of the other ribosyl protons are those of Gatlin and Davis (9), acid hydrolysis product obtained from guanosinyl- and and the chemical shift values are in good agreement. deoxyguanosinyl-MAB show that it is N-(guan-8-yl)-MAB. However, the shift values for the other protons in guanosine Hence these dye-nucleosides contain guanine residues sub agree better with the data of Katz and Penman ( I 3) than stituted at position 8 and MAB residues substituted on the with those of Gatlin and Davis (9). amino nitrogen. In the spectrum of guan-8-yl-MAB the N-9 and N-l

838 CANCER RESEARCH VOL. 35

protons of guanine appear as broad absorptions at 12.06 the spectrum of guanosinyl-MAB could not be detected and 10.61 Ã´,respectively, and each integrated for 1 proton. because of the lower amount of compound available. In the The aromatic protons of MAB are seen as 4- and 5-proton spectrum of guan-8-yl-MAB the N-I proton was clearly multiplets centered at 7.84 and 7.55 5. The guanine amino visible at 10.61 Ã´.The nibosyl C-l' proton of guanosinyl group appears as a 2-proton broad singlet at 6.23 Ã´.A sharp MAB wasseenas a doublet at 5.48Ã´.Interferenceby water singlet at 3.48 a is attributable to the N-methyl group. and the low concentration ofcompound made assignment of In the spectra of guanosinyl-MAB the methyl protons the remaining nibosyl protons in the guanosinyl-MAB appeared as a sharp singlet at 3.30 Ã´.In the spectra obtained spectra difficult to accomplish. The amino protons of these protons could not be integrated due to interference by guanosine appeared as a broad absorption at 6.65 Ã´.which a broad absorption band from a trace of water in the di disappeared after exchange with D2O. methyl sulfoxide. The absorption of the N-I proton was The most notable feature in the 100- and 220-MHz not observed, but this is not surprising. In the spectrum of spectra of guanosinyl-MAB was the lack of a sharp singlet @ guanosin-8-yl-AAF the N-l proton exhibited a very broad near 8 which could be attributed to a C-8 proton, as seen in absorption band at 10.95 Ã´that was barely visible. It is the spectrum ofguanosine. Such a signal was also lacking in probable that the absorption of the corresponding proton in the spectra of guanosin-8-yl-AAF and guan-8-yl-MAB. In the spectrum of an equimolar mixture of guanosine and MAB (not shown), the guanosineC-8 proton was seenas a sharp singlet at 7.93 Ã´downfield from the MAB aromatic multiplet of protons at 7.73 Ã´. The doublet at 6.88 Ã´was assigned to 2 of the aromatic protons of the MAB residue. The coupling constant, J = Ca. 9, of this doublet was identical to that of the 2-proton doublet at 6.67 Ã´in the spectrum of MAB after exchange with D20 (see above). The remaining 7 aromatic protons of the MAB residue were assigned to the multiplets centered at 7.84 and 7.54 Ã´.However, relative to the C- I' proton, these multiplets together integrated for more than 7 protons. All of the preparations of guanosinyl-MAB gave spectra that integrated for about 1 to 2 protons more than required in this region. The base of the aromatic region was broad and remained so after exchange with D20. Relative to the 2 protons at 6.88 Ã´,integration of the multiplets at 7.54 and 7.84 Ã´was not altered after exchange with D2O. The extra absorption in this region may be due to impurities contain ing nonlabile protons. The above observations make it unlikely that a C-8 proton, missing as noted above from the region near 8 Ã´, could be present in these 2 multiplets. A C-8 proton ex changeable with D20, if present, would have indicated N-7 substitution by the MAB residue since Tomasz (40) has shown the C-8 proton in 7-methylguanosine to exchange immediately with water. Tomasz attributed the extreme lability of the C-8 proton to the inner-salt structure of 7-methylguanosine. Furthermore. the C-8 proton of 7- methylguanosine appears as a singlet at 9.46 c5(spectrum not shown). Substitution at position 7 of guanosine with formation of an inner-salt structure thus causes a downfield shift of the resonance of the C-8 proton. A similar situation occurs in the spectrum of 7-methylguanosine iodide (not shown), where the C-8 proton was observed at 9. 19t5.In the spectrum of guanosinyl-MAB there was a total absence of any absorption in this region. Thus, in sum, a comparative analysis of the NMR proton spectra discussed above provided no evidence for the presence of a C-8 proton in guanosinyl-MA B. Comparative Analysis of the â€˜@CNMRSpectra of Guano Chart 3. The proton NMR spectra in dimethyl sulfoxide-d1 (DMSO sinyl-MAB and Related Compounds d,) of MAB. guanosine, N-(guanosin-8-yl)-MAB. N-(guan-8-yl)-MAB, and N-(guanosin-8-yl)-AAF. The numbers designate the chemical shifts in Further evidence for the substitution of the MAB residue aunitsorppmrelativetotetramethylsilane. at C-8 of the guanosine residue in guanosinyl-MAB was

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provided by â€˜3CNMR analysis. The chemical shifts and assignments for the latter 2 compounds are known (12). The spectral correlations for MAB, guan-8-yl-MAB, guano 3 bands farthest downfield in the MAB spectrum were sinyl-MA B, guanosine, 7-methylguanosine, guanosin-8-yl assigned to the 3 nitrogen-substituted aromatic carbons. AAF, and AAF are shown in Chart 4. The other 5 bands accounted for the remaining 9 ring The assignments for the various carbon atoms of MAB carbon atoms. are tentative and are based on correlations between the The assignmentsfor guanosineare those listed by John spectra of MAB, azobenzene, and N-methylaniline. The son and Jankowski ( 12) and Mantsch and Smith (26). The 5 bands downfield were assigned to carbon atoms in the 150 Inn 50 guanine residue. The nibose carbon atoms gave rise to the 5 IIIIIII T I I R= bands farther upfield. The 3 bands between 150and 160Ã´in the spectrum of ,@ OH OH@ guanosin-8-yl-AAF can be assigned to carbon atoms 2, 4, Iâ€”, b ,r@q c and 6 of the guanosine residue. Six bands were seen between 138and 144t5,and7 bandswere seenbetween I15and 128 I N-METHYL+AMIN@ZOOENZENE t5. In the spectrum of AAF 5 bands and 7 bands were seen (MAB) 6@fb in approximately these 2 regions, respectively, and these @ 1' bands thus represent the 12 ring carbon atoms of AAF. In the spectrum of guanosin-8-yl-AAF I of the 6 bands be tween 138 and 144 6 can therefore be assigned to carbon 8 N-(GUAN -8-YL)- MAB @ 6248,fb I'd @:s' of guanosine. Relative to guanosine there has thus been a downfield shift of the carbon 8 absorption from 135.5 Ã´to T'H2:Ã´@ksome position between 138 and 144. Thus a downfield shift of the carbon 8 absorption from 135.5 Ã¶would be expected N-(GUANOSIN-8-YL)-MAB 0 in the spectrum ofguanosinyl-MAB ifthe amino nitrogen of MAB replaced the C-8 hydrogen in the guanine residue. The absenceof a band in the region near 135 Ã´in the iiiI I iiTi1 spectrum of guanosinyl-MAB was therefore indicative of GUANOSINE 00'CH3 substitution by nitrogen on carbon 8. No absorption at all 1 N @â€˜..a was observed in the region 131 to 144 t5.The possibility that 2 @ H@N@'N substitution occurred at another position in the molecule 111 1[1'III R and that the carbon 8 absorption shifted upfield is unlikely, 7-METHYL-GUANOSINE ru +mâ€”,+5 since the spectral patterns in the aromatic regions of / f.L@' I I@4@ guanosinyl-MAB and guan-8-yl-MAB were very similar. In iiâ€˜@9@the upfield region ( I I 3 to 131 t5)there were 6 bands in each @@@ II ll@@ II N-(GUANOSIN- 8-YL)-AAF spectrum. Five of these bands accounted for 9 of the ring carbon atoms ofthe MAB residue. as was the case for MAB (see above), the 6th being the carbon 5 absorption of guanosine. Each spectrum had 4 downfield absorptions @ I I@ I @I@I1i@â€¢11 !@â€˜ I 2 -ACETYLAMINOFLUORENE I I I I (AAF) between 149 and 155 Ã´.Band assignments in this region are 50 00 50 0(TMS) not as clear as in the case of guanosin-8-yl-AAF. Carbon atoms 2, 4, 6, and 8 of the guanyl residue and the 3 carbon PPM atoms adjacent to the nitrogen atoms of MAB all absorb in Chart 4. A correlation chart showing the observed chemical shifts in this region, and the chemical shifts of some of these carbon the 13C NMR spectra in dimethyl sulfoxide-d, (DMSO-d6) of MAB. atoms must be identical. N-(guan-8-yI)-MA B, N-(guanosin-8-yl)-MAB. guanosine. 7-methylguano The spectrum of 7-methylguanosine is unambiguous and sine. N-(guanosin-8-yI)-AAF. and AAF. The chemical shifts are given in b shows that the carbon 8 absorption is not materially shifted units or ppm relative to tetramethylsilane (TMS). Dashed lines. probable locations of the methyl carbon absorptions (a) of N-(guan-8-yl)-MAB and from the position of that in guanosine. The absorptions of N-(guanosin-8-yl)-MAB: these absorptions were masked by the absorp carbon atoms 2, 4, and 6 and the absorption of carbon 5 tions of the methyl carbon atoms in the solvent in the relatively dilute showed shifts downfield and upfield, respectively, in the solutions of these poorly soluble compounds. The digital instrument data inner salt. thatare plottedareas follows:MAB. 29.3.111.2.121.6,125.0,129.0, The assignments of the various carbon atoms of the 129.2. 142.7, 152.4. 152.9: N-(guan-8-yl)-MAB. I 14.6. I 19.3, 122.1, 123.5, nibose residue in guanosinyl-MAB are based on the spec 129.2, 130.6, 146.3. 148.0, 151.9. 152.8: N-(guanosin-8-vl)-MAB, 61.97, trum ofguanosine. Relative to guanosine the 2'-band shifted 70.60,85.76,87.68,113.8,114.2,121.9,23.9.129.1,130.1,144.7,144.9, upfield and was coincident with the band for carbon 3'. An 150.1. 153.9: guanosine. 61.33. 70.29. 73.60. 85.12. 86.29. I 16.5, 135.5, analogous situation occurred in the guanosin-8-yl-AAF 151.2, 153.5, 156.7: 7-methylguanosine. 35.5. 61.0, 70.2, 74.4, 86.0, 88.9. spectrum. The methyl carbon absorptions of guanosinyl 108.0. 134.2. 161.2. 162.2. 164.4: N-(guanosin-8-@l)-AAF. 22.49, 36.35, 61.93. 70.73, 86.24. 87.73. 88.00. 115.1, 120.0. 122.8, 124.4, 124.9. 126.3. MAB and guan-8-yl-MAB did not appear as distinct bands 126.6,138.3,139.7.140.0.141.1,143.1,143.6.150.2,153.5.156.1,170.6: in the spectra and were presumably masked by the strong AAF. 24.1. 36.5. I 15.8. I 17.8. I 19.2. 119.9. 124.8. 125.8, 126.5. 136.1. absorption of the methyl carbon atoms of the solvent. 138.4. 141.0, 142.6. 143.6, 168.1. dimethyl sulfoxide-d6. The aforementioned labeling experi

840 CANCER RESEARCH VOL. 35

ments, however, showed the presence of an intact N-methyl Attempts to Synthesize N-(Guanosin-8-yl)-MAB group in guanosinyl-MAB. Considerable effort was devoted to devising an unambigu ous synthesis for N-(guanosin-8-yl)-MAB, so that the UV Absorption Spectra of Guanosinyl-, Deoxyguanosinyl-, identity of the guanosinyl-MAB from the reaction of and N-(Guan-8-yl)-MAB N-benzoyloxy-MAB and guanosine could be established by direct comparison. The major approach was to condense an The approximate molar absorbances of guanosinyl- and 8-halogenated guanosine directly with MAB. Thus, at deoxyguanosinyl-MAB were calculated from the electronic tempts were made to cause both 8-bromo- and 8-iodoguano absorption spectra and the radioactivity of the nucleoside sine to react with MAB in the presence or absence of base dye products isolated by thin-layer chromatography from under a variety of experimental conditions. Efforts were reaction mixtures that contained [8-'4C]guanosine, [8- also made to condense the N-sodio derivative of MAB, â€˜4C]deoxyguanosine, or [prime ring-3HJ-N-benzoyloxy prepared by reaction with sodium hydride in dimethyl MAB of known specific activity, while those for N-(guan-8- sulfoxide, with the halogenated guanosines. Direct reaction yl)-MAB were determined from the spectra of known of 8-oxoguanosine with MAB in an enamine-type of weights of compound. In 88% formic acid, which was used reaction and condensation of N-methylaniline with 8- because of the pool solubility of these compounds in halogenated guanosines to obtain a product that could be alcoholic HC1 solution, the molar absorbances were 2.0 x coupled with benzene diazonium chloride were also tried. l0@(333 nm) and I .9 x l0@(300 nm) for guanosinyl-MAB, None of these attempted syntheses was successful in that no 2.0 x l0@ (333 nm) and 1.9 x l0@ (300 nm) for deox product with the chromatographic properties of a nucleo yguanosinyl-MAB, and 2.4 x l0@(324 nm) and 2.5 x l0@ side-dye derivative was obtained. (298 nm) for N-(guan-8-yl)-MAB. At 515 nm each of these compounds had a molar absorbance of 7 to 10 x l0@ immediately after solution, and this value dropped to 2 to 4 Conclusion x l0@on standing at room temperature for short times. These latter values are in strong contrast to the high molar The above chemical, radiochemical, and spectroscopic absorption coefficient of 5.3 x l0@ for MAB at 515 nm. studies demonstrate that the major nucleoside-dye products These results suggest that the dye-nucleosides contain a formed by the reaction of N-benzoyloxy-MAB with guano group on the amino nitrogen of the dye residue that greatly sine and deoxyguanosine are N-(guanosin-8-yl)- and N- inhibits its resonance in acid. This would be consistent with (deoxyguanosin-8-yl)-M A B, respectively. Thus N-ben a guan-8-yl substitution of the amino nitrogen of MAB. zoyloxy-MAB, an ester of the hydroxylamine N-hydroxy The double bond on carbon 8 would be expected to reduce MAB, reacts with guanosine and deoxyguanosine in a the basicity of the amino nitrogen of the dye residue. manner analogous to that previously observed with N- In ethanol or methanol the approximate molar absorb acetoxy-2-acetylaminofluorene, an ester (or anhydride) of ance for both guanosinyl-MAB and deoxyguanosinyl-MAB the hydroxam ic acid, N-hyd roxy-2-acetylam inofluorene was 2. 1 x l0@at 260 nm. The values observed for the molar (19). absorbance at 369 nm were 1.9 x l0@ and 1.6 x l0@, respectively. However, after these data had been obtained, it ACKNOWLEDGM ENTS was observed that the intensity of the absorption maximum of these dyes at 369 nm in ethanol solution increased when the dyes were kept in the dark for several hr and then The authors are grateful to Dr. Koji Nakanishi (Department of Chemistry, Columbia University) for helpful discussions on the NMR decreased on exposure to the usual overhead room fluores studies and to David Swenson (McArdle Laboratory for Cancer Research) cent lighting or on repeated scans in the spectrophotometer. for the mass spectrum determinations of the silylated derivatives. Dr. The intensity ofthe absorption maximum at 260 nm did not Schmall thanks Dr. I. Bernard Weinstein and Dr. Dezider Grunberger change under these conditions. With maximum dark adap (Institute of Cancer Research. Columbia University) for interest and tation the ratio of the amount of light absorbed at 369 nm to encouragement. The excellent technical assistance of Janice Parker and that absorbed at 260 nm was 1.06 for both the guanosinyl Barbara Butler is acknowledged with thanks. and deoxyguanosinyl dyes. After exposure to room lighting for 2.5 hr these ratios were about 0.8. In methanol solution N-(guan-8-yl)-MAB showed absorption maxima at 383 nm REFERENCES (e = 2.0 x l0@), 297 nm (e = 2.5 x l0@), 254 nm (shoul der), and 229 nm (e = 2.9 x l0@).The above values were I. Barry, E. J., Malejka-Giganti, D., and Gutmann, H. R. Interaction of obtained after the solution had been stored in the dark for Aromatic Amines with Rat-Liver Proteins in Vivo. III. On the several days. The intensities of these absorption maxima Mechanism of Binding of the Carcinogens. N-2-Fluorenylacetamide also varied according to the amount of light exposure. The and N-Hydroxy-2-fluorenylacetamide, to the Soluble Proteins. Chem.-Biol. Interactions, 1: 139 155, 1969. absorption spectrum of MAB was not altered by these ex 2. Bartsch, H., Dworkin, C.. Miller, E. C., and Miller, J. A. Formation posures to light, but under some conditions AB and some of of Electrophilic N-Acetoxyarylamines in Cytosols from Rat Mam its derivatives change from a predominantly trans configu mary Gland and Other Tissues by Transacetylation from the Carcino ration to a mixture of the cis-trans isomers under the influ gen N-Hydroxy-4-acetylaminobiphenyl. Biochim. Biophys. Acta. 304: ence of light (4, 5, 11). 42 55, 1973.

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3. Bartsch, H., Dworkin, M., Miller, J. A., and Miller, E. C. Electro aminoazobenzene. II. Identity of Synthetic 3-(Homocystein-S-yl)-N- philic N-Acetoxyaminoarenes Derived from Carcinogenic N- methyl-4-aminoazobenzene with the Major Polar Dye P2b. Biochem Hydroxy-N-acetylam inoarenes by Enzymatic Deacetylation and istry,7.1889-1895,1968. Transacetylation in Liver. Biochim. Biophys. Acta. 286: 272298, 22. Lin, J.-K., Miller, J. A., and Miller, E. C. Studies on Structures of 1972. Polar Dyes Derived from the Liver Proteins of Rats Fed N-Methyl-4- 4. Brode, W. R., Gould, J. H., and Wyman. G. M. The Relation between aminoazobenzene. Ill. Tyrosine and Homocysteine Sulfoxide Polar the Absorption Spectra and the Chemical Constitution of Azo Dyes. Dyes. Biochemistry, 8: 1573 1582, 1969. xxv. Phototropismand Cis-TransIsomerismin Aromatic Azo 23. Lin. J.-K.. Miller, J. A., and Miller, E. C. Structures of Hepatic Compounds. J. Am. Chem. Soc.. 74: 4641 -4646, 1952. Nucleic Acid-bound Dyes in Rats Given the Carcinogen N-Methyl-4- 5. Brode, W. R., Gould, J. H.. and Wyman, G. M. The Relationship aminoazobenzene. Cancer Res., 35: 844 850, 1975. between the Absorption Spectra and the Chemical Constitution of Azo 24. Lotlikar, P. D., Scribner, J. D., Miller. J. A., and Miller, E. C. Dyes. XXVI. Effect of Solvent and of Temperature on the Cis-Trans Reaction of Esters of Aromatic N-Hydroxy Amines and Amides with Isomerization ofAzo Dyes. J. Am. Chem. Soc., 75: 1856-1859, 1953. Methionine in Vitro: A Model for in Vivo Binding of Amine 6. DeBaun, J. R.. Miller, E. C., and Miller, J. A. N-Hydroxy-2- Carcinogens to Protein. Life Sci., 5: 1263 1269, 1966. acetylaminofluorene Sulfotransferase: Its Probable Role in Car 25. Maher, V. M., Miller, E. C., Miller, J. A., and Szybalski, W. cinogenesis and Protein-(methion-S-yl) Binding in the Rat Liver. Mutations and Decreases in Density ofTransforming DNA Produced Cancer Res., 30. 577 595, 1970. by Derivatives of the Carcinogens 2-Acetylaminofluorene and N- 7. DeBaun, J. R., Smith. J. Y. R.. Miller, E. C., and Miller, J. A. Methyl-4-aminoazobenzene. Mol. Pharmacol., 4. 41 I -426, 1968. Reactivity in Vivo of the Carcinogen N-Hydroxy-2-acetylamino 26. Mantsch, H. H., and Smith, I. C. P. Fourier-Transformed â€˜3CNMR fluorene: Increase by Sulfate Ion. Science, 167: 184- 186, 1970. Spectra of Polyuridylic Acid, Uridine, and Related Nucleotidesâ€”The 8. Delia, T. J., and Brown, G. B. Purine N-Oxides. XVII. The Oxidation Use of 31P0C'3C Couplings for Conformational Analysis. Biochem. ofGuanine at Position 7. J. Org. Chem., 31. 178-181. 1966. Biophys. Res. Commun., 46: 808@8l5, 1972. 9. Gatlin, L.. and Davis, J. C.. Jr. Comparison of Ribose and Deox 27. Marroquin, R. F., and Farber, E. The in Vivo Labeling of Liver yribose Nucleosides by NMR and Deductions Regarding Ribose and Ribonucleic Acid by p-Dimethylaminoazobenzene-l'-C'4. Proc. Am. Deoxyribose Nucleic Acids. I. Tautomeric Form. J. Am. Chem. Soc., Assoc. Cancer Res., 4. 41, 1963. 84: 44f,44470 1962. 28. Miller, E. C., Butler, B. W., Fletcher, T. L., and Miller, J. A. 10. Gutmann, H. R., Malejka-Giganti. D.. Barry, E. J., and Rydell, R. E. Methylmercapto-4-acetylaminostilbenes as Products of the Reaction On the Correlation betweenthe HepatocarcinogenicityoftheCarcino of N-Acetoxy-4-acetylaminostilbene with Methionine and as Degrada gen, N-2-Fluorenylacetamide, and Its Metabolic Activation by the tion Products of Liver Protein from Rats Given N-Hydroxy-4- Rat.Cancer Res.,32:1554 1561,1972. acetylaminostilbene. Cancer Res.. 34. 2232-2239, 1974. I I. Ishikawa, N., Namkung, M. J., and Fletcher, T. L. Fluorinated Azo 29. Miller, E. C., and Miller, J. A. Biochemical Mechanisms of Chemical Dyes. I. Synthesis and Spectral Properties of 3,5-Difluoro-4-N- Carcinogenesis. In Ft. Busch (ed). The Molecular Biology of Cancer, methylaminoazobenzene, 2,6-Difluoroacetanilide, and Related Com pp. 377-402. New York: Academic Press. Inc., 1974. pounds. J. Org. Chem.. 30: 3878-3882, 1965. 30. Miller, J. A., and Baumann, C. A. The Determination of p-Dimeth 12. Johnson, L. F., and Jankowski, W. C. Carbon-l3 NMR Spectra. New ylaminoazobenzene. p-M onomethylaminoazobenzene, and p-Amino York: Wiley-lnterscience, 1972. azobenzene in Tissue. Cancer Res., 5: 157-161, 1945. 13. Katz, L., and Penman, S. Association by Hydrogen Bonding of Free 31. Miller, J. A., and Miller, E. C. The Metabolic Activation of Nucleosides in Non-Aqueous Solution. J. Mol. Biol., 15: 220- 231, Carcinogenic Aromatic Amines and Amides. Progr. Exptl. Tumor 1966. Res.,II:273-301,1969. 14. King. C. M. Mechanism of Reaction, Tissue Distribution, and 32. Neumann, H.-G., Metzler, M., and TÃ¶pner. W. Organotropy and Inhibition of Arylhydroxamic Acid Acyltransferase. Cancer Res., 34: Aromatic Amine Metabolism. Abstracts ofthe Second Meeting of the 1503-1515,1974. European Association for Cancer Research, Heidelberg, Oct. 2-5, 15. King, C. M., and Phillips, B. Enzyme-catalyzed Reaction of the 1973, p. 159. Carcinogen N-Hydroxy-2-fluorenylacetamide with Nucleic Acid. 33. Poirier, L. A., Miller, J. A., Miller, E. C.. and Sato, K. N-Benzoyloxy Science,159:1351-1353,1968. N-methyl-4-aminoazobenzene: Its Carcinogenic Activity in the Rat 16. King, C. M., and Phillips. B. N-Hydroxy-2-fluorenylacetamide: and Its Reactions with Proteins and Nucleic Acids and Their Reaction of the Carcinogen with Guanosine, Ribonucleic Acid, Constituents in Vitro. Cancer Res., 27: 1600- 1613, 1967. Deoxyribonucleic Acid. and Protein following Enzymatic Deacetyla 34. Roberts, J. J. The Binding ofMetabolites of 4-Dimethylaminoazoben tion and Esterification. J. Biol. Chem., 244: 6209-6216, 1969. zene and 2-Methyl-4-dimethylaminoazobenzene to Hooded Rat Ma 17. Kriek, E. On the Mechanism of Action of Carcinogenic Aromatic cromolecules during Chronic Feeding. In: E. D. Bergmann and B. Amines. I. Binding of 2-Acetylaminofluorene and N-Hydroxy-2- Pullman (eds.), The Jerusalem Symposia on Quantum Chemistry and acetylaminofluorene to Rat-Liver Nucleic Acids in Vitro. Chem. Biochemistry. Physicochemical Mechanisms of Carcinogenesis, Vol. Biol. Interactions. 1: 3â€”I7, 1969. I, pp. 229-236. Jerusalem: Israel Academy ofSciences and Humani 18. Kriek, E. On the Mechanism of Action of Carcinogenic Aromatic ties,1969. Amines. II. Binding of N-Hydroxy-N-acetyl-4-aminobiphenyl to 35. Roberts, J. J., and Warwick, G. P. The Covalent Binding of Rat-Liver Nucleic Acids in Vivo. Chem.-Biol. Interactions, 3: 19-28, Metabolites of Dimethylaminoazobenzene, $-Naphthylamine and 1971. Aniline to Nucleic Acids in Vivo. Intern. J. Cancer, i. 179- 196, 1966. 19. Kriek, E., Miller, J. A., JuhI, U., and Miller, E. C. 8-(N-2- 36. Scribner, J. D., Miller, J. A., and Miller, E. C. 3-Methylmercapto-N- Fluorenylacetam ido)-guanosine, an Arylamidation Reaction Product methyl-4-aminoazobenzene: An Alkaline Degradation Product of a of Guanosine and the Carcinogen N-Acetoxy-N-2-fluorenylacetamide Labile Protein-bound Dye in the Livers of Rats Fed N.N-Dimethyl-4- in Neutral Solution. Biochemistry. 6: 177-182, 1967. aminoazobenzene. Biochem. Biophys. Res. Commun., 20: 560-565, 20. Lawley, P. D., and Brookes, P. Further Studies on the Alkylation of 1965. Nucleic Acids and Their Constituent Nucleotides. Biochem. J., 89: 37. StÃ¶hrer, G., Corbin, E., and Brown, G. B. Enzymatic Activation of the 127-138,1963. Oncogen 3-Hydroxyxanthine. Cancer Res., 32: 637-642, 1972. 21. Lin, J.-K., Miller, J. A., and Miller, E. C. Studies on the Structures of 38. Tada, M., and Tada, M. 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Jen-Kun Lin, Bernard Schmall, Ian D. Sharpe, et al.

Cancer Res 1975;35:832-843.

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