Chem. Res. Toxicol. 1994, 7, 784-791
784
Synthesis of Multiply-Labeled [15N3,13C1]-8-O~~-Substituted Purine Bases and Their Corresponding 2’-Deoxynucleosides Richard H. Stadler,* Andreas A. Staempfli, Laurent B. Fay, Robert J. Turesky, and Dieter H. Welti Nestec Ltd., Nest16 Research Centre, Vers-chez-les-Blanc,P.O. Box 44, CH-1000Lausanne 26, Switzerland Received June 13, 1994@
Stable isotope-labeled analogues of oxidatively modified purine bases are required as internal standards for accurate quantitation of free radical induced damage in DNA using the isotopedilution G C N S technique. For this reason, we report on a facile and expedient method to synthesize the isotope-labeled oxidized DNA bases 8-oxoguanine (&oxo-Gua, 5a) and 8-oxoadenine (8-oxo-Ade, 5b). Both routes have in common the introduction of two exocyclic 15N isotopes simultaneously by halogen displacement of chlorine-substituted pyrimidines with [l5N1benzylamine. Debenzylation is achieved by either catalytic hydrogenation or treatment with aluminium chloride in benzene. An additional isotope is incorporated by nitrosation with 15Nlabeled sodium nitrite. Cyclocondensation of the triamines with 13C-labeledurea then affords 5a and 5b in overall yields of 34% and 27%, respectively, and each with four isotope labels and a t least 99 atom % excess. A further one-step enzyme catalyzed coupling of the C8 adducted purines with 2’-deoxyribose furnishes the isotope-labeled 2’-deoxynucleosides 2‘-deoxy-7,8dihydro-8-oxoguanosine (8-oxo-dGuo) and 2’-deoxy-7,8-dihydro-8-oxoadenosine (8-oxo-&do).
Introduction In vivo oxidative cellular damage is caused by reactive oxygen species that are generated after exposure to oxidizing agents or ionizing radiation, or during normal cellular oxidative events which contribute to aging (1,2) and age-related diseases such as cancer (3). It is well established that free radical mediated attack on DNA leads to characteristic base modifications, such as the formation of oxygen adducted purines, thymidine glycol, and ring-opened base adducts (4, 51, which may be involved in mutagenic and carcinogenic processes (6, 7). Of particular relevance as a biomarker to measure oxidative DNA damage is the C8 oxidized nucleoside 2’deoxy-7,8-dihydro-8-oxoguanosine (8-oxo-dGuo),l generated endogenously by the highly reactive and electrophilic hydroxyl radical (8). This base lesion is present at cellular “background levels in both nuclear (9) and mitochondrial DNA (IO). Site specific modified DNA templates have shown that the oxidized bases 8-oxo-dGuo (1I) and also 2’-deoxy-7,8-dihydro-8-oxoadenosine (8-oxoa d o ) (12) lead to DNA miscoding. In addition, 8-0x0dGuo may play a role in carcinogenesis due to its mutagenicity in bacterial (13)and mammalian cells (14). There are numerous analytical methods at our disposal today to ascertain the levels of 8-oxo-dGuo in tissues and body fluids, such as 32P-postlabeling(151,HPLC with EC (9, 161, and GCMS (17, 18). However, none of these
* To whom correspondence should be addressed. *Abstract published in Advance ACS Abstracts, October 1, 1994. Abbreviations: EI, electron impact; SIM, selected-ion monitoring; RP-HPLC, reversed-phase high-performance liquid chromatography; EC, electrochemical detection; 8-oxo-Gua, 8-oxoguanine; 8-oxo-Ade, 8-oxoadenine; 8-oxo-dGuo,2’-deoxy-7,8-dihydro-8-oxoguanosine; 8-0x0dAdo, 2’-deoxy-7,8-dihydro-8-oxoadenosine; Thy, thymine; dThd, thymidine; TPase, thymidine phosphorylase; PNPase, purine nucleoside phosphorylase; BSTFA, N,O-bis(trimethylsily1)trifluoroacetamide; HRMS, high-resolution mass spectra; TMS, tetramethylsilane; exch, DzO exchangeable; FAB, fast atom bombardment. 0893-228x/9#2707-0784$04.50J0
methods can accurately measure the Ybase-line”levels of this oxidatively modified base due to the lack of internal standards. Recently, Dizdaroglu et al. (19) have reported the use of the stable isotope-labeled purine bases 8-oxo-Gua and 8-oxo-Ade to quantify these lesions in DNA by stable isotope-dilution mass spectrometry with selected-ion monitoring (SIM). However, to date only one method has been reported on the synthesis of isotope-enriched 8-oxodGuo as an authentic standard for quantitation by GCJ MS-SIM (20). This procedure entails six steps and introduces a single l80atom at the C8 position, furnishing the oxo-adducted nucleoside with an isotopic purity of 93.4 atom % excess. In this paper we now report a novel and expedient approach to synthesize the purine bases [2-amino,7,9l5N,8J3C1Sa and [6-amino-7,9-15N,8-13C]5b and their corresponding 2’-deoxyribonucleosides, each with four isotopic labels and an isotopic purity of greater than 99 atom % excess.
Materials and Methods Chemicals. Caution: Sodium nitrite is toxic and should be handled accordingly. Benzamide (99% 15N-labeled)and urea (99% 13C-labeled) were purchased from Cambridge Isotope Laboratories (Innerberg, Switzerland). Sodium nitrite (99%15Nlabeled) and the commercially available nonlabeled pyrimidines 4,6-dichloropyrimidine, 4,6-diaminopyrimidine hemisulfate monohydrate, 4,5,6-triaminopyrimidine sulfate, 2,4,64richloropyrimidine, 2,4-diamino-6-hydroxy-pyrimidine, and 6-hydroxy2,4,5-triaminopyrimidinesulfate were from the Aldrich Chemical Co. (Buchs, Switzerland). Thymidine (dThd), thymidine phosphorylase (TPase, EC 2.4.2.4), purine nucleoside phosphorlyase (PNFase, EC 2.4.2.1), and Sephadex LH-20 were purchased from Sigma Chemical Co. (Buchs, Switzerland). Nonlabeled Sa was from Janssen Chimica (Geel, Belgium). 8-Oxo-dGuowas synthesized according to standard procedures (21)with a purity of at least 98% as determined by HPLC and
0 1994 American Chemical Society
Stable Isotope-Labeled Oxidized DNA Bases GC/MS analysis. All other reagents were of analytical grade and were used without further purification. Apparatus. Proton (360 MHz) and 13C (90 MHz) NMR spectra were recorded on a Bruker AM-360 spectrometer with tetramethylsilane (TMS) as a n internal shift standard. Fast atom bombardment (glycerol matrix) and electron impact (EI) mass spectra were obtained on a Finnigan MAT 8430 instrument. High-resolution mass spectral data (EI-mode) were collected on the molecular ion using perfluorokerosene as the reference. GC/MS was carried out on a Hewlett Packard Model 5972 mass-selective detector interfaced to a HP 5890 Series I1 gas chromatograph. The mass spectrometer (E1 mode) was operated at a n ion source temperature of 180 "C with total ion monitoring to check the purity of the synthesized analogues. Analytical RP-HPLC employed a Hewlett Packard Model 1050 Ti series chromatograph equipped with a HP 1050 W/vis detector and an Antec Decade electrochemical detector with a n integrated pulse dampener and oven chamber set at the specified temeperature. Semipreparative RP-HPLC was carried out using a Hewlett Packard Model 1090 liquid chromatograph with a n H P photodiode array detector. Ultraviolet spectra were recorded on a Perkin Elmer Lamda 5 UV/vis spectrophotometer. Melting points were obtained on a Biichi 510 melting point apparatus and are uncorrected. GCMS Analysis. Separations were performed using a Rtx5 (Restek Corp., Bellefonte, PA) column (20 m x 0.18 mm, film thickness 0.2 pm) with He as carrier gas a t a flow rate of 30 c d s . The oven program was set at 128 "C for 1 min t o 220 "C a t 10 W m i n , resting 1 min, and then increasing to 300 "C at 40 W m i n and resting for 2 min. Injections were performed in the splitless mode. The compounds 5a, 5b, and their nonlabeled analogues were trimethylsilylated in tightly closed screw cap vials (Reacti-Vials, Pierce Chemical Co., Rockford, IL) using a mixture of 25 pL of BSTFA, 15 yL of acetonitrile, and 10 pL of pyridine at a reaction temperature of 130 "C for 25 min. After adjustment to room temperature, the derivatized products were injected directly without further treatment. HPLC Systems. System 1. RP-HPLC using a Macherey & Nagel Nucleosil 100-5 ym C18 column, 250 x 8 x 4 mm, column temperature 35 "C, solvents: A = 50 mM ammonium acetate, pH 5.5 with acetic acid, B = 100% methanol. Flow was initially isocratic at 0.8 m u m i n with 100% A for the first 10 min, then increasing to 20% B over 20 min and 50% B over 5 min, and remaining at 50% B for 5 min. t~ (min) of 5a, 11.09; Thy, 12.86; dThd, 24.84; 8-oxo-dGu0, 27.13; EC with a glassy carbon electrode operated at 35 "C, potential set a t +0.65 V versus the AgCLXC1 reference electrode, detection set a t 5 PA. Absorbance monitored at 290 nm. System 2. RP-HPLC using a Supelco LC-8-DB column, 5 ym, 250 x 4.7 mm, column temperature 34 "C, isocratic flow a t 1.0 m u m i n with 12% methanol and 50 mM citric acid buffer, pH 5.0 adjusted with solid sodium hydroxide. t~ (min) of Thy, 5.05; 5b, 5.5; dThd, 6.7; 8-oxo-ado, 12.6. EC with a glassy carbon electrode operated a t 34 "C, potential set a t f0.85 V versus the AgCyKCl reference electrode, recording set at 5 yA. Absorbance (W)was monitored a t 275 nm. Semipreparative HPLC. Purification of the labeled nucleosides was carried out using a semipreparative Macherey & Nagel column (Nucleosil5 pm C18,200 x 10 mm) at ambient temperature. Chromatographic conditions for 8-oxo-dGuo purification were 100%50 mM ammonium acetate (pH 5.3) over the first 10 min and then a linear gradient going to 20% methanol over 30 min, and increasing to 50% methanol over 10 min. The flow rate was 2.5 m u m i n , and peaks were monitored a t 290 nm. Chromatographic conditions for the purification of 8-oxo-ado used the same semipreparative column as stated above for 8-oxo-dGuo. The gradient started with 80% 50 mM ammonium acetate (pH 5.5) and 20% methanol over the first 15 min and was then increased to 50% methanol over 5 min, and going back to initial conditions over 10 min. The flow rate was 1.5 m u m i n , and peaks were monitored a t 275 nm. Enzymatic Coupling. This reaction was carried out essentially as described by Chapeau and Marnett (22)with the
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 786 following modifications. For 8-oxo-dGuo synthesis, the starting aglycon 5a was dissolved in a 0.1 M solution of potassium hydroxide (0.5 mg/mL) and a part of this stock solution diluted with a hot 50 mM potassium dihydrogen phosphate and adjusted to pH 8.0 with 50 mM dipotassium hydrogen phosphate to give a final concentration of 5a of 0.167 mM. For the enzymic incubation mixture, 4 mL (0.67 pmol) of this solution was added to 1 mL of hot MezSO under stirring to afford a clear solution of Sa (0.13 mM) containing 20% Me2SO. After adjustment to ambient temperature, dThd (25 ymol), TPase (26.8 units), PNPase (45.5 units), and sodium azide (final concentration 0.05%) were added. The mixture was incubated a t 38 "C for varying times, and the progress of the reaction was monitored by RP-HPLC employing System 1. The enzymic synthesis of 8-oxo-dAd0 was carried out under similar conditions but without MezSO in the incubation mix and with a final 5b concentration of 0.55 mM. Due to the lower turnover of 5b to its deoxynucleoside, the amount of PNPase was increased to 60 units in the same reaction volume (5 mL, 2.75 pmol of 5b) as described for Sa. The progress of the reaction was followed by RP-HPLC using System 2. [16NJBenzylamine. This compound was synthesized according to a published procedure (23). Solid [15Nlbenzamide(1.5 g, 12.4 mmol) was added t o a dry etherLiAlH4 slurry (1g in 250 mL) under stirring. The reaction mix was refluxed for 4 h and then left stirring at room temperature overnight. The reaction was terminated by careful addition of 1 mL of water, 3 mL of sodium hydroxide (1N), and finally 1-2 mL of water until all reactivity had ceased. The residue was separated by filtration and washed with 2 x 200 mL of dry ether. The combined filtrates were concentrated in vacuo (45 "C) to afford 1.23 g (92%)of [16N]benzylamine,which was employed directly without further purification. 4-Hydroxy.2,6-dichloropyrimidine (la). This compound was synthesized by selective base hydrolysis of 2,4,6-trichloropyrimidine (18.3 g, 0.1 mol) according to the previously described procedure (24, 25). Yield: 6.93 g (42%); mp 170-171 "C [lit. mp 170-171 "C (25)]. HRMS on the molecular ion calcd for C4HzNzOC12, mlz 163.95442, found 163.95426. 4 H y d r o x y - [ 2 , 6 1 s N ] b i s ~ ~ ~ o(b). ) p The ~~e free base of ['5N]benzylamine (0.96 g, 9 mmol) was added to dry la (0.3 g, 1.8 mmol) and the mixture heated in a glass vial a t 145 "C on a thermostated heater block for 25 min. After reaching room temperature, the white solid was suspended in hot water (5 mL) and filtered, and the residue was washed with 20 mL of hot water. The aqueous filtrate contains residual nonreacted benzylamine, a part of which was recovered by concentration in vacuo (45 "C) and by TLC on silica preparative plates (Silica 60 F254, Merck, 2 mm thickness) in solvent chlorofordmethanol/aqueous ammonia (80:20:1). Benzylamine ( R f =0.34) was scraped off the plate and eluted with methanol. After solvent removal in uucuo (40 "C), the labeled base was taken up in a small volume of ethanoVHC1 and concentrated under vacuum, upon which benzylamine crystallizes out as its hydrochloride. Typical yield of benzylamine hydrochloride was 0.24 g, 1.7 mmol, 31% recovery of the unreacted excess material. The bis(benzy1amino)pyrimidine was crystallized from ethanol, affording 0.499 g of 2a (90% yield based on 4-hydroxy-2,6dichloropyrimidine and 44% yield based on benzylamine); mp 191 "C. 1H NMR (MezSO-ds)6 9.77 ppm, lH, broad, exch; 7.357.17 ppm, ca. 10H, superposed multiplets; ca. 7.08 ppm, lH, broad d, exch, coupling to 15N observed, ~ J Nca. H 94 Hz (from 15N spectrum), left half of signal hidden under phenyl proton H 92 Hz; 4.43 ppm, 2H, signals; 6.69 ppm, lH, br d, exch, ~ J Nca. d slightly broad, apparent 3 J 5.3 Hz ~ (reduced ~ ~because of line broadening); ca. 4.42 ppm, lH, slightly broad; 4.29 ppm, 2H, broad. I3C NMR (MezSO-ds)quaternary carbons: 6 163.57 ppm, d d, JCN19.4,2.6 Hz; 162.76 ppm slightly broad, ca 153.68 ppm, broad d, JCNca. 23 Hz and undeterminable due to signal broadening; ca. 140 ppm, very broad; 139.37 ppm; CH carbons: 128.21 ppm, 2C; 128.09 ppm, 2C; 127.25 ppm, 2C; 126.95 ppm, 2C, slightly broad; 126.77 ppm; 126.44 ppm, slightly broad; ca. 75.7 ppm, broad; methylene carbons: ca. 44.0 ppm, broad; 43.28
Stadler et al.
786 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
glass tube. The progress of the reaction was monitored by TLC ppm, d, JCN10.9 Hz. HRMS on the molecular ion calcd for analysis (chloroformlmethanolaqueousammonia, 140:60:1.5) C&1&2l5N2O, m / z 308.14213, found 308.14165. and terminated after disappearance of the starting material (Rf 4-Hydroxy.[2,6-15N]diaminopyrimidine (3a). A 10%pal= 0.95) and appearance of a new spot at Rf = 0.6. After 6 h the ladium-carbon catalyst (0.4 g) was added to a solution of 2a mixture was poured into 5 mL of cold dilute aqueous ammonia (0.35 g, 1.14 mmol) in 10 mL of ethanol/water/hydrochloric acid, (2%v/v) and spun for 2 min (Eppendorfcentrifuge, 14 000 rpm). 1 M (15:5:2vlv). The reaction mixture was hydrogenated (1-2 The organic layer and the precipitated aqueous residue were atm) at 45-50 "C for 20 h under rapid stirring. The catalyst separated, and the benzene layer was washed a second time was removed by filtration and the clear filtrate was concentrated with water. The combined aqueous layers were loaded onto a to dryness in vacuo (40 "C) to afford the hydrochloride salt of Sephadex LH-20 column (25 x 4 cm), eluting 3b with water 3a as a white precipitate (148 mg, 80% yield). HRMS on the and affording 40 mg (70%yield) of the free base as a white molecular ion calcd for C4H&15N20, m l z 128.04823, found precipitate after lyophilization. HRMS on the molecular ion 128.04830. calcd for C4H,N2l5N2, m l z 112.05332, found 112.05345. 4-€Iydro~y-[2,5,6J~Nl triaminopyrimidine(4a). This re[4,5,6J6N]Triaminopyrimidine(4b). The free base of 3b action was carried out essentially as described (26)with slight (78 mg, 0.7 "01) was dissolved in 1.5 mL of 1 N hydrochloric modifications. The hydrochloride salt of 3a (0.13g, 0.79 "01) acid. The solution was kept at 0-5 "C under stirring and 15Nwas dissolved in 5 mL of 10 % acetic acid. The solution was labeled sodium nitrite (100 mg in 0.2 mL of cold water) added kept on ice (5 "C) under stirring and 15N-labeledsodium nitrite dropwise. The clear green solution was kept a t 0-5 "C for added portionwise (0.2 g in 0.5 mL of cold water). After 1 h, another 45 min and then solid sodium hydrogen carbonate the pink precipitate which had formed was removed by cenadded until the reaction had ceased. The resulting thick bluish trifugation (Eppendorf centrifuge, 14 000 rpm, 2 min), washed precipitate was collected by centrifugation (14 000 rpm, 1 min) with cold water (5 mL), and suspended in 4 mL of hot water and washed with cold water. The clear green supernatant was (ca. 60 'C) under stirring. Sodium dithionite (0.3 g) was added discarded and the pellet suspended in 1mL of hot water (60in portions and the mixture spun down (14 000 rpm, 2 min). 70 "C). Solid sodium dithionite (100 mg) was added and the The supernatant was kept on ice and the resulting cream-like solution acidified by addition of 0.5 mL of 50% sulfuric acid. precipitate washed with cold water and acidified with 2 N Particulate matter was removed by filtration through a cotton sulfuric acid. A final wash with cold water afforded 0.14 g (73% plug. Pure 4b was crystallized out on ice and was washed with yield) of the sulfate of 4a as a white powder after lyophilization; cold water, affording 95 mg (61%yield) after lyophilization; mp mp >300 "C. HRMS on the molecular ion calcd for C4H7N215N30, 2300 "C. HRMS on the molecular ion calcd for C4H7N216N3, m l z 144.05617, found 144.05672. m I z 128.06125, found 128.06112. [2.amin0,7,9-~~N,8-~~C18-Oxo-guanine (Sa). l3C-Labeled [6amino,7,9-15N,&13Cl&Oxoade~e (5b). This compound was added t o solid 4a (40 mg, 0.165 urea (40 mg, 0.66 "01) was synthesized as described above for Sa by cyclization of 4b mmol) and the mixture heated to 155 "C in a thermostated (21 mg, 0.094 mmol) with 13C-labeledurea (23 mg, 0.37 mmol). heater block for 25 min. After reaching room temperature, the The solid mixture was heated a t 155 "C in a glass vial on a yellow residue was dissolved in 2 mL of warm 1 N potassium thermostated heater block for 30 min. m e r cooling, the solid hydroxide. To this clear yellow solution was added 0.1 g od material was dissolved in 1 N sodium hydroxide (0.5 mL) and activated charcoal, and the suspension was agitated on a n activated charcoal (0.1 g) added. The mixture was agitated for Eppendorf shaker for 15 min. Charcoal was removed by 10 min on a n Eppendorf shaker and charcoal removed by centrifugation (Eppendorf, 14 000 rpm for 2 min) and filtration centrifugation (14 000 rpm, 2 min) and filtration (0.45 pm). The (0.45pm filters). The clear filtrate was acidified to pH 5.5 with clear solution was acidified with 2 N sulfuric acid and left on 1 N hydrochloric acid, and the solution was left at 4 "C for 3 h, ice for 2-3 h. 5b gradually crystallized out and was washed resulting in a white precipitate which was washed first with with cold water and subsequently lyophilized. The mother water and then with methanol. Yield: 22 mg (64%);mp '300 liquor was subjected to semipreparative HPLC using the identi"C. HRMS on the molecular ion calcd for C413C~H5Nz16N302, c a l conditions as described for 8-oxoAde purification. The peak mlz 171.03879, found 171.03863. corresponding to 8-oxoAde was collected, concentrated in vacuo [4,6-15N1Bis(benzylaino)pyrimidine (2b). [16NlBenzyla t 45 "C, and subsequently lyophilized. Total yield: 0.064 mmol amine (0.96 g, 9 mmol) was added to lb (300 mg, 2.01 mmol) (68%);mp '300 "C. HRMS on the molecular ion calcd for and the mixture heated to 145 "C on a thermostated heater block C413C1H5N2"N30, 155.04387, found 155.04391. for 2.5 h in a sealed vial. After reaching ambient temperature, the resulting solid residue was suspended in ethanol, filtered, [2smino,7,9-15N,8-13C18-Oxo-dGuo. After incubation, the and washed with ethanol (20 mL). The ethanolic filtrate was labeled nucleoside was purified from the enzyme mixture by concentrated under vacuum (40 "C) and unreacted benzylamine diluting the 5 mL reaction mix 1:4with water and charging two recovered by preparative TLC as described for 2a. Qpical pre-equilibrated (methanol, then water) (2-18 EC Chromabond recovery of [15Nlbenzylamine hydrochloride was 0.21 g (1.5 cartridges (1000 mg) with equal volumes (10 mL) of the diluted mmol, 30% of the total unreacted benzylamine). reaction mix. After penetration by gravity-induced flow, the columns were washed with 4 mL of water and the aqueous The labeled bis(benzy1amino)pyrimidine was crystallized on effluent was discarded. The desired nucleoside as well as the ice to afford 562 mg of 2b (95%yield based on lb,and 52%yield unreacted starting material was eluted with 50% methanoV based on benzylamine), mp 232-234 "C [lit. mp 234-235 "C water (6 mL each column), and the combined effluents of the (27)l. lH NMR (Me2SO-de)6 7.91 ppm, 1H; 7.32-7.18 ppm, ca. two columns were concentrated in vacuo and subsequently 10H,superposed multiplets; ca. 7.15 ppm, lH, d t, coupling to lyophilized. The white residue was redissolved in 50%methanol 15N observed, ~JNH 91 Hz (from 16Nspectrum), left half of signal (2 mL), and 500 pL aliquots were injected onto a semipreparahidden under phenyl proton signals; 5.38 ppm, lH, slightly J 5.4 Hz ~ ~ tive~RP-HPLC column using the conditions described above. The broad; 4.36 ppm, 4H,d slightly broad, apparent 3 unreacted excess purine base Ba, t~ 15.2 min, and 8-oxo-dGuo, (reduced due to line broadening). 13C NMR (MezSO-&) quat~ 32 min, were collected and lyophilized. The multiply-labeled ternary carbons: 6 162.25 ppm, d, ~ J C19.4 N Hz; 140.07 ppm, s nucleoside was quantified spectrophotometrically in 10 mM slightly broad; CH carbons: 157.43 ppm, t, 3 J c ~ >= 2.6 Hz; ammonium acetate buffer, pH 7.0 with aqueous ammonia, 128.11 ppm, 2C; 126.92 ppm, 2C, slightly broad; 126.45 ppm; and yielded 0.40 ca. 81.3 ppm, very broad; methylene carbons: 43.57 ppm, ~ J C N employing E = 9.7 mM-l at 1 = 293 nm (28), ca. 10 Hz. HRMS on the molecular ion calcd for C I ~ H I & ~ ~ N Z , pmol(60%) of 8-oxo-dGuo. FAB/MS [M HI+, calcd 288.09395, found 288.09644. m l z 292.14721, found 292.14701. [6amino,7,9-15N,8-13C18-Ox~-dAd~. The crude reaction [4,6-15NlDiaminopyrimidine (3b). Aluminium chloride mix was diluted with water (1:4) and Sep-packed as described (0.18 g, 1.35 mmol) was added to a suspension of 2b (100 mg, above for 8-oxo-dGuo, but this time eluting the basdnucleoside 0.51 "01) in 2 mL of benzene at room temperature under rapid with 95% methanol containing 2 mM acetic acid. After lyostirring in a tightly closed (screw capped with a Teflon septum)
+
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 787
Stable Isotope-Labeled Oxidized DNA Bases
.t5. Scheme 1
R1
a: PdGIH2
N ~ 1 5 N m b : ~ ~ ~ :
CI
R
R2-&
l a , ~ 1 OH; = ~2 = CI l b , R1 = CI: R2 = H
b AlCl-&Hg
NHCHZPh
RZA~
NH2
Za, R1 =OH; R2 = l5NHCHZPh
sa, ~ 1 OH; = ~2 = 1
2b, R1 = 15NHCH2Ph: Rz = H
3b, R1 = 15NHz: Rz = H
4a, R1 = OH; Rz = 15NH2
sa, ~ 1 OH; = ~2 = 1
4b, R1 = 15NH2: R2 = H
5b, R1 = 15NHz: R2 = H
philization, the residue was redissolved in 50%methanoywater (2 mL) and injected onto a semipreparative HPLC column with on-line diode array detection using the separation conditions described above. Excess 5b, t~ 12.3 min, and 8-oxo-ado, t~ 19.8 min, were collected and lyophilized. Determination of converted levels of 8-oxo-Ade to 8-oxo-ado was done by peak integration (UV/electrochemically)of the nucleoside and extrapolation based on a standard curve of 8-oxo-ado which was recorded under identical HPLC conditions. This gave a relative yield of ca. 8 pg of 8-oxo-&do. FABMS [M HI+ calcd 272.09903, found 272.09861.
+
Results Our strategy for the synthesis of multiply-labeled modified purines involves the introduction of two 15Nlabels simultaneously by displacement of the two chlorine atoms of 4-hydroxy-2,6-dichloropyrimidine (la)and 4,6dichloropyrimidine (lb)with benzylamine (Scheme 1). The synthesis of 15N-labeledbenzylamine was performed by reduction of commercially available 15N-labeled benzamide with lithium aluminium hydride in ether, employing the free base immediately without additional purification (23). The starting dichloropyrimidine la was synthesized from 2,4,6-trichloropyrimidineby selective base hydrolysis (24), and the reaction conditions were optimized as described in a previously published procedure to yield a 1:l ratio of la to its 4,6-dichlorosubstituted isomer (25). Complete amination of the 4-hydroxy-2,6-dichloro-substituted pyrimidine la was found to be dependent on the molar ratio of the reactants as well as the temperature and duration of the reaction. Optimal reaction conditions for halogen displacement of la with [15N]benzylamine were at 145 "C for 25 min. Taking into account the stoichiometry of the reaction, in which 2 mol of benzylamine react with 1mol of la,[15N]benzylamine is employed in a 2.5-fold molar excess. Under these conditions, crystalline 4-hydroxy-[2,6-l5N1bis(benzy1amino)pyrimidine (2a),mp 190 "C, is isolated i n 90% yield2 based on la. Due to the relatively expensive isotopically labeled reagent, we attempted to isolate the unreacted excess benzylamine after completion of the reaction. Approximately 30-35% of the unreacted labeled benzylamine can be recovered from the Yields are reported as isolated and based on average ranges of three trials, f i s t with nonlabeled compounds and then with the isotopelabeled analogues.
5
~
~
5
~
~
~
~
reaction mixture by TLC and subsequently crystallized as its hydrochloride salt. The yield of 2a, based upon the amount of [l5N1benzylamineemployed, is on the order of 44% and, in general, can be considered satisfactory for benzylation reactions. The identity of the novel compound 2a was established by lH and 13CNMR spectroscopy, showing all the characteristic signals of a 4-hydroxy2,6-dibenzylated pyrimidine. Cleavage of both benzylic groups to give 4-hydroxy[2,6-l5N1diaminopyrimidine (3a)in 80% yield was accomplished by hydrogenation over a 10% palladiumcarbon catalyst a t 45-50 "C (1-2 atm) and a reaction time of 20 h. However, if the reaction is performed a t ambient temperatures (20-25 "C), the yields are only about 40-45% due to incomplete N-debenzylation. A third exocyclic nitrogen isotope was introduced into the pyrimidine nucleus by treatment of 3a with I5N-labeled sodium nitrite (26), which affords 4-hydroxy-[2,6-l5N1diamin0-5-[~~N]nitrosopyrimidine. This colored intermediate was reduced without prior isolation in the same reaction mixture by treatment with solid sodium dithionite to give 4-hydro~y-[2,5,6-~~N]triaminopyrimidine (4a) in satisfactory yield (73%). The 8-oxopurine was then constructed by fusion of the triply-labeled triaminopyrimidine 4a with a 4-fold molar excess of [13C]urea at elevated temperature (155 "C) on a heater block for 25 min. This condensation reaction goes smoothly and leads to [2-amin0,7,9-~~N,8-~~C18-oxoguanine (5a)in an overall yield of 34%. An analogous route was followed to synthesize 15NP3Clabeled 5b,which was completed in a total of four facile steps. The key intermediate, [4,6-15N]bis(benzylamino)pyrimidine (2b),was prepared from 4,6-dichloropyrimidine (lb)and [ 15N]benzylamine. Optimal reaction conditions were achieved using a ca. 2.3-fold molar excess of benzylamine (2 mol of benzylamine react with 1 mol of lb)and heating the reaction mixture at 145 "C for 2.5 h. The product 2b crystallizes easily from ethanol (mp 232-234 "C) and is obtained in good yield (95% based upon lb). This represents an improvement over the published procedure (27) which afforded 40-45% of the desired dibenzylamine after heating the reaction mix on a steam bath for 3 h. Similarly, a part of the unreacted excess [l5N1benzylamine can be recovered (30%)by TLC and crystallization as its hydrochloride salt. This gives 2b in 52% yield based upon labeled benzylamine, com-
Stadler et al.
788 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
parable to the recoveries recorded for the benzylation of Abundance (~10') 4xTMS la. Spectroscopic analysis of 2b by lH and 13C NMR showed characteristic signals for two benzylamine moieties at the C4 and C6 positions of the pyrimidine ring. However, reductive cleavage of the benzylic groups of 501 2b as described above for 2a resulted in a mixture of undefined products. Conditions using oxidative reagents such as ruthenium oxidelsodium periodate, as reported recently for the N-debenzylation reaction leading to [6~mino-~~N]-Y-deoxyadenosine (291,also remained without success. Conversion to [4,6J5N1diaminopyrimidine (3b)was finally accomplished in 70% yield under relatively mild conditions by Lewis acid catalyzed N-debenzylation with aluminium chloride in benzene (30) at ambient temperature for 6 h. The product [4,6-l5N1diaminopyrimidine (3b)partially crystallizes from the reaction mixture in benzene and is the major compound as determined by TLC, with only traces of the monobenzylated intermediates. However, purification of the free diamine proved rather difficult, probably due to contaminating salts that were not visible by TLC analyAbund8nce (~10s) sis. Column chromatography on LH-20 removed all 73 B 444 impurities, eluting 3b with water. 6 I450 A third exocyclic 15N atom was introduced into the 4 molecule as described for [7J5N1adenine (31)and above for the synthesis of 4a. The free base of 3b is treated 2 with a 2-fold molar excess of 15N-labeledsodium nitrite 0 in dilute hydrochloric acid to give the colored nitrosated intermediate [4,6-15Nldiamino-5-[15Nlnitrosopyrimidine. mh Again, reduction with sodium dithionite is accomplished in the same reaction mixture without having to isolate Figure 1. GUMS analysis of trimethylsilylated Sa: (A) the nitroso intermediate and affords 4b in 61% yield. unlabeled compound; (B)isotope-labeled analogue. Top: Excerpt of the total ion chromatogram of B; bottom: E1 mass spectrum. However, it is important that the diaminopyrimidine is present as the free base and not as the sulfate salt which, labeled nucleoside entails a single solid-phase extraction in our hands, led to very poor yields of the triaminopystep which removes MezSO and polar constituents, rimidine in the range of 25-30%. Finally, ring closure followed by semipreparative RP-HPLC which gives 8-oxot o the imidazole was performed in a single step as dGuo in 60% isolated yield (Scheme 2). described for the route to Sa by cyclization with a 4-fold molar excess of [13Clurea,giving [6-amino,7,9-l5N,8-l3C1- Similarly, 5b was incubated with thymidine, TPase, 5b in 68% yield for this step and 27% overall yield. and PNPase but without MezSO due to its solubility The mass spectra (GC/MS, E1 mode) of the trimethylunder the given incubation conditions. However, it was silylated modified bases and their stable isotope-labeled surprising that for this compound the relative conversion analogues correspond to previously published data (19), to its corresponding nucleoside reached only 2% even depicting a fragmentation pattern with an intense moafter prolonged incubation times of up to 72 h, as lecular ion (M+) and a characteristic (M - 15)+ ion determined by RP-HPLC with EC (Figure 3B). Neverresulting from the loss of a methyl radical. As anticitheless, we were able to purify ca. 8 pg of the multiplypated, the isotope-enriched analogues Sa and 5b show a labeled nucleoside by semipreparative RP-HPLC, which mass shift of 4 Da relative to the unlabeled compounds, showed the characteristic U V absorption spectrum of in accord with their extent of labeling. Characteristic 8-oxo-dAdo and coeluted on RP-HPLC System 2 with ions were recorded at m l z 459 (M+)and 444 (M - 15)+ nonlabeled reference material. To further support the for tetra-trimethylsilylated Sa (Figure 11, and 371 (M+) structure of the 8-oxo nucleoside, the isolated material and 356 (M - 15)+for 5b (Figure 2) with three trimethwas analyzed by FABMS. In comparison, 8-amino-2'ylsilyl groups. Based on GCMS measurements, both deoxyadenosine has been isolated in 21% yield starting compounds revealed an isotopic purity of greater than from the aglycon by utilizing Escherichia coli encapsu99 atom % excess, with no visible overlap of the molecular lated cells as coupling catalysts (32). ions of the isotope-labeled analogues with the natural abundance isotope peaks of the unlabeled oxo bases. Discussion Previously reported (22)PNPase catalyzed coupling of The 15N/13C-labeledpurine base syntheses described adducted purines with deoxyribose 1-phosphate prompted here are facile, high-yield reactions that do not require us to attempt this reaction with Sa and 5b. In the case tedious purification and chromatographic procedures. of the former, a limiting factor was found to be its poor The isotope-labeled reagents are employed in 2- to 4-fold solubility in the aqueous buffer at slightly alkaline pH, which permitted a maximum substrate concentration of molar excess, emphasizing efficient usage of the 15Nand I3C sources. This route also provides isolable amounts 0.13 mM in the reaction mix, even in the presence of 20% of the C8 substituted 2'-deoxynucleoside analogues, MeZSO. Nevertheless, the relative conversion of 5a to which are synthesized in a 6ingle enzymic step that does its 2'-deoxynucleoside reached 85% after 48 h at 38 "C not necessitate tedious protection and deprotection meaas monitored by HPLC (Figure 3A). Extraction of the
I
I
I
I/
Stable Isotope-Labeled Oxidized DNA Bases
I
Abundance (x105)
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 789
1
3xTMS
A 15 "A
8 - 0 ~I o - d G ~ o
1 1
1 8-oxo-Gua
A
I
1
I
time (min)
Abu;rnce
I 21
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L
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1"
11
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20
25
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Time (min.)
B 8-oxo-Ade
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" "
200
0
I
mlz
I 1'
I
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I '
,-+,
250
I "
300
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I
350
400
Figure 2. W/MS analysis of trimethylsilylated 5b: (A) unlabeled compound; (B)isotope-labeled analogue. Top: Excerpt of the total ion chromatogram of B; bottom: E1 mass spectrum.
sures. In addition, the labeled bases 5a and 5b are stable under the given reaction conditions, and excess substrate can easily be recovered from the enzymic mixture by a simple Sep-pack procedure followed by semipreparative HPLC. We have also applied this synthetic route to construct the multiply-labeled bases guanine and adenine,3 by treatment of the 15N-enriched triaminopyrimidines 4a and 4b, respectively, with 13C-labeled sodium formate (33). This approach could provide multiply-labeled oligomeric DNA analogues (341,which could serve as biophysical probes to study DNA as well as protein-nucleic acid interactions (26). The continued interest and the indisputable need to measure oxidative damage to DNA both in vivo and in vitro demand accuracy and reproducibility of the applied analytical methodologies. Most of the reports pertaining to the in vivo and in vitro measurements of 8-oxo-dGuo in tissues and body fluids employ either the HPLC-EC or the GCMS technique. Both analytical methods have intrinsic advantages and, as noted in recent reviews (18, 351, do not provide comparable values of "background" levels of certain oxidized DNA bases. As an example, base-line levels of 8-oxo-dGuo measured by HPLC-EC in commercial calf thymus DNA have been reported to range between 8 and 70 8-oxo-dGuo/106DNA bases (36391, compared to 159 and 318 8-oxo-Gud106DNA bases measured by the GC/MS technique (40-42). In this sense, the HPLC-EC method tends to underestimate and the GCMS method t o overestimate the levels of 8-oxodGuo. Such discrepancies between the published levels R. H. Stadler, unpublished observations.
i u L I
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'
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.
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.
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.
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.
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.
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.
.
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.
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Figure 3. RP-HPLC-EC profiles showing (A) the formation of 8-oxo-dGuo from Sa in a TPasePNPase catalyzed reaction over time, HPLC conditions as described under system 1; and (B) the formation of 8-oxo-dAdo from Sb in a TPasePNPase catalyzed reaction over time, HPLC conditions as described under system 2.
Scheme 2 0
0
HO
of certain oxidized DNA bases may, in part, be attributable to the choice of the DNA hydrolysis methods, i.e.,
,
,
l
~
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790 Chem. Res. Toxicol., Vol. 7,No.6, 1994
either enzymatic or acidic (18).Enzymatic hydrolysis of DNA under neutral conditions apparently leads to a higher recovery of 8-oxo-dGuo by up to 80% compared t o hydrolysis at an acidic pH of 5.1, resulting in elevated levels of 8-oxo-dGuo that approach those measured by the GC/MS method (43). Furthermore, discrepancies between the two analytical methods have also been reported in the quantitation of modified pyrimidine bases, exemplified by 5-hydroxy-2'-deoxycytidine and 5-hydroxy2'-deoxyuridine. Steady-state levels of these bases were measured in calf thymus DNA, revealing 8-fold higher values when quantified by G U M S as compared to HPLCEC (44). The accurate quantification of oxidized DNA bases can only be achieved by using internal standards. In the case of the GC/MS technique, tri- and tetra-isotopically labeled analogues of oxidized DNA bases, including also 8-oxoGua and Ei-oxo-Ade, have been prepared, albeit without description or literature citation of the synthetic procedures (19). Such internal standards including the isotopically labeled nonmodified purine bases can be added t o the DNA at an early stage of isolation, and with isotope-dilution mass spectrometry reflect the true levels of oxidized DNA bases present, compensating for potential artifact formation during DNA workup and derivatization. Thus, the isotope-labeled analogues prepared in this report enable the accurate quantification of oxidized purines in DNA following the GC/MS technique. Recent experiments to determine 8-oxo-Gua formation in vivo have for the first time employed the electrochemically active compound 2,6-diamino-8-oxopurine as an internal standard for the accurate quantification of DNA base lesions by the HPLC-EC technique (45).Experiments are now underway in this laboratory to compare the GC/MS and the HPLC-EC methods in quantifylng oxidized DNA bases using identical DNA samples and the appropriate internal standards.
Acknowledgment. We thank Dr. Jean Cadet, Commissariat a 1' Energie Atomique, Grenoble, for the kind gift of 2'-deoxy-7,8-dihydro-8-oxoadenosine, Dr. J.-L. Ravanat and Mr. E. Gremaud for recording GC/MS spectra, and Mrs. F. Arce Vera for recording NMR spectra.
References (1)Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993) Oxidants,
antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U S A . 90,7915-7922. Harman,D. (1981) The agingprocess. Proc. Natl. Acad. Sci. U S A . 78,7124-7128. Fraga, C. G., Shigenaga, M. K., Park, J.-W., Degan, P., and Ames, B. N. (1990)Oxidative damage to DNA during aging: 8-hydroxy2'-deoxyguanosine in rat organ DNA and urine. Proc. Natl. Acad. Sci. U S A . 87,4533-4537. Halliwell, B. (1993) Oxidative DNA damage: meaning and measurement. In DNA and Free Radicals (Halliwell, B., and Aruoma, 0. I., Eds.) pp 67-79, Ellis Horwood Publishers, New York. Halliwell, B., and Aruoma, 0. I. (1991) DNA damage by oxygenderived species. Its mechanism and measurement in mammalian systems. FEBS Lett. 281, 9-19. Breimer, L. H. (1990) Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage. Mol. Carcinog. 3, 188-197. Kuchino, Y., Mori, F., Kasai, H., Inoue, H., Iwai, S.,Miura, K., Ohtsuka, E., and Nishimura, S. (1987) Misreading of DNA templates containing 8-OH-dG at the modified base and at adjacent residues. Nature 327, 77-79. Kasai, H., and Nishimura, S. (1984) Hydroxylation of deoxyguanosine at the C8 position by ascorbic acid and other reducing agents. Nucleic Acids Res. 12, 2137-2145.
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Stable Isotope-Labeled Oxidized DNA Bases Gaffney, B. L., Kung, P.-P., and Jones, R. A. (1990) Nitrogen-15labeled deoxynucleosides. 2. Synthesis of [7JSN]-labeled deoxyadenosine, deoxyguanosine, and related deoxynucleosides. J.Am. Chem. SOC.112,6748-6749. Cadet, J., and Weinfeld, M. (1993) Detecting DNA damage. Anal. Chem. 65,675-682. Park, J. W., and Floyd, R. A. (1992) Lipid peroxidation products mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free Radical Biol. Med. 12, 245-250. Aiyar, J., Berkovits, H. J., Floyd, R. A., and Wetterhahn, K. E. (1990) Reaction of chromium(VI) with hydrogen peroxide in the presence of glutathione: reactive intermediates and resulting DNA damage. Chem. Res. Toxicol. 3, 595-603. Lu, L. J. W., Tasake, F., Hokanson, J. A., and Kohda, K. (1991) Detection of 8-hydroxy-2'-deoxyguanosinein deoxyribonucleicacid by the 32P-postlabellingmethod. Chem. Pharm. Bull. 39, 18801882. Mouret, J. F., Polverelli, M., Sarrazini, F., and Cadet, J. (1991) Ionic and radical oxidations of DNA by hydrogen peroxide. Chem.Biol. Interact. 77, 187-201. Aruoma, 0. I., Halliwell, B., and Dizdaroglu, M. (1989) Iron iondependent modification of bases in DNA by the superoxide radical-
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 791 generating system hypoxanthindxanthine oxidase. J. Biol.Chem. 264, 13024-13028. (41) Aruoma, 0. I., Halliwell, B., Gajewski, E., and Dizdaroglu, M. (1989)Damage to the bases in DNA induced by hydrogen peroxide and ferric ion chelates. J . Biol. Chem. 264, 20509-20512. (42) Aruoma, 0. I., Halliwell, B., Gajewski, E., and Dizdaroglu, M. (1991) Copper-ion-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem. J. 272, 601-604. (43) Frenkel, K., Zhong, Z., Wei, H., Karkoszka, J., Patel, U., Rashid, K., Georgescu, M., and Solomon, J. J. (1991) Quantitative high performance liquid chromatography analysis of DNA oxidized in vitro and in vivo. Anal. Biochem. 196, 126-136. (44) Wagner, J. R., Hu, C.-C., and Ames, B. N. (1992) Endogenous oxidative damage of deoxycytidine in DNA. Proc. Natl. Acad. Sci. U S A . 89,3380-3384. (45) Ravanat, J.-L., Stadler, R., and Turesky, R. (1994) In vivo detection of 8-oxoguanine in DNA by using HPLC with electrochemical detection and 2,6-diamino-8-oxopurine as internal standard. Deauville Conference 94,13th International Symposium on Microchemical Techniques and 2nd Symposium on Analytical Sciences, Montreux, Switzerland.
