Trapping of a Cross-Link Formed by a Major Purine Adduct of a

Apr 15, 2013 - These data indicate that there are a minimum of two populations of cross-links, one that forms rapidly, in minutes, and another that gr...
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Trapping of a Cross-Link Formed by a Major Purine Adduct of a Metabolite of the Carcinogen N‑Nitrosomorpholine by Inorganic and Biological Reductants Niangoran Koissi and James C. Fishbein* Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, United States S Supporting Information *

ABSTRACT: 3-Hydroperoxy-N-nitrosomorpholine in buffered aqueous media in the presence of calf thymus DNA was treated with a phosphine reductant to generate the transient αhydroxynitrosamine and subsequent diazonium ion that alkylated the DNA, as previously reported. Subsequent addition of hydride donors, for 30 min, followed by acid hydrolysis of the mixture allowed detection and quantification of 6-(2-{2-[(9H-purin-6yl)amino]ethoxy}ethoxy)-9H-purin-2-amine, the reduced crosslink formed from deposition, via the diazonium ion, of a 3oxapentanal fragment on O6-Gua, and condensation with N6Ade, presumably in the vicinity. Decreasing the temperature of the reaction mixtures and decreasing the pH modestly increased the yields of the trapped cross-link. Among three borohydride reductants, NaNCBH3 is superior, being ∼4 times more effective on a molar basis, as opposed to a hydride equivalent basis, than NaBH4 or Na(AcO)3BH. For trapping with NaNCBH3, it is deduced that the reaction likely occurs with the iminium ion that is in protonic equilibrium with its conjugate base imine. In an experiment in which the hydroperoxide was decomposed and NaNCBH3 was introduced after various periods of time, the amount of cross-link was observed to increase, nearly linearly, by ∼4-fold over 1 week. These data indicate that there are a minimum of two populations of cross-links, one that forms rapidly, in minutes, and another that grows in with time, over days. Reduced nicotinamide cofactors and ascorbate are observed to effect reduction (over 3 days) of the cross-links, confirming the possibility that otherwise reversible cross-links might be immortalized under biological conditions.

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the specific case involving the N6-Ade in Scheme 1. Indeed, evidence of cross-linking reactions has been obtained, and various conditions that affect trapping yields of cross-links using borohydride reductants have been examined. Observations of the kinetics of trapped cross-link evolution with time indicate that there are at least two populations of cross-links: those that are rapidly formed, in minutes, and a larger pool of cross-links that form over days. Finally, it is reported that there are biological reagents that are capable of “immortalizing” otherwise reversible cross-links.

INTRODUCTION N-Nitrosomorpholine (1) is a potent liver and respiratory tract carcinogen in rodents.1−3 There are numerous avenues of exposure to N-nitrosomorpholine in the human environment, and it is formed in humans endogenously.4−13 A major pathway of metabolism in the rat is hydroxylation α to the N-nitroso functionality, likely mediated by P450 enzymes, as indicated in Scheme 1.14,15 The α-hydroxy-N-nitrosomorpholine (2) metabolite rapidly decomposes to a diazonium ion capable of alkylating DNA.16 Purine alkylation by the diazonium ion in Scheme 1 has been reported, and the pattern of adduct distribution is qualitatively similar to that observed for the simpler methyl- and ethyldiazonium ions.17 A major adduct observed in reactions with DNA is that from capture of the diazonium ion by the O6 atom of Gua. Unlike the adducts of simple alkyldiazonium ions, those derived from direct reaction of the diazonium ion of N-nitrosomorpholine, and indeed the diazonium ions of all cyclic nitrosamines, contain a pendant aldehyde moiety. The promiscuity of this functional group is such that it was anticipated that it might participate in DNA cross-linking, possibly transiently, even involving, as it were, more than one “player”. In this work, we sought to obtain evidence of such liaisons by attempting to trap cross-links using reductants, as indicated for © 2013 American Chemical Society

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EXPERIMENTAL PROCEDURES

Materials. Except as noted, chemicals, reagents, and solvents obtained from commercial sources were of analytical or synthetic grade and were used as received. Deionized water was obtained in house and filtered for HPLC and LC/MS experiments. Solvents such as methylene chloride, acetonitrile, ethyl acetate, and hexanes were dried and distilled from calcium hydride. Methanol was distilled from magnesium turnings. Ether and tetrahydrofuran were dried and distilled from sodium. Methanol and acetonitrile used in HPLC and LC/MS experiments were purchased as “HPLC grade” solvents and Received: December 31, 2012 Published: April 15, 2013 732

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used for this analysis entailed initially 0% acetonitrile for 5 min followed by a 10 min linear gradient to 10% acetonitrile and then a 10 min linear gradient to 30% acetonitrile, which was kept isocratic for 5 min followed by a 5 min linear gradient to the initial condition. A period of 15 min of equilibration time was allowed between injections. Synthesis of the 6-(2-{2-[(9H-Purin-6-yl)amino]ethoxy}ethoxy)-9H-purin-2-amine Reduced Cross-Link Standard. The series of reactions indicated in Scheme 2 were used for the synthesis of an authentic standard of a prospective cross-linked base pair that could be stabilized in DNA by reduction and isolated after acid hydrolysis, as described in further detail, below. 2-[2-(2-Hydroxyethoxy)ethyl]isoindoline-1,3-dione (3a). To a solution of 2-(2-chloroethoxy)ethanol (5 mL, 47.4 mmol) in N,Ndimethylformamide (35 mL) was added potassium phthalimide (8.8 g, 47.51 mmol), and the reaction mixture was stirred for 20 h at 120 °C. The reaction mixture was left to cool, concentrated, and then diluted with 300 mL of dichloromethane. This solution was washed four times with 20 mL each time of water and once with 20 mL of saturated brine and then dried over anhydrous magnesium sulfate. The solvent was removed, and the residue was purified by flash column chromatography on silica gel (3:2 hexane/ethyl acetate mixture) to give the desired compound as a white solid (8.91 g, 76.13%) having an Rf of 0.10 (solvent, 3:2 hexane/ethyl acetate mixture). 1H NMR (CDCl3, 400 MHz): δ 7.85 (dd, 2H, J = 5.96, 3.24 Hz), 7.72 (dd, 2H, J = 5.96, 3.20 Hz), 3.92 (t, 2H, J = 5.48 Hz), 3.76 (t, 2H, J = 5.48 Hz), 3.72− 3.66 (2H, m), 3.61 (t, 2H, J = 5.52 Hz), 2.54 (t, 1H, J = 5.50 Hz). 13C NMR (CDCl3, 400 MHz): δ 168.60, 134.11, 132.10, 123.39, 72.25, 68.47, 61.81, 37.60. 2-[2-(2-{[2-Amino-9-((2R,4S,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}tetrahydrofuran2-yl)-9H-purin-6-yl]oxy}ethoxy)ethyl]isoindoline-1,3-dione (4). The TBDMS-protected guanine nucleoside (3) (443 mg, 0.89 mmol) was stirred at 0 °C with triphenylphosphine (2 equiv, 467 mg) and 2[2-(2-hydroxyethoxy)ethyl]isoindoline-1,3-dione (2 equiv, 420 mg) in 45 mL of dry THF. To this was added, dropwise, diisopropyl azodicarboxylate (DIAD, 2 equiv, 0.36 mL). The reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was evaporated to dryness. The mixture was dissolved in ether, and the precipitate was filtered off. The residue was dried again, redissolved in CH2Cl2, washed with water (10 mL) and saturated NaHCO3 (10 mL), dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude product was purified on a silica gel column with a hexane/ethyl acetate mixture (3:2) as the eluant to give 370 mg (58% yield) of compound 4 as a light brownish oil. 1H NMR (400 MHz, CDCl3): δ 7.84 (s, 1H, H-8), 7.78−7.65 (m, 4H, phthal), 6.29 (t, 1H, H-1′, J = 9.19 Hz), 4.87 (br, 2H, NH2), 4.56 (q, 3H, H-10, H-3′), 3.95 (q, 1H, H-4′), 3.87 (q, 4H, H-11, H-13), 3.81−3.73 (m, 4H, H-14, H-5′, H-5″), 2.55−2.50 (m, 1H, H, H-2′), 2.35−2.29 (m, 1H, H-2″), 0.89 [s, 18H, Si-C(CH3)3], 0.08 (s, 6H, SiCH3), 0.06 (s, 6H, Si-CH3). 6-[2-(2-Aminoethoxy)ethoxy]-9-((2R,4S,5R)-4-[(tertbutyldimethylsilyl)oxy]-5-{[(tert-butyldimethylsilyl)oxy]methyl}tetrahydrofuran-2-yl)-9H-purin-2-amine (5). To a solution of 4 (300 mg, 0.768 mmol) in THF (10 mL) was added hydrazine hydrate (0.1 mL, 2 mmol), and the solution was stirred at room temperature overnight. The resulting red solution contained a precipitate that was filtered over Celite and discarded. The filtrate was dissolved in 20 mL of CH2Cl2, washed with water (10 mL) and saturated NaHCO3 (10 mL), dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by silica gel chromatography with 2% MeOH in CH2Cl2 to provide 110 mg (45% yield) of compound 5 as a colorless oil. 1H NMR (400 MHz, CDCI3): δ 7.88 (s, 1H, H-8), 6.30 (t, 1H, H-1′, J = 9.19 Hz), 4.88 (br, 2H, NH2), 4.63 (t, 2H, H-10, J = 4.60 Hz), 4.56 (q, 1H, H-3′), 3.97 (q, 1H, H-4′), 3.86 (t, 2H, H-11), 3.81−3.68 (m, 4H, H-14, H-5′, H-5″), 3.57 (t, 1H, H-13), 2.86 (t, 2H, CH2-NH2), 2.55 (m, 1H, H-2′), 2.33 (m, 1H, H-2″), 0.89 [ds, 18H, Si-C(CH3)3], 0.09 (s, 6H, Si-CH3), 0.06 (ds, 6H, Si-CH3). 9-((2R,5R)-4-[(tert-Butyldimethylsilyl)oxy]-5-{[(tertbutyldimethylsilyl)oxy]methyl}tetrahydrofuran-2-yl)-6-[2-(2-

Scheme 1. Formation and Distribution of Cross-Links Derived from Activation of N-Nitrosomorpholine (1), or Reduction of 11, to Metabolite 2 in the Presence of DNA Followed by Reduction (H−) and Acid Hydrolysis To Yield Cross-Linked Nucleobases 9 and Un-Cross-Linked 10

were not further purified. Calf thymus DNA was purchased from commercial sources and purified after precipitation with ethanol by three successive washings and precipitations, at 0 °C, with a 70% ethanol/30% water mixture (v/v). Analytical Determinations. 1H NMR and 13C NMR spectra were acquired using a JEOL AS 400 (400 MHz) instrument. Measurements of pH were performed using an Orion pH meter (model SA720), with a combination electrode. Two point calibrations, using commercially available standards, were conducted prior to recording pH values. High-resolution mass spectra were obtained at the University of Maryland, Baltimore County Center for Biological Mass Spectrometry. HPLC. Some purification steps employed a Waters 2695 liquid chromatograph with a diode array UV detector monitoring at 260 nm using a Phenomenex Luna column (250 mm × 4.6 mm, 1 mL/min) and the following gradient elution method. The two solvents were 10 mM aqueous ammonium acetate (pH 6.9) and acetonitrile. The gradient entailed initially 5% acetonitrile for 5 min and then a 20 min linear gradient to 40% acetonitrile followed by a 20 min linear gradient to 60% acetonitrile, which was kept isocratic for 5 min followed by a 5 min linear gradient to the initial condition. A period of 15 min of equilibration time was allowed between injections. LC/MS/MS analyses of cross-links were conducted using a Water Micromass ZQ triple quadrupole mass spectrometer interfaced with a Waters 2695 HPLC system equipped with a BDS Hypersil C18 analytical column (150 mm × 2.1 mm, 0.2 mL/min). The gradient 733

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Scheme 2. Synthetic Scheme for Formation of Reduced Cross-Linked Nucleobases Gua and Ade

HPLC) to produce 8 as a faintly red solid in 90% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.30 (s, 1H, H-8), 8.18 (s, 1H, H-2), 8.05 (s, 1H, H-8), 7.77 (br, 1H, NH), 6.40 (br, 2H, NH2), 6.31 (t, 1H, H-1′), 6.17 (t, 1H, H-1′), 5.29 (d, 1H, OH), 5.26 (d, 1H, OH), 5.16 (t, 1H, OH), 4.98 (t, 1H, OH), 4.49 (t, 2H, H-10), 4.39 (q, 1H, H-3′), 4.34 (q, 1H, H-3′), 3.85 (q, 1H, H-4′), 3.80−3.75 (m, 3H, H-4′, H-11), 3.63−3.45 (m, 8H, H-13, H-14, 2 × [H-5′ and H-5″]), 2.67 (m, 1H, H-2′), 2.54 (m, 1H, H-2′), 2.26−2.17 (m, 2H, 2 × H-2″). HRMS (FAB+): m/z calcd for C24H32N10O8 [M + H]+ 589.2484, found 589.2473 6-(2-{2-[(9H-Purin-6-yl)amino]ethoxy}ethoxy)-9H-purin-2amine (9). Compound 8 (20 mg, 0.034 mmol) was dissolved in HCl (1 mL, 5 M) and 5 mL of water. The mixture was stirred at 80 °C for 30 min and then cooled and neutralized with NaOH (1 mL, 5 M). The mixture was purified by HPLC using the buffered aqueous acetonitrile gradient (see HPLC) to yield 10.52 mg (87%). 1H NMR (400 MHz, DMSO-d6): δ 12.36 (s, 1H, NH), 8.16 (s, 1H, H-8), 8.03 (s, 1H, H-8), 7.75 (s, 1H, H-2), 6.20 (br, 2H, NH2), 4.50 (t, 2H, CH2), 3.78 (t, 2H, CH2), 3.64 (m, 4H, 2 × CH2). λmax = 241 and 282 nm. 13C NMR (400 MHz, DMSO-d6): δ 160.56, 160.13, 153.60, 152.81, 149.98, 144.42, 138.70, 140.35, 120.52, 108.69, 69.45, 68.89, 65.29. HRMS (FAB+): m/z calcd for C14H16N10O2 [M + H]+ 357.1537, found 357.1527. 2-{2-[(2-Amino-9H-purin-6-yl)oxy]ethoxy}ethanol (10). To 0.176 mmol of the precursor aldehyde17 in 1 mL of water was added NaBH4 (10 mg). Stirring was continued for 10 min. The reaction mixture was neutralized and purified by HPLC as a single peak (90:10 H2O/MeCN mixture) on a 5 μm, C18, 250 mm × 21.2 mm semipreparative column to yield the product as a white solid (70%). 1H NMR (D2O): δ 8.38 (1H, s), 4.77 (2H, m), 3.99 (2H, m), 3.73 (4H, m). 13C NMR (D2O): δ 160.0, 156.2, 150.6, 142.3, 106.6, 71.8, 68.3, 67.4, 60.3. HRMS: m/z calcd for [M + H]+ C9H14N5O3 240.1091, found 240.1090. Trapping Cross-Links. Standard Protocol. A solution of 3hydroperoxy-N-nitrosomorpholine [HOONOMO, 11 (Scheme 1)]16 in acetonitrile was added to a buffered aqueous solution [0.05 M 70% anion cacodylic acid buffer (pH 6.9)] containing tris(2-carboxyethyl)-

{[9-((2R,4S,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-{[(tertbutyldimethylsilyl)oxy]methyl}tetrahydrofuran-2-yl)-9Hpurin-6-yl]amino}ethoxy)ethoxy]-9H-purin-2-amine (7). A solution of 5 (330 mg, 0.567 mmol) in ethanol (10 mL) was added to a solution of 5′,3′-di-O-TBDMS-6-chlorodeoxyadenosine (6) (0.85 g, 1.7 mmol) in 40 mL of ethanol. The mixture was heated slowly to 78 °C and stirred overnight under reflux. The solvent was removed in vacuo and the residue dissolved in 20 mL of CH2Cl2, washed with water (20 mL) and saturated NaHCO3 (20 mL), dried over Na2SO4, filtered, and evaporated to dryness under reduced pressure. The mixture was purified by silica gel chromatography with a CH2Cl2/ MeOH mixture (9:1) to give 220 g (37% yield) of compound 7 as a light red oil. 1H NMR (400 MHz, CDCI3): δ 8.33 (s, 1H, NH), 8.03 (s, 1H, H-2), 7.88 (s, 1H, H-8), 6.42 (t, 1H, H-1′), 6.30 (t, 1H, H-1′), 4.97 (br, 2H, NH2), 4.65 (t, 2H, H-10), 4.56 (m. 2H, 2 × H-3′), 4.00− 3.94 (dq, 2H, 2 × H-4′), 3.89−3.68 (m, 10H, H-11, H-13, H-14, 2 × [H-5′ and H-5″]), 2.64−2.55 (m, 2H, 2 × H-2′), 2.43−2.30 (m, 2H, 2 × H-2″), 0.89 [ds, 36H, 4 × Si-C(CH3)3], 0.09 (s, 12H, 4 × Si-CH3), 0.06 (ds, 12H, 4 × Si-CH3). 13C NMR (400 MHz, CDCI3): δ 160.98, 159.34, 154.80, 153.67, 153.04, 138.03, 137.53, 132.23, 123.11, 128.62, 128.43, 120.26, 115.60, 87.98, 87.70, 84.29, 83.73, 71.97, 70.02, 69.18, 65.64, 62.85, 41.14, 40.82, 26.08, 25.86, 18.38, 18.05, −4.60, −4.77, −5.34, −5.43. (2R,5R)-5-(2-Amino-6-{2-[2-({9-[(2R,4S,5R)-4-hydroxy-5(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-6-yl}amino)ethoxy]ethoxy}-9H-purin-9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-ol (8). Compound 7 (50 mg, 0.048 mmol) was dissolved in THF (2 mL), and to this mixture was added 0.5 mL of a 1 M solution of tetrabutylammonium fluoride (TBAF) in THF (0.5 mmol); this mixture was stirred at room temperature for 1 h. The colorless solution became purple in this time. The solvents were removed under reduced pressure, and the residue was dissolved in water (10 mL) and washed twice with CH2Cl2 (10 mL). The crude compound was extracted into the aqueous layer. The compound was purified by HPLC using a buffered aqueous acetonitrile gradient (see 734

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phosphine hydrochloride (TCEP) from a freshly neutralized stock and calf thymus DNA. The final concentrations were 10 mM αHOONOMO, 10 mM TCEP, and ∼0.5 mg/mL calf thymus DNA. The reaction mixture was stirred at room temperature for 30 s, during which the evolution of gas was observed. A solution of freshly prepared hydride donor (for details, vide inf ra) was added. The time of exposure to the reductant varied (for details, vide inf ra). In most experiments that used boron hydride reductants, after being exposed for 30 min, the solution was acidified to 0.1 M HCl and subjected to mild acid hydrolysis (30 min at 80 °C). The solution was cooled briefly, neutralized with NaOH (pH 7), and separated into three aliquots. One aliquot was used for LC/MS analysis of total O6-Gua alkylation that generated un-cross-linked 10 (Scheme 1) by monitoring for the MH+ ion at m/z 240.1. A second aliquot was used for LC/MS analysis of the reduced cross-link [9 (Scheme 1)] with monitoring for the MH+ ion at m/z 357.17, isolation, and quantification by analysis of a daughter fragment as described in Results. The third aliquot was used for determination of the total amount of Gua by LC with UV detection. The products of the reaction were identified by co-elution with authentic standards under two different elution protocols and verified occasionally by spiking a replicate injection with the standard. Product yields were calculated on the basis of interpolation from standard curves. Effect of Varying the Reducing Agent on Cross-Link Trapping. Calf thymus DNA was incubated as previously mentioned with 10 μM HOONOMO and TCEP at 21 °C. Cross-link trapping was conducted via addition of 10 mM NaBH4, NaCNBH3, or NaBH(OAc)3 and various concentrations of other organic and natural reductants, including dithiothreitol, NADH, NADPH, ascorbate, and GSH. Effect of the Variation of Temperature on Cross-Link Trapping. Calf thymus DNA was incubated as previously mentioned with 10 μM HOONOMO in 250 μL of cacodylic buffer (pH 6.9) at 21, 37, and 50 °C, and trapping at the same temperature employed treatment with NaBH4 or NaNCBH3 for 30 min. Effect of pH on Cross-Link Trapping. The reaction was conducted generally according to the standard protocol with the exception that the values of the solution pH were 5.0, 6.8, and 8.5 and were maintained by 0.055 M cacodylic acid buffers. Reduction of the crosslinks was effected by treatment with 10 mM NaCNBH3 for 30 min. Time Course of A−G Cross-Link Formation. Cross-link formation was monitored over 7 days. A batch reaction mixture was prepared and the reaction carried out as described in Standard Protocol using a final concentration of hydride donor NaCNBH3 of 10 mM. Aliquots were removed at various times and subjected to reduction (10 mM NaCNBH3 for 30 min) and hydrolysis as described in Standard Protocol.

Figure 1. LC/MS chromatogram and fragmentation pattern of the reduced cross-link, 9 (Scheme 1). (A) The bottom chromatogram is the signal monitored at m/z 357.3 of the synthetic standard [9 (Scheme 1)]. The top chromatogram is the signal monitored at m/z 357.3, starting at 22.5 min, of the experimental sample derived from the treatment of calf thymus DNA, as detailed in Experimental Procedures. (B) Fragmentation of the m/z 357.3 species at 23.9 min. Proposed structures are assumed on the basis of the nominal masses.

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RESULTS The yield of the un-cross-linked O6-Gua adduct, detected after reduction and depurination as 10 (Scheme 1), was 0.31% (±0.03), on the basis of four determinations. Evidence of cross-links formed by the aldehyde function of the O6-Gua adduct formed in the capture of the diazonium ion derived from α-hydroxy-N-nitrosomorpholine was sought in reactions with calf thymus DNA that were similarly subjected to reduction, depurination, and LC/MS analysis. This was guided by authentic standard 9 (Scheme 1) that was synthetically generated. A typical LC/MS profile is indicated in Figure 1A, where the upper chromatogram, in which monitoring at [M + H] m/z 357 was initiated at 22.5 min, is the experimental while the lower chromatogram is that of the synthetic standard. Collision-induced dissociation of the [M + H]+ m/z 357 parent ion of the standard in a triple quadrapole instrument gave rise to two major fragment ions with nominal masses of [M + H]+ m/z 162 and 206 (Figure 1B). These masses are consistent with structures depicted Figure 1B, presumably because of the cleavages indicated by the hash marks in Scheme 3. The most

intense ion, at m/z 162, was used for quantification. For reduction by borohydride reagents, the reduction was typically conducted for 30 min on the basis of the following observation. Comparison, at 21 and 37 °C, of reduction by 25 mM NaBH4 for 30 and 120 min generated yields of the reduced cross-link that decreased