Photochemistry and Photobiology Vol. 55, No. 6, pp. 823-830, 1992 Printed in Great Britain. A11 rights reserved

0031-8655/92 S05.00+0.00 Copyright @ 1992 Pergamon Press Ltd

THE PHOTOCHEMISTRY OF 5-METHYLCYTOSINE AND 5-METHYL-2‘-DEOXYCYTIDINE IN AQUEOUS SOLUTION LECHCELEWICZ* and MARTIN D. SHETLARt Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143, USA

(Received 2 October 1991; accepted 23 November 1991) Abstract-The nucleobase 5-methylcytosine (I) is a minor component of eukaryotic DNA thought to be important in regulation of gene expression. The photochemical reactions of this nucleobase and its 2’-deoxyribonucleoside, 5-methyl-2‘-deoxycytidine(11), in water have been studied. These reactions lead, respectively, to 3-amino-2-methylacrylamidine(Ib) and 3-(2-erythro-~-pentopyranos-l-yl)amino2-methylacrylamidine (IIb) as the main photoproducts. The structure of the photoproducts was established by spectroscopicmethods (‘H and ‘”C NMR, UV spectroscopy, electron impact and liquid secondary ion mass spectrometry); in the case of Ib, confirmatory evidence was obtained by chemical methods (photolysis of 5-methyl[2-13C]cytosine, hydrolysis of N-carbomethoxy-3-amino-2-methylacrylamidine and reaction of Ib with 1,l’-carbonyldiimidale to give I). The quantum yield for formation of Ib was determined to be 1.8 x at pH 7.5 while the quantum yield for formation of IIb has a lower value of 0.2 x lo-” at pH 7.5. These quantum yields depend strongly on pH and reach maximum values of 2.0 x loW3at pH 7.0 (Ib) and 0.6 x lo-” at pH 5.0 (IIb). The mechanism of formation of Ib (or IIb) is proposed to involve nucleophilic attack of water on the C-2 position of photoexcited I (or 11), followed by ring opening and decarboxylation of an intermediate carbamic acid.

becomes saturated (Wang, 1976; Cadet and Vigny, 1990). Although an early report indicated that a The nucleobase 5-methylcytosine (5-MeCyt) is a photoreaction occurred, relatively few studies have minor component of most eukaryotic DNA. Evibeen made of photoreactions of 5-MeCyt and its dence has accumulated over the years that “methylnucleosides in water or aqueous inorganic buffers. ation” of CpG dinucleotides is involved in the reguAs early as 1960, Wierzchowski and Shugar (1960) lation of gene expression (Adams and Burdon, pointed out that irradiation of 5-MeCyt in buffered 1984; Razin et al., 1984; Holliday et af., 1990; Clawaqueous solution (pH 8.5) leads to the formation of son et af., 1990). 5-Methylcytosine (5-MeCyt)S has a photoproduct with an increased extinction coefbeen also found in transfer RNA (McCloskey and ficient and red-shifted,,,A (285 nm), as compared Nishimura, 1977; Apirion, 1984), ribosomal RNA to the parent compound; the product itself could (Apirion, 1984; Clawson et al., 1990), and messennot be isolated for further characterization. The ger RNA (Apirion, 1984). The photochemical reacsame workers indicated that irradiation of 5-methyltions of uracil, thymine and cytosine, the three 2’-deoxycytidine (5-MedCyd) at pH 7 led to only a major pyrimidine bases occumng in nucleic acids, small increase of absorption in the range between have been extensively studied during the past 30 270 and 310 nm, while at pH 9 there was an increase years (Wang, 1976; Cadet and Vigny, 1990). It has in absorbance over the whole region of the specbeen established that among the main phototrum. Ehrlich and Dove (1983) examined the photoreactions of these pyrimidine bases are dimerizreactivity of 5-MedCyd at 254 nm in water, both in ation, to form cyclobutane dimers and (6,4) ice and in fluid solution, and indicated that there pyrimidine-pyrimidinone type products, and, in the was insignificant formation of photohydrate or case of uracil and cytosine, reaction with water to photodimer. These authors also remarked on a form photohydrates in which the 5,6-double bond small increase in UV absorption at wavelengths to the red of the parent absorption during the *Visiting scientist from the Faculty of Chemistry, Adam irradiation of 5-MedCyd. There is also a report on Mickiewicz University, Poznan, Poland during the the photoreactions of 5-MeCyt residues contained course of the work reported here. in DNA, when irradiated in aqueous media (Ehrlich tTo whom correspondence should be addressed. et al., 1986). Little formation of cyclobutane type $Abbreviations: DMU, 1,3-dimethyluracil; EI MS, electron impact mass spectrometry; SMeCyt, 5-methylcyto- photodimers from the 5-MeCyt residues was sine; SMedCyd, 5-methyl-2‘-deoxycytidine;HPLC, observed, in contrast to the high yield of this type high performance liquid chromatography; LSIMS(+), of photoproduct from thymine residues. Bama et liquid secondary ion mass spectrometry in the positive ion mode; TMS, tetramethylsilane; TSP, 3- al. (1988) isolated two photoproducts by reversed phase HPLC from the formic acid hydrolysate of (trimethylsilyl)propionic-2,2,3,3-d4acid, sodium salt. INTRODUCTION

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LECHCELEWICZ and MARTIN D. SHETLAR

irradiated (A = 254 nm o r A = 302 nm) D N A containing 5-MeCyt labeled with tritium in the methyl group. O n e product was suggested to be monomeric whereas the second was thought t o result from cyclobutane-type photodimerization with an adjacent cytosine. However, the authors did not present any spectroscopic evidence relevant to the structure of these photoproducts. Recently, it has been shown that both 5-MeCyt and 5-MedCyd undergo photoisomerization via a Dewar intermediate to give 3-ureidoacrylonitriles (Shaw and Shetlar, 1990). This reaction occurs cleanly in acetonitrile; it also takes place in water, although with smaller quantum yields. In other work, it has been found that 5-MeCyt forms ring opened photoproducts with alcohols (Shaw and Shetlar, 1989) and aliphatic amines (Shetlar et al., 1988). Recently, it has been shown that 5-MedCyd also reacts with ethylamine to form opened ring products (Hom, 1991). Analogous opened ring structures are almost certainly the precursors of the photoexchange products formed in the photoreaction of 5-MeCyt with L-lysine (Donvin et al., 1988). It is interesting t o note that U V light can activate gene expression (see the discussion in Barna et al., 1988, and the references therein); this is possibly the result of photoinduced chemical changes in the 5-MeCyt residues. For example, Bednarik et al. (1990) have recently presented evidence that “DNA methylation” preserves the latent state of human immunodeficiency virus (HIV), which can be activated by U V light. In the present paper, we report our work on the isolation and characterization of products obtained from photochemical reactions of 5-MeCyt and 5MedCyd in water. MATERIALS AND METHODS

nitrogen (99.997%, Liquid Carbonic, Chicago, IL) through the solution to be irradiated. Ultraviolet spectra were run on a Cary 118C spectrophotometer (Varian Instruments, Palo Alto, CA) or measured “on the fly” using the rapid detection and data analysis capabilities of the Hewlett-Packard 1040A diode array HPLC detector. Secondary ion mass spectra were done on a Kratos MS50 (Ramsey, NJ), and electron impact mass spectra were run on a Kratos MS-9. ‘H and ‘’C NMR spectra were obtained on a General Electric GN-500 NMR spectrometer or General Electric QE-300 NMR spectrometer (Fremont, CA). Samples run in aqueous solvent were adjusted to pH 7.0 and twice rotovapped from D,O; they were then redissolved in 100% D 2 0 from Aldrich; TSP was used as the internal standard. Samples run in nonaqueous solution were dissolved directly in deuterated solvent, using TMS as an internal standard. Irradiation of 5-methylcytosine in water at 254 nm. A solution of 5-methylcytosine (I) (2 mM, 360 mL) in distilled water at pH 7.2 (pH was adjusted with aqueous sodium hydroxide solution), contained in two 180 mL quartz vessels, was irradiated at 4°C for 40 h. After this time, about 70% of the original I had reacted. The mixture was concentrated by rotary evaporation to a small volume (-3 mL) and filtered through a Millipore HAWP filter (Bedford, MA). The solution was then chromatographed on the preparative Hamilton PRP-1 HPLC column, using 2 mM aqueous lithium chloride eluent (pH 7.2) at a flow rate of 3.5 mumin; detection was at 285 nm. Because the product was slightly contaminated by I after the first HPLC pass, it was rechromatographed on the same column. The product, whose structure was established as Ib, was isolated in 32% yield, based on consumed I. (See Scheme 1 for product structures which, for illustrative purposes, are drawn in the E form.) Before spectroscopic analysis, Ib was desalted on a Hamilton PRP-1 column, using water as eluent. (In general, the HPLC peaks corresponding to acrylamidine type photoproducts were quite broad and, in the case of Ib, tailed significantly; this was true for each of the columns we employed in this work.) The purified I b was spectroscopically characterized; the resulting data are given below: UV(H,OgH 7.5): A,,, 285 nm, emax 14800, Amin 240 nm, emin 1500 (data at room temperature for a mixture of two isomers containing predominately one isomeric form; see Results and Discussion). ‘H NMR (D,O, TSP): 6 1.69 (s, 3H, CH3), 7.41 (5, lH, C’-H). ‘H NMR (CD’CN,TMS): 6 1.64 (s, 3H, CH’), 5.68 (broad s, 2H, C3-NH,), -, 7.02 (broad s. 3H. amidine), 7.31 (t, lH, J = 11 Hz). I3C NMR (D20, vs dioxane): 6 9.2 (9, CH,), 89.9 (s, C2), 146.6 (d, C3), 166.6 (s, C’). EI MS, m/z (rel. int.): !%(lob), 93(11), 83(84).

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General remarks. Chemicals were obtained from Sigma Chemical Company, St. Louis, MO (5-methylcytosinehydrochloride and 5-methyl-2‘-deoxycytidine), Aldrich Chemical Company, Milwaukee, WI (3-bromo-2-methylacrylonitrile and 1,l ‘-carbonyldiimidazole) and Medical Isotopes Inc., Concord, NH (ureaJ’C). Solvents for HPLC were from Fisher Scientificand NMR solvents were from Aldrich. Preparative HPLC was done on a Whatman Irradiation of 5-methyl-2’-deoxycytidine in water at 254 Partisil ODS-3 (9.2 x 250 mm, 10 pm particle size) reverse phase column (Clifton, NJ), or on a Hamilton PRP-1 nm. A solution of 5-methyl-2‘-deoxycytidine(11) (2 mM, reverse phase (4.5 x 200 mm) column (Reno, NV). Ana- 360 mL) in distilled water at pH 6.3 (pH was not adjusted), lytical HPLC was carried out on Whatman Partisil ODS- placed in two 180 mL quartz vessels, was irradiated at 4°C 3 reverse phase columns (4.7 x 100 mm, 5 pm particle for 80 h as described above. After this time, about 30% size), or a Hamilton PRP-1 reverse phase (4.5 x 100 of the original Il had reacted. The mixture was concenmm). A Rainin Rabbit ternary-gradient pumping system trated to a small volume (-3 mL) and filtered through a (Emeryville, CA) and a Hewlett-Packard 1040 A diode Millipore HAWP filter. The solution was then chromatoarray detector (Palo Alto, CA) completed the system used graphed on the preparative Hamilton PRP-1 HPLC colfor HPLC. Preparative irradiations were carried out at A umn using 2 mM lithium chloride (pH 7.2) at a flow rate = 254 nm in Vycor shielded 180 mL quartz reaction vessels of 3.5 mumin as eluent; detection was at 285 nm. Because placed in a Southern New England Ultraviolet Company the product had residual contamination with 11, the chroRP-100 reactor (Hamden, CT) housed in a cold room matography was repeated on the same column. (See Scheme (maintained at 4°C). Samples used for quantum yield 1 for structures.) The final product, a mixture of the measurements and pH profile determination were two anomers of 3-(2-erythro-~-pentopyranos-l-yl)amino-2irradiated eight at a time in 10 mL quartz tubes shielded methylacrylamidine(IIb), was isolated in 36% yield, based by Vycor filters and mounted in a “merry-go-round” on consumed 11. Before spectroscopic analysis, IIb was apparatus. Deoxygenation was carried out by bubbling desalted on the Hamilton PRP-1 column, using water as

Photochemistry of 5-methylcytosine

I,RrH 11, R

= 2'-DwxyrIbose

111

825

Is,RrH Ib, R = H Ila, R = P'-Deoxyriboso lib, R I P'Deoxyrlbore

Scheme 1. The probable reaction pathway followed by 5-methylcytosine and 5-methyL2'deoxycytidine when irradiated in water.

eluent; no resolution of the anomeric forms of IIb was observed. The resultant data are as follows: UV(H20, pH 7.5): A,,, 285 nm, emar 14700, Ami, 240 nm, em," 1800 (data for a mixture of pyranoid anomers at room temperature). 'HNMR (D20, TSP): 6 1.75 (s, CH,), 1.76 (s, CH,), (CH, signals integrated in ratio about 1:3.4), 1.25-2.36 (m, H2', H2", a-and p-pyranosyl), 3.83 (m. H4', a-pyranosyl), 3.61-4.01 (m, H3',H4', H5', H5", a- and p-pyranosyl), 4.20 (m,H3', p-pyranosyl), 4.66 (dd, HI', J,,*, = 2.6 Hz, J,.,- = 10.1 Hz, a-pyranosyl), 4.96 (dd, HI', J,,,, = 8.9 = 2.8 Hz,p-pyranosyl), 7.38 (s, l H , C3-H). Hz, I3C NMR (D,O, vs dioxane): 6 10.1 (CH,), 33.7 (C,'), 66.9, 68.2, 68.5, 85.4 (C4'), 93.3 (C*), 145.9 (C3), 167.4

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(Cl).

LSIMS( +), m/z 216(MH+), 238(M+Na+). EI MS, mlz (rel. int.) 200(2), 182(10), 164(18), 152(58), 136(42), 123(100), 117(22), 110(67), 99(17), 83(54). Synthesis of 5-methyl[2-'JC]cytosine. For this synthesis, we used a modification of the procedure of Tarsio and Nicholl (1957) for the synthesis of cytosine. To a solution of sodium (0.38 g, 16.4 mmol) in dry 1-butanol (30 mL) were added urea-W (0.50 g, 8.2 mmol, 99% 13C) and 3bromo-2-methylacrylonitrile (1.20 g, 8.2 mmol). The reaction mixture was refluxed for 3 h and evaoporated under reduced pressure. The residue was dissolved in I M sulfuric acid (30 mL) and the mixture was stirred for 30 min at room temperature. The mixture was neutralized with 1 M sodium hydroxide, evaporated to a small volume (-5 mL) and filtered through a Millipore HAWP filter. Preparative HPLC of the mixture on a Whatman Partisil ODS-3 reverse phase column, using 5 mM sodium chloride as eluent, afforded 5-methyl[2-13C]cytosine (0.58 g, 56% yield), whose NMR spectroscopic data are given below. 'H NMR (CD,OD): 6 1.90 (s, 3H, CH,), 7.27 (d, l H , J ~ z H= ~ 7 Hz, C"-H). 'T NMR (CD,OD): 6 159.6 (d, JczH6 = 7 Hz, C'). Irradiation of 5-rnethyl[2-'3C]cytosine in water at 254 nm. A solution of 5-methyl[2-13C]cytosine (2 mM, 360 mL) in distilled water at pH 7.2 (the pH was adjusted with aqueous sodium hydroxide solution), placed in two 180 mL quartz vessels, was irradiated at 4°C for 40 h as described above. After this time, about 70% of the 5methyl(2-13C]cytosinewas reacted. The mixture was concentrated to a small volume (-3 mL) and filtered through a Millipore HAWP filter. The solution was then chromatographed (twice) on preparative Hamilton PRP-1 HPLC column using 2 mM lithium chloride (pH 7.2) at a flow rate of 3.5 mL/min as eluent; detection was at 285 nm. The product Ib was isolated in 34% yield based on consumed I. The spectroscopic data (in particular, the I3C NMR) of the product from this experiment were identical with those obtained for Ib isolated from an irradiated aqueous solution of unlabeled 5-methylcytosine.

Hydrolysis of N-carbomethoxy-3-aminoacrylamidine. A solution of N-carbomethoxy-3-amino-2-methylacrylarnidine (5 mg, 0.032 mmol), obtained as described by Shaw and Shetlar (1989), in distilled water at pH 6.3 was stirred for 20 h at room temperature. The reaction mixture was analyzed by HPLC on an analytical Whatman Partisil ODS-3 reverse phase column, using 12 mM NaH,P04 (SOY0, pH 7.2)-methanol (ZOO/,) at a flow rate of 1.5 mL/min as eluent; detection was at 285 nm. After 20 h, about half of the starting material had disappeared and was found to have been converted to 5-methylcytosine and 3-amino-2-methylacrylamidine (Ib) (in a ratio of approximately 3:l). In the HPLC separation, 5-methylcytosine eluted with a retention time of 1.3 min; 3-amino-2-methylacrylamidine followed after 2.1 min and, finally, N-carbomethoxy-3-amino-2-methylacrylamidineeluted after 3 min. Reaction of 3-amino-2-methylacrylamidine (Ib) with 1, 1 '-carbonyldiimidazole. To a solution of 3-amino-2methylacrylamidine ( 5 mg, 0.05 mmol) in dry dimethyl sulfoxide (1 mL) was added 1,l'carbonyldiimidazole (10 mg, 0.06 mmol).The reaction mixture was stirred at room temperature for 1 h. Then a second portion of 1,l'-carbonyldiimidazole (10 mg, 0.06 mmol) was added and the mixture was stirred for 24 h. The mixture was diluted with water (2 mL) and analyzed by HPLC on a Whatman Partisil ODS-3 reverse phase column, using 12 mM NaH,PO, (80%. pH 7.2)-methanol (20%) at a flow rate of 1.5 mUmin as eluent; detection was at 272 nm. Essentially the only detectable compound was 5-methylcytosine (yield: greater than 90%). HPLC separation of isomeric forms of Ib and ZZb. After irradiation of purified Ib at 4°C (see below), two isomeric forms of Ib could be separated on an analytical Whatman Partisil ODS-3 HPLC column using 10 mM NaH,PO, (80%. pH 7.2)-methanol (20%) at a flow rate of 1.5 mUmin as eluent (detection at 285 nm); their individual UV spectra were obtained using the Hewlett-Packard diode array detector. Because the isomers rapidly interconvert, it was not possible to isolate them in pure form. The major isomer, at room temperature, had a retention time of 3.4 min and the following UV par15 700,Amin 240 nm,em," ameters: Amax = 285 nm, c, 900. (The c values were determined as described in the next paragraph.) The minor isomer of Ib had a retention = 291 time of 1.7 min and the following UV data: A,, nm, emax 10300, Ami, 241 nm, cmin 800. (It should be noted that the HPLC column used in this experiment was a different column than that used in the previous section describing the results on the hydrolysis of N-carbomethoxy-3-aminoacrylamidine,being almost new; this fact, along with the somewhat different concentration of phosphate in the aqueous component of the eluent, probably accounts for the difference in retention times for the major

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LECHCELEWICZ and MARTIN D. SHETLAR

isomer of Ib in these two experiments.) To separate the two corresponding isomeric forms of Ub, obtained after UV irradiation of purified IIb, a different eluent was used: 10 mM NaH,PO, (W%Fmethanol (10Y0).The separation was not as good as in case of Ib and estimation o f t values was not possible for each isomer; we saw no evidence for separation of the two pyranoid forms for either isomer. The major isomer, at room temperature had a retention time of 3.9 min with Amax at 285 nm and A,,, at 240 nm. The minor isomer of IIB had a retention time of 4.9 min and had a A,,, of 288 nm and Ami, at 241 nm. Determination of rhe t of Ib via use of the isosbestic point of the isomeric f o r m . When Ib or Ub was irradiated with 254 nrn light, it was found that the two isomeric forms of Ib (Ub) have isosbestic pionts at 300 nm (Ib) and 302 nm (Ub). The t values for Ib and IIb were determined at these isosbestic points as described below. An 0.2 mM solution of I (or 11) at pH 7.5 was irradiated with 254 nm light at 4°C in a UV cell in front of a germicidal lamp until about 7% of I (or II) was consumed. The amount of reacted I (or U) was determined by HF'LC on an analytical Whatman Partisil ODS-3 column. It was assumed that an equal amount of Ib (or IIb) was formed. Then t was calculated on the basis of the increase of UV absorption at the isosbestic point. (The small absorbances of I and H at the isosbestic points were corrected for.) We calculated the isosbestic point values to be 8200 (Ib) and cm 7000 (IIb). From these values we calculated and t,,, of the isomers of Ib, using spectra of the individual isomers obtained from application of the Hewlett-Packard HPLC diode array detector (see above). Quantum yield measurements. Quantum yields at 254 nm were measured, as described in detail by Shaw and Shetlar (1990), using the production of 6-hydroxy-5,6dihydro-l,3-dimethyluracil (DMU hydrate) from 1,3dimethyluracil as the actinometric reaction. The formation of DMU hydrate was followed by HPLC, using the Hewlett-Packard diode array detector. Aqueous solutions, 0.2 mM in I (or 11) at pH 7.5, were used. The amount of Ib (or Ub) was calculated from the increase in UV absorbance at the isosbestic point of the two isomeric forms of Ib (or Ilb). All quantum yields were measured using irradiation times such that only small conversions (less than 7%) of parent compound to product occurred. Determination of pH profiles for formation of I b and Ilb. Solutions that were 0.2 mM in concentration of I (or 11) were used. The pH was adjusted with aqueous sodium hydroxide or hydrochloric acid. The quantum yields were determined relative to that obtained at pH 7.5, as described above. All samples were kept on ice until analysis. Irradiation of I and Il at 313 nm, with and without acetone photosensitization. Two 0.2 mM samples of I (or U), one in water and the second in water-5% acetone and contained in 3 mL quartz cuvettes, were irradiated at equivalent positions in front of a Pyrex shield, using a lamp emitting principally at 313 nm. The samples were concentrated and then analyzed by HPLC. Irradiation of I and II at 254 nm in the presence and absence of oxygen. Two samples of 0.2 mM I (or H) in water were prepared at pH 7.5. One sample was deoxygenated by passage of nitrogen for 30 min,the second sample was left saturated with air. The two samples were simultaneously irradiated at 254 nm on a Vycor shielded merrygo-round, as described under General Remarks, and analyzed by HPLC.

methylacrylamidine (Ib) as the main photoproduct. Formation of small amounts of 2-methyl-3-ureidoacrylonitrile (Shaw and Shetlar, 1990) and some uncharacterized minor products was also detected by HPLC. (See Scheme 1 for the structures of the compounds discussed in this section.) The photoproduct was isolated from the reaction mixture by HPLC in 32% yield, based on consumed I, as described in Materials and Methods. Support for the assignment of the structure of Ib was obtained from 'H NMR, 13C NMR, mass spectrometric and UV data, as well as from experiments on the photolysis of 5-MeCyt-2-13C, studies of the hydrolysis of N-carbomethoxy-3-amino-2-methylacrylamidine and reaction of Ib with 1,l'-carbonyldiimidazole to give 5-MeCyt. One possibility that we carefully considered when making our structural assignment was that the photoproduct had the structure Ia, rather than Ib. While carbamic acid can exist in the gas phase (van den Berg et al., 1987), it is known that carbamic acid and its N-substituted derivatives are unstable in solution, spontaneously losing carbon dioxide to give the corresponding amine (March, 1985). However, Ia has the carbamic acid moiety attached to a conjugated system that could possibly stabilize it. Indeed, much of the spectroscopic data below is consistent with both structures. The high resolution electron impact mass spectrum of Ib showed the base ion peak at mlz 99.0799 corresponding to a molecular formula C4H9N3.This suggested that rupture of the 5-MeCyt ring occurred, accompanied by loss of the carbonyl moiety during the photochemical reaction leading to Ib; however, we could not rule out the loss of the carbonyl in the mass spectrometer. It was not possible to obtain the mass spectrum of Ib using the LSIMS technique, due to its high hydrophilicity. The 'H NMR spectrum was consistent with assignment of the structure as Ib, but again could not rule out Ia. When recorded in DzO, it showed a singlet at 7.41 ppm assigned to the olefinic proton at the 3 position and a singlet at 1.69 ppm assigned to the methyl group attached to the C-2 position. The 'H NMR spectrum of Ib, run in CD3CN, revealed additional features, as compared to that in DzO. In particular the H-3 proton appeared as a triplet centered at 7.31 ppm (J = 11 Hz) due to its coupling to 3-amino group protons, which themselves gave a broad singlet at 5.68 ppm. Selective decoupling of the signal at 5.68 ppm caused the collapse of the triplet at 7.31 ppm into a singlet. The amidine protons appeared as a broad singlet centered at 7.02 ppm. The I3C NMR spectrum of Ib, run in D 2 0 , indicated the presence of four nonequivalent carbon atoms in the molecule. Carbon signals at 89.9 ppm RESULTS AND DISCUSSION and 166.6 ppm, singlets in the non-decoupled specPhotoreaction of 5-methylcytosine in water trum, were assigned to quaternary carbon atoms COur results indicate that irradiation of 5-MeCyt 2 and C-1 respectively. A quartet at 9.2 ppm was (I) in water with 254 nm UV light gives 3-amino-2- ascribed to the methyl group carbon and a doublet

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Photochemistry of 5-methylcytosine

827

at 146.6 ppm to C-3. As with the mass spectrometric and proton NMR data, the 13C NMR does not unambiguously rule out the carbamic acid structure Ia for the isolated photoproduct. The lack of a carboxyl carbon signal might be a result of a weak signal due to a long relaxation time (although pulse delays of 15 s were used). To resolve this problem, we synthesized 5-methyl[2-13C]cytosinefrom ureaI3C and 3-bromo-2-methylacrylonitrile (see Materials and Methods) and irradiated this compound as before in water. It was established that 5methyl[2-13C]cytosinegave a photoproduct with the same properties as that obtained from unlabeled 5MeCyt, including an identical 13C NMR spectrum. The lack of an observable carboxyl carbon signal in the 13C NMR spectrum forces the conclusion that the loss of the carbonyl moiety of 5-MeCyt occurs during photochemical reaction or rapidly thereafter and strongly supports the proposed structure. Another line of evidence supporting Ib as the correct structure of the 5-MeCyt photoproduct comes from a study of the reaction of this compound 0.2 with 1,l'-carbonyldiimidazole.Although 3-amino62 min 2-methylacrylamidine has not been previously 0 rnin described, there is information available in the literature about 1-substituted 5-aminoimidazole-4Oe0 250nm 300nm 350nm carboxamidines (Montgomery and Thomas, 1972; k,nm Meyer et al., 1974; Fujii et al., 1989), which are structurally related. This class of compounds undergoes cyclization reactions with 1,l'-carbonyl- Figure 1. UV spectral curves generated when an 0.2 mM diimidazole to give 9-substituted isoguanosines aqueous solution of I at pH 7.5 was irradiated in the cold (Meyer et al., 1974). We have similarly found that (4°C) in front of a Vycor shielded germicidal lamp for 0, 2, 6, 14, 30 and 62 min. 3-amino-2-methylacrylamidine reacts with 1,l'-carbonyldiimidazole in dry dimethyl sulfoxide to yield almost exclusively I. Evidence that Ia is labile and as a product, instead gives Ib. This suggests that Ia, decarboxylates to form Ib came from a study of the even if initially formed as a product, is too unstable hydrolysis of N-carbomethoxy-3-amino-2-methyl-to be isolated under our conditions. acrylamidine (obtained as described by Shaw and Shetlar 1989). We have found that this compound Ib consists of a mixture of two isomers that are undergoes two slow parallel reactions in water, one thermally and photochemically interconvertible of them being ring closure to 5-MeCyt and the other hydrolysis of the methyl ester function. Ester Irradiation of an 0.2 mM aqueous solution of I hydrolysis should give N-carboxy-3-amino-2-methyl-at pH in the cold (4°C) over a 62 min period generacrylamidine (Ia) as the product. However, the ated the set of UV spectral curves shown in Fig. product that is isolated after hydrolysis is, in fact, 1. There was a small increase in absorption and 3-amino-2-methylacrylamidine (Ib). This implies movement of A,, to the red, relative to the absorpthat Ia, when formed by other than photochemical tion maximum of I; a considerable increase of means, rapidly decarboxylates to form Ib and absorption at higher wavelengths was also observed. strongly suggests that Ia is not an isolatable inter- When the irradiated solution was allowed to stand mediate in the photochemical reaction pathway at room temperature, a significant increase of absorption at ,,,A was observed (Fig. 2), leading from I to Ib. In summary, the spectroscopic evidence is all con- accompanied by a slight shift of,,A to the blue; at sistent with the structure of the main photoproduct the same time, the absorption at higher wavelengths from the reaction of 5-methylcytosine in water being decreased. Reirradiation of the latter solution in the Ib. The lack of a detectable carboxyl resonance in cold (4°C) caused the absorption spectrum to revert the I3C NMR spectrum of the photoproduct to one similar to that found after the initial obtained from 5-MeCyt-2J3C indicates that Ia is irradiation in the cold. Irradiation of Ib at 4"C, after not a viable alternative structure. Finally, the isolation from its parent reaction mixture by HPLC hydrolysis of N-carbomethoxy-3-amino-2-methyl-at room temperature, resulted in a decrease in the acrylamidine, which would be expected to yield Ia absorbance at the spectral maximum and appear-

LECHCELEWICZ and MARTIND. SHETLAR

828

spectral curves similar to those shown in Fig. 1 was generated. This suggested that similar photochemistry could be occurring in the two systems. Irradiation of 5-methyl-2'-deoxycytidine (2 mM) 1.4 2 min in water with 254 nm light was found to give 3-(2erythro-D-pentopyranos-l-yl)amin0-2-methylacrylamidine (IIb) as the main photoproduct. The photo1.2 product IIb was isolated from the reaction mixture by HPLC chromatography in 36% yield, based on consumed 11, as described in Materials and 1 .o Methods. The proposed structure of IIb is supported by a 8 5 large body of 'H NMR, 13C NMR, mass spectro.e 0.8 metric and UV data. The high resolution positive LSIMS of IIb gave a protonated molecular ion (MH') peak at mlz 216.1343 (100% intensity) cor0.6 responding to a molecular formula C,HI8N3O3.The expected (M+Na+) peak at mlz 238 was also clearly seen. The electron impact mass spectrum of IIb did 0.4 not show a molecular ion peak. The peaks with the highest masses, at mlz 200 and mlz 182, were assigned to (M*-CH3) and (M*-CH3-HZO), 0.2 respectively, on the basis of high resolution data. The 'H NMR spectrum of IIb, recorded in DzO, 20 rnin showed characteristic signals of the 3-amino-2methylacrylamidine moiety: a singlet at 7.38 ppm, 0.0 250nm 300nm 350nm corresponding to the olefinic proton at the 3 poslimn ition, and two singlets at 1.76 ppm and 1.75 ppm (in ratio about 3.4: 1) assigned to the methyl group Figure 2. UV spectral curves obtained when an 0.2 mM at the 2 position. The presence of these two methyl aqueous solution of I at pH 7.5 was irradiated in the cold resonances suggests the presence of two isomeric (4°C) in front of a Vycor shielded germicidal lamp for 62 forms of IIb. The splitting patterns of the proton min and was then allowed to warm at room temperature. (After 20 min, the sample had reached room temperature). signals in the sugar moiety (Shetlar et al., 1991 and references therein) indicates that IIb exists in aSpectra were run after 2, 6, 14 and 20 min. and p-pyranosyl forms. The anomeric proton signal ance of stronger absorbance at higher wavelengths; assigned to the predominant a-pyranosyl form of an isosbestic point was observed at 300 nm. These IIb appeared as a doublet of doublets centered at observations indicate that photoproduct Ib exists as 4.66 ppm with coupling constants Jle2,= 2.6 Hz and isomeric forms, presumably E and Z, that can be JIe2..= 10.1 Hz. The anomeric proton signal of the interconverted photochemically. Furthermore, one p-pyranosyl form was observed at 4.96 ppm as a of these forms (that with the longer,,,A and lower doublet of doublets with coupling constants J1'2, = value oft,,,) is thermally unstable with respect to 8.9 Hz and JlsY = 2.8 Hz. The I3C NMR spectrum the other. Chromatographic studies of irradiated of IIb, run in DzO, revealed four carbon signals solutions of Ib at room temperature showed that it corresponding to the acrylamidine moiety. The sigwas possible to separate the putative isomers on a nals at 145.9 ppm and 167.4 ppm were assigned, Whatman Partisil ODS-3 column [eluent: 10 mM respectively, to quaternary carbon atoms C-3 and NaH2P04(80%)-methanol (20%)] and obtain their C-1. The signal at 93.3 ppm was attributed to carbon individual UV spectra, using the Hewlett-Packard C-2 and that at 10.1 ppm to the methyl carbon. The diode array detector; however they could not be remaining five carbon signals were ascribed to the isolated individually in pure form (see Materials and major a-pyranosyl form of IIb. In particular, the Methods). We did not see evidence for a second signal at 33.7 ppm was assigned to carbon atom Cisomeric form in the NMR spectrum of Ib. Our 2' and that at 85.4 ppm to C-4'. results thus indicate that the isomer with the shorter It appears that opening of the pyrimidine ring of ,,,A and higher emax is predominant in amount, at 5-methyl-2'-deoxycytidinecauses rearrangement of least after workup for chromatography. the 2'-deoxyribofuranose moiety into 2'-deoxyribopyranose. There are precedents for this type of Photoreaction of 5-methyl-2'-deoxycytidine in rearrangement. For example, an analogous sugar water rearrangement occurs after sodium borohydride When an 0.2 mM aqueous solution of I1 at pH reduction of cytidine photohydrate; this reaction 7.5 was irradiated in the cold (4"C), a set of UV leads to the formation of the ring opened compound 1.6

20 min

Photochemistry of 5-methylcytosine

829

It is reasonable to assume that a neutral water molecule reacts with an excited species of 5-MeCyt or 5-MedCyd that has an electron deficient centre at C-2 to form a tetrahedral intermediate 111 (see Scheme 1). However, other factors must also play 0 0 a role if the detailed pH profiles of Fig. 3 are to be 0 0 understood. One of these factors could be the pK, of the excited state species of 5-MeCyt or 5-MedCyd 0 0 involved in the reaction. Proceeding further along the pathway of the proposed detailed mechanism, 0 0 the resulting tetrahedral intermediate 111 ring opens to give the unstable carbamic acid Ia (Ira), which then rapidly loses carbon dioxide to form the amidine Ib (IIb). The experiments supporting these latter segments of the pathway, namely experiments 2 4 6 8 10 12 on the hydrolysis of N-carbomethoxy-3-amino-2methylPH acrylamidine, are described in Materials and Figure 3. The pH dependences of quantum yields for for- Methods. In particular, the non-detectability of a mation of Ib from 5-MeCyt (0.2 mM in water) and IIb peak corresponding to Ia, following a process that from 5-MedCyd (0.2 rnM in water). should readily generate it, indicates that Ia can function as a precursor to I b and further suggests 1 -N-(~-ribopyranosyl)-3-N-(y-hydroxypropyl)urea that loss of carbon dioxide by Ia is a process that occurs on a faster time scale than that of HPLC (Miller and Cerutti, 1968). experiments designed to detect it. The photoreaction of 5-MeCyt with water to form Quantum yield studies I b is not photo-sensitized by acetone. Similarly, the The quantum yield for the photoreaction of 5- presence of oxygen had no significant effect on the MeCyt (0.2 mM) with water to form Ib was deter- rate of producton of Ib. These observations suggest mined to be 1.8 x at pH 7.5. As shown in Fig. that a triplet state is not involved in the production 3, this quantum yield depends strongly on pH and of Ib. In drawing this conclusion, however, we are reaches a maximum of 2.0 X at pH 7.0. We making the not unreasonable assumption that the also measured the quantum yield for the corre- triplet energy level of acetone lies above that of sponding photoreaction of 5-MedCyd (0.2 mM) to the lowest triplet energy level of 5-MeCyt (whose form IIb at pH 7.5 and found it to have a value of energy has, evidently, not been determined) and 0.2 x However, in this case, the pH profile that 5-MeCyt triplet states have long enough life(Fig. 3) shows that the quantum yield reaches a times such that the rate constant for oxygen quenchmaximum value of 0.6 x at pH 5.0. These ing is large enough to insure inactivation before values can be compared to a quantum yield of 2.8 reaction takes place. x obtained by Saito et a/. (1983) for the ring opening reaction in the thymidine (0.2 mM)- Acknowledgements-Research support from the NIH methylamine (10mM) system at its pH of maximum (Grant GM 23526) is gratefully acknowledged. We wish to thank Drs. John Cashman, Kellie Hom and Anthony reactivity, and 1.0 x obtained by Shetlar et Shaw for their helpfulness during the course of this investial. (1991) for the ring opening reaction in the 5- gation. Also acknowledged is the Bio-organic, Biomedical bromouracil (2 mM)-ethylamine (100 mM) system Mass Spectrometry Resource (A.L. Burlingame, Director), supported by NIH Division of Research at pH 10.0. Resources Grant RR01614. Mechanistic aspects

It appears likely that the photoreactions of 5MeCyt and 5-MedCyd with water proceed via a mechanism with similarities to those involved in the photoreactions of 5-methylcytosine, cytosine and related compounds with alcohols (Shaw and Shetlar, 1989) and amines (Shetlar et al., 1988;Horn, 1991). The first step, in a plausible mechanism for these reactions, is a combination of proton transfer and nucleophilic attack on the C-2 position of the excited pyrimidine base, followed by cleavage of the C2-N1 bond to form a ring opened product.

REFERENCES

Adam, R. L. P. and R. H. Burdon (1984) Molecular Biology of DNA Methylation. Springer-Verlag, New York. Apirion, D. (Editor) (1984) Processing of RNA. CRC Press, Boca Raton, FL. Barna, T., J. Malinowski, P. Holton. M. Ruchirawat, F. F. Becker and J.-N. Lapeyre (1988) UV-induced photoproducts of 5-methylcytosine in a DNA sequence context. Nucleic Acids Res. 16, 3327-3339. Bednarik, D. P., J. A. Cook and P. M. Pitha (1990) The role of DNA methylation in HIV latency. In Nucleic Acid Methylation (Edited by G. A. Clawson, D. B. Willis, A. Weissbach and P. A. Jones), pp. 153-169.

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Wiley, New York. stituted derivatives of adenosine cyclic 3‘,5’-phosphate. 1. Am. Chem. SOC. 96, 4%2-4966. van den Berg, K. J., C. B. Lebrilla, J. K. Terlouw and H. Schwarz (1987) Generation of carbamic acid Miller, N. and P. Cerutti (1968) Structure of the photohydration products of cytidine and uridine. Proc. Natl. (NH,C02H) and its radical cation as stable species in Acad. Sci. USA 59, 34-38. the gas phase. Chimia 41, 122-124. Cadet, J. and P. Vigny (1990) The photochemistry of Montgomery, J. A. and H. J. Thomas (1972) Nucleosides of 2-azapurines and certain ring analogs. J: Med. Chem. nucleic acids. In Bioorganic Photochemistry (Edited by 15, 182-186. H. Morrison). Vol. 1, pp. 1-272. Wiley, New York. Clawson. G. A., D. B. Willis, A. Weissbach and P. A. Razin, A., H. Cedar and A. D. Riggs (Editors) (1984) DNA Methylation: Biochemistry and Biological SignifiJones (Eds) (1990) Nucleic Acid Methylation. Wiley, cance. Springer-Verlag, New York. New York. Dorwin, E. L., A. A. Shaw, K. Hom, P. Bethel and M. Saito, I., H. Sugiyama and T. Matsuura (1983) Photoreaction of thymidine with alkylamines. Application to selecD. Shetlar (1988) Photoexchange products of cytosine tive removal of thymine from DNA. J. Am. Chem. SOC. and 5-methylcytosine with N--acetyl-L-lysine and L105, 956-962. lysine. 1. Photochem. Photobiol. B 2 , 265-278. Ehrlich, M. and M.-F. Dove (1983) Photolysis at 254 nm Shaw, A. A. and M. D. Shetlar (1989) Ring-opening photoreactions of cytosine and 5-methylcytosine with of 5-methyldeoxycytidine. Photobiochem. Photoaliphatic alcohols. Photochem. Photobiol. 49, 267-271. biophys. 6 . 121-126. Shaw, A. A. and M. D. Shetlar (1990) 3-UreidoacryloniEhrlich, M., M.-F. Dove and L.-H. Huang (1986) Phototriles: novel products from the photoisomerization of lysis of methylated DNA. Phorobiochem. Photobiophys. cytosine, 5-methylcytosine and related compounds. J. 11, 7379. Am. Chem. SOC. 112, 7736-7742. Fujii, T., T. Itaya, T. Saito, K. Mohri, M. Kawanishi and Shetlar, M. D., K. Hom, S.Distefano, K. Ekpenyong and T. Nakasaka (1989) Purines. XXXII. Synthesis and ring J. Yang (1988) Photochemical reactions of cytosine and fission of 3,9-dialkyladenines. Chem. Pharm. Bull. 37, 5-methylcytosine with methylamine and n-butylamine. 1504-1513. Photochem. Photobiol. 41, 779-786. Holliday, R., M. Monk and 5. E. Pugh (1990) DNA Shetlar, M. D., R. B. Rose, K. Hom and A. A. Shaw Methylation and Gene Regulation. The Royal Society, (1991) Ring opening photoreactions of 5-bromouracil London. and 5-bromo-2’-deoxyuridine with selected alkylamines. Hom, K. (1991) Photochemical reactions of 2‘-deoxycytiPhotochem. Photobiol. 53, 59544W. dine, 2’-deoxyuridine and related pyrimidine nucieos- Tarsio, P. J. and Nicholl (1957) Preparation of cytosine. ides and nucleobases with ethylamine. Ph. D. Disser1. Org. Chem. 22, 191-193. tation, University of California, San Francisco. Wang, S. Y. (Editor) (1976) Photochemistry and PhotoMarch, J. (1985) Advanced Organic Chemistry, p. 786. biology of Nucleic Acids, Vol. 1. Academic Press, New Wiley. New York. York. McCloskey, J. A. and S. Nishimura (1977) Modified Wienchowski, K. L. and D. Shugar (1960) Further studies nucleosides in transfer RNA. Acc. Chem. Res. 10, on the photochemistry of pyrimidines, with special refer403-410. ence to 5- and 6-substititued derivatives in relation to Meyer, R. B., D. A. Shuman and R. K. Robins (1974) photoreactivation in the T-even bacteriophages. Acta A new purine ring closure and the synthesis of 2-subBiochim. Polon. 7 , 63-84.