Photochernrriry and Phorohrology. Vol. 29. pp. 253 259 0 Ptrgiimon Pre\* Lid.. 197’). Printcd in Grwt B r i h i n
PHOTOCHEMICAL AND FREE RADICAL INITIATED REACTIONS OF 1,3-DIMETHYLTHYMINE WITH ISOPROPANOL MARTIND. SHETLAR Department of Pharmaceutical Chemistry, School of Pharmacy. University of California. San Francisco, CA 94143, U S A . (Receiced 17 January 1978: ucceptrd 13 June 1978) Abstract-A photochemically induced reaction of 1,3-dimethylthymine(DMT) with isopropanol leads to the formation of four alcohol adducts. The products have been identified as the cis and /runs isomers of 5,6-dihydro-1,3-dimethyll-6-(2-hydroxy-2-propyl) thymine (I and II), 2.4-diaza-8-hydroxy2.4,6.8-tetramethylbicyclo[4.2.0]octan-1,3-dione (HI), and 5,6-dihydro-1,3-dimethyl-6-(2-oxo-l-propyl)thymine (IV). An acetone photosensitized reaction of DMT with isopropanol gives the same products in a similar relative yield distribution. In both of these reactions, cyclobutane dimers of DMT are produced as well. Free radical reactions of 2-hydroxyisopropyl radicals with DMT, initiated by decomposition of di-r-butyl peroxide, leads to formation of only one of the cis and trans isomers described above. along with 1,3-dimethyl-5-(2-hydroxy-2-methyl1-propy1)uracil (V). INTRODUCTION
The photoreactivity of thymine and its derivatives toward isopropanol and other alcohols is an area of importance to those studying the photochemistry of deoxyribonucleic acid (DNA). For example, the alcoholic amino acid threonine is one of the commonly occurring amino acids found in proteins in intimate contact with DNA in the living cell. The observations that histones (Martinson et a/., 1976), DNA polymerase (Markovitz, 1972), and RNA polymerase (Strniste and Smith, 1974), along with other proteins, can be crosslinked photochemically t o DNA raises the possibility that alcoholic amino acids, such as threonine, may be involved in the crosslink. The aliphatic alcohol isopropanol can be regarded as an analog of the amino acid side chain of threonine. While there are a number of studies of the photoreactivity of the purine bases adenine and guanine, along with their derivatives, toward isopropanol (for a review, see Elad, 1976), there is relatively little work reported on the photochemical reactions of the DNA base thymine and its derivatives toward isopropanol. Leonov et a/. (1973) and Leonov and Elad (1974) have studied the acetone sensitized photoadditions and the di-t-butyl peroxide (DBP) induced free radical additions of isopropanol t o thymine and its derivatives and found that products of structure type V were produced. Havron et a/. (1976) reported that both the 5’ and 3’ thymidine monophosphates form 6-~-hydroxyisopropyl adducts of structure type I or I1 when free radical reaction of the monophosphate with isopropanol is induced with > 300nm. Shetlar, DBP decomposed with light of i. in 1975, reported at the Williamsburg Symposium on “Protein and Other Adducts t o DNA” that 1,3-dimethylthymine (DMT) added isopropanol photochemically t o form products of structure type I and
11: similarly adducts were also reported to be formed when DBP was decomposed photochemically at i> 290 nm in the presence of D M T and isopropanol. In the present paper we report the details of our studies on the photochemical and free radical induced reactions of DMT with isopropanol. MATERIALS AND METHODS
1,3-dimethylthymine was synthesized according to the procedure of Davidson and Baudish (1926): gas liquid chromatography (GLC) indicated that it was pure. Isopropanol and acetone were Mallinkrodt spectroscopic grade and met specifications: di-r-butylperoxide (DBP) was a product of Matheson, Coleman & Bell and was used as received. A Hewlett-Packard 5700 A gas chromatograph equipped with a flame ionization detector was used for GLC analysis. Product mixtures were analyzed on a loft. x lj8 in. column of OV-17 on 100/120 mesh Supelcoport. At a temperature of 165’C. the peaks for DMT. 5,6-dihydro-1,3-dimethylthymine (VI) and the various addition products of DMT with isopropanol were all resolved: at 250 C the cyclobutane dimers of DMT were eluted in a reasonable time (10 min). Ultraviolet spectra were recorded on a Cary 118C spectrophotometer while I R spectra were obtained on a Perkin-Elmer 457. Fourier transform nuclear magnetic resonance (FT-NMR) spectra were r u n on a Varian XL-100 using tetramethyl silane (TMS) as an internal standard. Irradiations were carried out at ambient temperature using a Hanovia 450 W medium pressure mercury lamp in a water cooled quartz immersion well. For irradiations at i. > 260nm Corex filters were used to cut out the shorter wavelengths: the Corex filters were checked to be sure that they indeed did have the correct wavelength cutoff (Morrison and Maleski. 1972). For acetone sensitized reactions and for photolytic decomposition of DBP at i> 290nm. a Pyrex filter was used in the immersion well. Deoxygenation was achieved by bubbling prepurified oxygen-free nitrogen through the solution to be irradiated for 15 rnin. Agitation during photolysis was achieved using magnetic stirring. Liquid chromatography was carried out on a Whatman Partasil PXS-10 4.6 mm x 250 mm column using heptane:isopropanol:methanol mixtures. A n Altex 253
254
MARTIND.SHETLAR
100 pump and i i Chromatronix UV detector operating at 254 n m complctcd the high prcssurc liquid chromatography system.
products accounting for the rest, again as estimated by GLC. Material ,from several runs was combined and products were isolated using liquid chromatography in a multistage procedure. After injection of a 4.5 mg RESll LTS load (contained in 0.3m/ of isopropanol). an Plioroc,lii,rii;.s~,rr.!. of' D M T iri isoprnpirnol elution solvent of heptane: isopropanol :methanol Irradiation of deoxygenated solutions of DMT (l50:7:1.5V/V) was used for the first stage. At a flow (0.65mM) in isopropanol leads to formation of four rate of 9.9 m//min, I11 was eluted after 2.8 min folisopropanol adducts as well as at least three of the lowed by a fraction containing I. 11, and IV after dimethylthymine cycolbutane dimers. The progress of 3.6 min. Unreacted DMT followed at 4.5 min. Cyclothe rcaction could be followed by GLC. The following butane dimers could be eluted from the column, after GLC temperature program was found to have suffi- a number of runs were made. by washing with methcient resolution to allow detection of all of the photo- anol. The fraction containing I, I1 and IV was evaporproducts in one run: 165 C for 8min followed by ated to dryness, taken up in isopropanol to a concentration of about 15mg/m/, and injected on the ;I temperature increase of 16 /min until 280C is reachcd. whereupon a constant temperature is main- column in 0.2 m/ portions. In this second stage heptaincd for 4 min. Under these conditions. using 250 C tane:isopropanol:methanol(150:2:0.3 V/V) was used inject ion and detector temperatures. DMT was eluted as the eluent at 9.9m//min. Product IV was eluted aftcr 2.15 min followed by I (3.1 rnin). I1 (3.6min). 111 after 7min while a broad doublet peak containing (4.4 min). IV (5.1 min). and the three cyclobutane 1 and I1 eluted during the time period between 10 and dimcrs (11.9. 13.7 and 14.3 min). respectively. [An 22 min. It was necessary to rechromatograph the fracauthentic sample of DMT cyclobutane dimer mixture tion containing IV. after evaporation. using the same was prepared according to Morrison et trl. (1968).] solvent in order to obtain a highly pure sample of For preparative purposes loom/ of the 0.65mM IV. The fraction containing I and I1 was evaporated solution was divided among eight 15 m/ quartz tubes to dryness, taken up in isopropanol to a concenand these tubes. after deoxygenation. were irradiated tration of approximately 15 mg/m/, and chromatofor 15 h on the Corex shielded Hanovia reactor. After graphed as above. using the same solvent and sample this period of time. about three-fourths of the DMT injections of 0.1 m/. Three fractions were collected: had disappeared as estimated by GLC. Using the fact the first contained 11, the second a mixture of 1 and that all four adducts resemble one another very 11. and the third I. The structures of photoproducts I. 11. 111, and IV closely in atomic composition and mol wt. the area under the peaks of the GLC trace at 135'C were used were established using UV spectroscopy, IR specto cstimate the relative amounts of the four adducts troscopy, NMR spectroscopy. and mass spectrometry. I -1V. The results are 1(37",,). 11(14nJ, 1I1(3On,,). Molecular weights were established using low resolulV( IY",,).These figures must be regarded as approxi- tion chemical ionization mass spectrometry with isomate iis there is some overlap between the peaks cor- butane as the reactant gas: molecular formulae were responding to 111 and IV and considerable overlap determined using high resolution chemical ionization mass spectrometry with methane as the reactant gas. for I and 11. In the case of photoproduct I. the mol wt was found to be 214 and the high resolution mass spectral data was consistent with molecular formula of C,,-,HI8N2O3 (predicted protonated peak mass: 215.1395, found: 215.1383). The FT-NMR in CDCI, I or il I or II gave the following peaks: 63.22, 3H and 63.09. 3H. singlets (N-CH3). 63.16, IH. doublet (6-CHI, a pair of almost coinciding doublets, one with comM e x N 5 t ' 0 ponents 61.275 and 61.224 and the other with com$, CH2-C-CH, N H ponents at 61.275 and 61.217. 9H (5-CH-CH3) and Me Me 6-C-C(CH3)20H. Although additional weak peaks 111 IV appeared in the region between 6 = 2.7 and 3.0. probably corresponding to 5-CH resonances. we cannot assign these with certainty: both the 5-methyl protons and the proton on the 6-carbon would be expected Me Me to split the resonance due to the 5-CH proton and V VI make it difficult to observe clearly. The IR spectrum in dry alcohol-free chloroform showed amide carAt this concentration and degree of conversion, the bony1 absorption at 1700cm-' and 1650cm-' with adducts I-IV account for about 80",, of the product the 1650cm-' peak significantly the stronger of the in the reaction mixture with cyclobutane dimer two. It also showed a strong broad absorption cen-
Photochemistry ofdimethylthymine in isopropanol tered at 3450cm-', corresponding to the O H stretch and absorption centered at 2950 cm -' corresponding to CH stretching. The I R spectrum also showed the 'isopropyl split' (Conley, 1972) centered at 1350 cmThe UV absorption spectrum showed no absorption maxima above 230 nm. Photoproduct I reacts readily with acetic anhydride in pyridine to give a compound with a different retention time on GLC. The product is presumably the acetate of I ; acetylations of products analogous to I in the y-ray irradiated system containing DMT and ethanol lead to acetates (Brown et d., 1966). All of the above data are consistent with I having one of the two structures shown above. Photoproduct I1 had a rnol wt of 214 and the high resolution mass spectrum pointed to a molecular 1 8 N 2 0 3(predicted protonated peak formula of C mass 215.1395, found: 215.1399). The following NMR peaks were obtained by FT-NMR in CDCI,: 63.17, 3H, singlet, and 63.10, 3H, singlet (N-CH3), overlapping doublets with peaks between 61.29 and 61.18, 9H (5-CH-CH3 and C(CHJ20H), 6 3.12, overlapped doublet, 1H (6-CH). Again the 5-CH absorption could not be assigned with certainty, although weak peaks appeared between 6 = 3.0 and 2.7 that probably correspond to this proton. The NMR spectrum of I1 in CDCI, on the Varian A-60 showed a broad peak at (52.0 which disappeared on shaking with D 2 0 : this is presumably due to the isopropyl hydroxy group. The IR spectrum of I1 in dry alcohol-free CHCl3 shows amide carbonyl absorption at 1705 cm-' and 1660cm-' with the 1660cm-' absorption being the stronger of the two. A broad OH stretching vibration is centered at 3450cm-': there is strong absorption centered at 2940 cm- corresponding to CH stretching with the 'isopropyl ,split' centered at 1335 cm-'. The UV spectrum has no maxima above 230nm. Photoproduct I1 readily reacts with acetic anhydride in pyridine to give a product with a different retention time on GLC: this product is presumably the acetate (see discussion above for photoproduct I). All of these data are consistent with I1 having one of the structures shown previously. The mol wt of photoproduct 111 was established as 212 and the high resolution mass spectral data was consistent with a molecular formula of C I 0 H l 6 N 2 O 3 (predicted protonated peak mass: 213.1239, found: 213.1253). The FT-NMR spectrum in CDCI, showed the following peaks: 63.19, singlet, 3H and 6 3.00, singlet, 3H (N-CH,), 6 1.44, singlet. 3H and 61.41, singlet, 3H (5-C-CH3) and (8-C-CH3), 63.20, singlet. 1H (5-CH), AB quartet with components centered at 62.39 and 61.87. 2H, J = 13 H, (7-CH-,). Morrison and Maleski (1972) have reported that the NMR of 2,4-diaza-2,4,6-trimethylbicyclo[4.2.0]octan-1,3-dione shows the 5-C-CH3 at 6 1.38 and that the center of the multiplet corresponding to the hydrogens at the 7 and 8 positions occurs at 62.04. The IR spectrum of I11 run in dry alcohol-free CHCl,, possesses amide carbonyl absorption at 1660cm-' and 1710cm-' with the 1660cm-'
'.
255
absorption being significantly stronger. A broad OH absorption is centered at 3450cm-' and a strong absorption centered at 2950 cm ' corresponds to CH stretching. The low resolution NMR, run on the Varian A-60. in CDCI,, showed a broad peak at 62.72 which disappeared on shaking with DzO, providing independent confirmation of the presence of the hydroxyl group. The UV absorption of 111 showed a maximum at 227.5 nm (6 = 2527) and a minimum at 215.5 nm (E = 2075). There is only slight tail absorption above 265 nm, but the absorbance rises sharply below 265 nm. having an E of 731 at 253.6 nm. Similar behavior is exhibited by the cyclobutane type dimers of thymine (Herbert et d.,1969). Irradiation of a 0.2 m M solution of I11 in isopropanol at 254 nm with a Hg germicidal lamp in a quartz spectrophotometer cuvette lead to rapid generation of the absorption spectrum of DMT. Within 30min almost quantitative conversion to DMT was accomplished, based on absorbance at i.= 269.0 nm. After evaporation of this photolyzed solution to dryness, the residue was taken up in 10 p/ of isopropanol and 1 p/ was injected into the GLC: a peak with the same retention time as DMT was eluted at three different temperatures. Coinjection with authentic DMT lead to the same conclusion. This behavior is, of course, that expected of cyclobutyl derivatives of pyrimidine bases. In summary, the spectral and photoreversal data are in complete agreement with that expected for structure 111. The mol wt of photoproduct IV was found to be 212 and the high resolution mass spectral data implied a molecular formula of C I o H l , N 2 0 , (predicted protonated peak mass: 213.1239, found: 213.1244). The FT-NMR spectra run in deuterated acetone (99.987;))displayed the following peaks: 6 3.03, singlet, 3H and 62.97, singlet. 3H (N-CH,). 62.16, singlet, 3H, (COCH,). 6 1.05, doublet, 3H, J = 7 H2 (5-CH-CH,), 63.93, slightly broadened quartet, 1H (6-CH on DMT ring), 62.70, complex multiplet, 2H. (CH-CH2-COCH,). In addition, a multiplet structure corresponding to the 5-CH on the DMT ring is overlapped by the N-CH, peaks. The FT-NMR spectrum in CDC13 showed this latter multiplet moved out partially from under the N-CH, peaks. taking the form of a IH broadened complex multiplet. The IR spectrum of I V showed no evidence of O H absorption at concentrations of up to 42, in dry alcohol-free CDCI,. There was strong absorption centered at 2920 cm-'. corresponding to CH stretching, as well as amide carbonyl absorption at 1660cm-' and 1710cm-'. However. in contrast to the IR spectra of I, 11. and 111, the 1710cm-' and 1660cm-' absorptions were of comparable intensity. This is attributed to the additional carbonyl contained in the acetone-like moiety of IV: acetone itself absorbs at 1710cm-'. The UV spectrum of IV shows absorption starting to rise at 340nm, having values of ~ ( 3 3 0= ) 8.7, ~ ( 3 0 0= ) 37.3 and ~ ( 2 7 0 = ) 89.7. The ~
256
MARTIND. SHETLAR
absorbance starts to rise sharply below 270 nm. having an E of 481 at 250nm. There is an absorption maximum at 217.5 nm (c = 4790) and an absorption minimum at 214nm ( E = 4740). The values of E between 330 nm and 270 nm are comparable to those for acetone. Treatment of IV with acetic anhydride in pyridine under very stringent conditions (100°C for I h) did not lead to reaction as evidenced by GLC and NMR. In summary. the spectral and chemical data are all in accord with that expected of structure 1V. Acetonc p/ioto.sriisiti=erl rrtrctions of
DM T nith isopro-
ptIlUJ/
The acetone photosensitized reaction of DMT with isopropanol at E. > 290 nm was carried out in deoxygenated 0.65 m M solution which was S',, by volume acetone. Within 6.5 h approximately 95",, of the DMT was converted to product. The GLC of the photoreaction mixture showed four adducts with the same retention times as I. 11, 111, and IV. The products were isolated and NMR confirmed the identity of the adducts formed in the direct photolysis and the acetone sensitized reaction. The relative distribution of yield of the four adducts in the acetone sensitized reaction and the unsensitized reaction was quite similar. as determined by GLC pattern of peak intensities. In addition to the photoadducts I-IV at least three cyclobutane type dimers were formed. along with a small amount of 5.6-dihydro-1,3-dimethyIthymine (VI) (less than 2",,). The ratio of total adduct to total dimer was estimated to be about 4:l by GLC. Thc, DMT-isoproptrnol Lidducts 1. 11. 111 trrrd prit iiorj. phot opt'oclitc.t s
Iv m?
Studies of the photochemistry of DMT in isopropanol. using GLC at 135'C as a monitor of reaction progress. were made at low percentage conversion of DMT to photoproducts to determine if I. 11. Ill and 1V were primary photoproducts. In the case of the unsensitized reaction at i. > 260 nm (0.65 mM in DMT). the products all appeared when less than 5",, of the DMT had been converted to product: furthermore. the pattern of relative intensities of the GLC peaks for 1, 11, 111. and IV was closely similar to that found after 75",, conversion. Progressive conversion to product percentages higher than 5",, maintained the same pattern of peak intensities. Thus. it may be concluded that I. 11. 111, and IV are all primary photoproducts. Similar results were obtained for the acetone sensitized reaction of DMT (0.65 mM) with isopropanol at i. > 290 nm: analogous conclusions can. therefore. be drawn for this situation as well. Concentrot ion qffecrs on the DM Tisopropanol photorruction
The photochemical reaction of DMT with isopropanol was run at a tenfold higher concentration of DMT than was normal in this study (6.5mM). After 42 h irradiation at i. > 260 nm approximately 900, of
the DMT was converted to product The DMT cyclobutane dimers were the predominant products. However, at least 1004, of the product consisted of adducts I, 11, 111 and IV. The distribution of photoadduct yields among the four adducts. as studied by GLC, was closely similar to that obtained for the same reaction in the 0.65 mM solutions described above. Thus. a tenfold concentration change in DMT appears to have no significant effect on the distribution of yields of adduct formed. In addition to the four adducts and cyclobutane dimers found in this more concentrated system, a small amount of V1 was detected by GLC. This is in contrast to the situation in the reaction mixture resulting from irradiation of DMT at 0.65 mM solution where VI was not detected. Free radical initiator induced chemistry in the DMTisopropanol system
DBP (di-t-butyl peroxide) is a free radical initiator which. upon photolysis with light of i. > 290nm. decomposes to form t-butoxy radicals. In isopropanol these radicals readily abstract hydrogen to form a-hydroxy isopropyl radicals. In an effort to determine the similarities and differences between the photochemistry of DMT in isopropanol and the chemistry induced by a-hydroxy isopropyl radical attack on DMT. we decomposed 10m/ of DBP in 190mf of isopropanol which was 6.5 mM in DMT, with light of i. > 290 nm. After 28 h the D M T was about 90:; converted to products. The reaction mixture. after evaporation, was taken up to a concentration of 30 mg/m/ in isopropanol and loads of 0.3 m/ were injected onto the Partasil PXS column using heptane:isopropanol: methanol (150:6:0.3) with the eluent pumped at a rate of 5m//min. Three peaks were detected by the UV monitor and collected. Fraction 1 contained photoadduct 11. identified by the identity of its NMR with that obtained from the DMT-isopropanol reaction mixture and by the fact that its retention time on GLC was the same as that of 11. Fraction 2 was identified as unreacted DMT containing some I1 as an impurity. Fraction 3 consisted of material whose structure was determined to be that described by structure V. Adduct V has been reported previously by Elad and co-workers (Leonov et ul.. 1973). but its spectral characteristics have not been recorded. We found that V has the following properties. The low resolution mass spectrum gives the expected mol wt of 212. The UV spectrum shows a maximum at 272.4nm ( E = 6595) and a minimum at 2380 ( E = 1450). The NMR spectrum, run on the Varian A-60 in CDC13 shows the following peaks: 63.35 and 63.38, both singlets and both 3H (N-CH,): 1.21 singlet. 6H (C(CH,),OH: 2.54: singlet, 2H (5-C-CH2-): 7.06 singlet, 1H (6-CH). The IR spectrum showed broad amide carbonyl absorption centered at 1650cm-' and a second amide carbonyl peak of lesser intensity at 1700cm-'. A broad peak, corresponding to the O H stretch was centered at
Photochemistry of dimethylthymine in isopropanol
3450 cm- ' while absorption corresponding to CH stretching occurred centered at 2990 cm- The 'isopropyl split' appeared in the spectrum centered at 1360cm-'. These data are all in accord with the structure V reported by Elad and co-workers for this compound. The relative amounts of I1 and V in the DBP initiated reaction mixture was determined by isolation and weighing in conjunction with NMR. In this experiment I1 and D M T were collected in one fraction and V in a second. Fraction 1 and 2 were weighed and the relative amounts of D M T and I1 in fraction 1 were determined by comparing integrated areas of peaks in the NMR spectrum of fraction 1. Under our reaction conditions the relative yields of the two products were 73% V and 27% 11. In the DBP initiated reactions which worked optimally (see below), there was no evidence of formation of I, I11 and IV. We also ran the same reaction at 0.65 mM in DMT-isopropanol systems using the same conditions. Again, I1 and V were the major products as evidenced by CLC and there was no evidence for formation of l, 111, and IV. The rate of reaction is much slower in these more dilute systems. In the absence of DBP, reaction did not occur. In some experimental runs on the DMT-DBP-isopropanol system, we found that the reaction product mixture changed with irradiation time. At low irradiation times (lOh), the reaction mixture was as described above. At longer times. photoproducts I, 111, and IV, as well as cyclobutane dimers, started to appear: after 28 h the reaction mixture strongly resembled that from a DMT-acetone-isopropanol reaction mixture with the amount of 11 exaggerated and, of course, V present. This is presumably due to the accumulation of acetone in the system due to the decomposition of t-butoxy radical to acetone and methyl radicals. Why this occurs in some runs and not in others is unclear.
'.
Possible occurrence of V irr the DMT-isopropanol sy.stern
Using the GLC at 180C. a search for the occurrence of V in irradiated DMT-isopropanol and DMT-acetone-isopropanol systems was carried out. At this temperature, V was eluted after 7.5 min. Injection of a l o p / sample of the reaction mixture (30 mg/m/) resulting from irradiation of D M T (6.5mM) in isopropanol indicated that V might be present in small amounts. The DMT-isopropanol reaction mixture was chromatographed by HPLC using the conditions established for the DMT-DBPisopropanol reaction mixture. The fraction eluting at the retention time of V showed slight absorbance according to the UV monitor and was collected. After evaporation, the collected material was taken up in isopropanol and injected onto the GLC: a peak with a retention time identical with an authentic sample of V was eluted from the column. This combination of identical retention times by both GLC and HPLC
251
is suggestive that a small amount of V may be formed in the photolyzed DMT-isopropanol system. However, because the amount is small compared to the amounts of the other photoproducts formed, it was not feasible to collect sufficient material to definitely establish that the product was indeed V. The DMT-acetone-isopropanol system (0.65mM) showed no evidence for formation of V. Chemical properties of I , 11, 111 and I V .
Using GLC, the stabilities of the photoadducts 1. 11, I l l and IV toward heat, acid, and base were determined. For these studies a reaction mixture containing I, 11, Ill and IV at a total photoadduct concentration of 20mg/m/ was used. Heating at 1 W C in distilled water for 15min had no effect on the total or relative amount of each adduct. Treatment with 1 N HCI at room temperature for 15min resulted in decomposition of I11 with an accompanying increase in amount of a compound with the same retention time as DMT. The photoadducts I, 11, and 1V appeared to be stable in this situation as well as to more extended treatment for 1.5 h. The GLC of the reaction mixture, when dissolved in 0.1 M NaOH and allowed to stand for 5 min, showed that 111 was again decomposed while a peak eluting with the same retention time as DMT appeared: the peak corresponding to IV was also decreased significantly in intensity. After 30min of this treatment, I and 11 appear to be stable. After 30min in 1 N NaOH. all photoadducts were decomposed. DlSCUSSlOY
Types of adducts formed Our results indicate that DMT shows an interesting set of photoaddition reactions in the presence of isopropanol. The photoaddition reactions of cx-methylz,B-cycloalkenones with alcohols do not appear to have previously been studied, although Dauben et al. (1968) have studied the cycloaddition (dimerization) reactions of 2-methylcyclohex-2-en-1-one.However, compounds 1 and 11 are the types of photoproduct which might be expected, based on the known photochemistry of other z,S-unsaturated cyclic ketones in alcoholic solution. For example, 2-cyclopentan-I-one adds isopropanol at the 3-position to form 3-(2-hydroxy-2-propy1)-cyclopentanone(Pfau rr ul., 1962) while 1,3-dimethyluracil adds isopropanol (Leonov rt a/., 1973). ethanol (Shetlar, 1974), and methanol (Shetlar, 1976) at the 6-carbon to form r-hydroxyalkyl adducts. Several studies of the free radical initiated reactions of DMT and related compounds in alcoholic solution have appeared in the literature. Brown rt a / . (1966) showed that cis and truns ethanol adducts, analogous to I and 11, were formed when DMT and thymine were irradiated with prays in aqueous solution containing ethanol. Zarebska and Shugar (1972) confirmed and extended these results. Elad and co-
258
MARTIND. SHETLAR
workcrs (Lconov ct d.. 1973: Elad. 1976) have re- from isopropanol to form the a-hydroxyisopropyl radical (Elad, 1976). This radical then attacks the ported that thc DBP initiated reaction of isopropanol with DMT and thymine leads to products of structure 6-carbon of a ground state DMT molecule to form type V : the acetone sensitized reaction of thymidine an adduct radical: the reaction is then completed by evidcntly leads to a similar type of adduct (Leonov abstraction of another H from an isopropanol to form 11. along with another a-hydroxyisopropyl radical. and Elad. 1974). Other types of products were not Thus, the potential for a free radical chain mechanism reported in these studies. More recently, Havron ef exists. (Note that excited states of DMT do not enter t r l . (1976) h a w found that at least one isomer of 5.6-dihydro-6-(2-hydroxy-2-propyl)-3’(5’)-thymidineinto this mechanism as DMT does not absorb signifimonophosphatc is formed from 3’(5’)-thymidine cant amounts of light under our reaction conditions monophosphatc when free radical reaction with iso- at 1 > 290 nm.) The experimental observations in the propanol is initiated with DBP. Adducts of structure DMT-isopropanol and DMT-DBP-isopropanol system are that I, rather than 11, is the major adduct type V werc not reported in this study. Reactions leading to the interesting compounds of of the 6-r-hydroxyalkyl type in the DMT-isopropanol system and that I. 111. and IV are not formed at all structure types 111 and IV appear to be unprecedented in reported studies of the photochemical reactions of in the DMT-DBP-isopropanol system. This rules out mechanisms for formation of I, I11 and IV that inr,/l-cycloalkenones with alcohols. volve attack on ground state DMT by r-hydroxyiso,M~,chtriisiic considerrit ions propyl radicals. Little can be said with certainty about the mechanAny mechanism proposed for formation of the photoadducts 1. 11. 111 and IV must take into account ism of formation of I1 in the DMT-isopropanol and DMT-acetone-isopropanol systems. The experiments the following observations. I . Adducts I, 11, 111. and IV are primary photo- in the DMT-DBP-isopropanol system certainly indicate that I1 can be formed by the chain type mechanproducts. both in the directly photolyzed DMT-isoism described above. but do not imply that this is propanol system and in the acetone sensitized reacthe only or even dominant pathway of formation. tion of DMT with isopropanol. With regard to possible alternative mechanisms of 2. The distributions of yields of photoadducts I. 11. I 1 1 and IV in the direct photolysis of DMT in isopro- formation of I and 11, it is interesting to note that panol and in the acetone sensitized formation of Fisher er cd. (1974) found that the photoaddition of cysteine to thymine proceeds by triplet state reaction ndducts in isopropanol display a close similarity. 3. Increase of the concentration of DMT in the di- which could be sensitized by acetone and that radical rectly photolyzed reaction from 0.65 mM// to combination reactions account for the main adduct. 6.5mM:/ does not have a significant effect on the rather than the type of mechanism involving attack of cysteinyl radicals on ground state thymine. pattern of relative photoadduct yields. A solid mechanistic proposal which incorporates the implications of the above observations must await further studies. One important type of mechanism can he ruled out. however. by considering the results from the DMT - isopropanol and DMT-DBP-isopropanol systems together. In the case of the DBP induced reaction of isopropanol with DMT to form 11, the probable sequence of reaction steps presumably starts with abstraction of H by the t-butoxy radical
Ackno,c/etfgenienrs-Research support from NIH Grant No. GM-23526 and from the Cancer Research Coordinating Committee of the University of California is gratefully acknowledged, Research support to the Magnetic Resonance Laboratory (Grant No. RR00892-01A1) of the University of California. San Francisco. from the NIH Division of Research Resources is also acknowledged. Helpful discussions of this work with Dr. Paul Ortiz de Montellano and Dr. Chi-Gen Lee are also gratefully acknowledged .
REFERENCES
Brown. P. E.. M. Calvin and J. F. Newmark (1966) Science 151, 68-70. Conley. R. T. (1972) f ~ f i t r r e tSprcrroscop!. l 2nd Ed.. p. 100. Allyn & Bacon. Boston. Davidson. D. and 0. Baudish (1926) J. Am. Client. Soc. 48, 2379-2383. Dauben. W. G.. G. W. Shaffer and N. D. Vietmeyer (1968) J. Orgnn. Clieni. 33, 406C4069. Elad. D. (1976) In Aging. Carcinogmesis. nntf Rotliririon Biology (Edited by K. C. Smith) pp. 243-260. Plenum Press. New York. Fisher. G. J.. A. J. Varghese and H. E. Johns (1974) Photochuni. Phorohiol. 20, 109-120. Hnvron. A,. J . Sperling and D. Elad (1976) Nircleic Acids Rrs. 3, 1715-1726. Herbert. M. A.. J. C . Le Blanc. D. Weinblum and H. E. Johns (1969) Phorocheti?. Phorohiol. 9, 33-43. Leonov. D. and D. Elad ( 1974) J. Am. Chmi. Soc. 96, 5635-5637. Leonov. D.. J. Salomon. S . Sasson and D. Elad (1973) P/iotocheni. Photobiol. 17, 465-468. Markovitz. A. (1972) Biochim. Biophys. Acra 281, 522-534. Martinson. H. G.. M. D. Shetlar and B. J. McCarthy (1976) Biocheniisiry 15, 200-2007. Morrison. H. and R. Maleski (1972) Mol. Plrofochem. 4, 507-511. Morrison. H.. A. Feeley and R . Kloepfer (1968) Clienr. Commun. 358-359.
Pholochemistry of dimethylthyrninc in isopropnol Pfau. M.. R. Dulon and M . Vilas (1962) Compt. R w d . 254, 1817 1818. Strniste. G. F. and D. A. Smith (1974) Biochemistry 13. 485-493. Shetlar. M. D. (1974) Terrlrhcdron Lett. 477-480. Shetlar. M. D. (1976) Photorhem. Photobiol. 24, 315-319. Zarebska. Z. and D. Shugar (1972) Inf. J . Rodiut. BifJ/.21, 101-114.
259
PHOTOCHEMICAL AND FREE RADICAL INITIATED REACTIONS OF 1,3-DIMETHYLTHYMINE WITH ISOPROPANOL MARTIND. SHETLAR Department of Pharmaceutical Chemistry, School of Pharmacy. University of California. San Francisco, CA 94143, U S A . (Receiced 17 January 1978: ucceptrd 13 June 1978) Abstract-A photochemically induced reaction of 1,3-dimethylthymine(DMT) with isopropanol leads to the formation of four alcohol adducts. The products have been identified as the cis and /runs isomers of 5,6-dihydro-1,3-dimethyll-6-(2-hydroxy-2-propyl) thymine (I and II), 2.4-diaza-8-hydroxy2.4,6.8-tetramethylbicyclo[4.2.0]octan-1,3-dione (HI), and 5,6-dihydro-1,3-dimethyl-6-(2-oxo-l-propyl)thymine (IV). An acetone photosensitized reaction of DMT with isopropanol gives the same products in a similar relative yield distribution. In both of these reactions, cyclobutane dimers of DMT are produced as well. Free radical reactions of 2-hydroxyisopropyl radicals with DMT, initiated by decomposition of di-r-butyl peroxide, leads to formation of only one of the cis and trans isomers described above. along with 1,3-dimethyl-5-(2-hydroxy-2-methyl1-propy1)uracil (V). INTRODUCTION
The photoreactivity of thymine and its derivatives toward isopropanol and other alcohols is an area of importance to those studying the photochemistry of deoxyribonucleic acid (DNA). For example, the alcoholic amino acid threonine is one of the commonly occurring amino acids found in proteins in intimate contact with DNA in the living cell. The observations that histones (Martinson et a/., 1976), DNA polymerase (Markovitz, 1972), and RNA polymerase (Strniste and Smith, 1974), along with other proteins, can be crosslinked photochemically t o DNA raises the possibility that alcoholic amino acids, such as threonine, may be involved in the crosslink. The aliphatic alcohol isopropanol can be regarded as an analog of the amino acid side chain of threonine. While there are a number of studies of the photoreactivity of the purine bases adenine and guanine, along with their derivatives, toward isopropanol (for a review, see Elad, 1976), there is relatively little work reported on the photochemical reactions of the DNA base thymine and its derivatives toward isopropanol. Leonov et a/. (1973) and Leonov and Elad (1974) have studied the acetone sensitized photoadditions and the di-t-butyl peroxide (DBP) induced free radical additions of isopropanol t o thymine and its derivatives and found that products of structure type V were produced. Havron et a/. (1976) reported that both the 5’ and 3’ thymidine monophosphates form 6-~-hydroxyisopropyl adducts of structure type I or I1 when free radical reaction of the monophosphate with isopropanol is induced with > 300nm. Shetlar, DBP decomposed with light of i. in 1975, reported at the Williamsburg Symposium on “Protein and Other Adducts t o DNA” that 1,3-dimethylthymine (DMT) added isopropanol photochemically t o form products of structure type I and
11: similarly adducts were also reported to be formed when DBP was decomposed photochemically at i> 290 nm in the presence of D M T and isopropanol. In the present paper we report the details of our studies on the photochemical and free radical induced reactions of DMT with isopropanol. MATERIALS AND METHODS
1,3-dimethylthymine was synthesized according to the procedure of Davidson and Baudish (1926): gas liquid chromatography (GLC) indicated that it was pure. Isopropanol and acetone were Mallinkrodt spectroscopic grade and met specifications: di-r-butylperoxide (DBP) was a product of Matheson, Coleman & Bell and was used as received. A Hewlett-Packard 5700 A gas chromatograph equipped with a flame ionization detector was used for GLC analysis. Product mixtures were analyzed on a loft. x lj8 in. column of OV-17 on 100/120 mesh Supelcoport. At a temperature of 165’C. the peaks for DMT. 5,6-dihydro-1,3-dimethylthymine (VI) and the various addition products of DMT with isopropanol were all resolved: at 250 C the cyclobutane dimers of DMT were eluted in a reasonable time (10 min). Ultraviolet spectra were recorded on a Cary 118C spectrophotometer while I R spectra were obtained on a Perkin-Elmer 457. Fourier transform nuclear magnetic resonance (FT-NMR) spectra were r u n on a Varian XL-100 using tetramethyl silane (TMS) as an internal standard. Irradiations were carried out at ambient temperature using a Hanovia 450 W medium pressure mercury lamp in a water cooled quartz immersion well. For irradiations at i. > 260nm Corex filters were used to cut out the shorter wavelengths: the Corex filters were checked to be sure that they indeed did have the correct wavelength cutoff (Morrison and Maleski. 1972). For acetone sensitized reactions and for photolytic decomposition of DBP at i> 290nm. a Pyrex filter was used in the immersion well. Deoxygenation was achieved by bubbling prepurified oxygen-free nitrogen through the solution to be irradiated for 15 rnin. Agitation during photolysis was achieved using magnetic stirring. Liquid chromatography was carried out on a Whatman Partasil PXS-10 4.6 mm x 250 mm column using heptane:isopropanol:methanol mixtures. A n Altex 253
254
MARTIND.SHETLAR
100 pump and i i Chromatronix UV detector operating at 254 n m complctcd the high prcssurc liquid chromatography system.
products accounting for the rest, again as estimated by GLC. Material ,from several runs was combined and products were isolated using liquid chromatography in a multistage procedure. After injection of a 4.5 mg RESll LTS load (contained in 0.3m/ of isopropanol). an Plioroc,lii,rii;.s~,rr.!. of' D M T iri isoprnpirnol elution solvent of heptane: isopropanol :methanol Irradiation of deoxygenated solutions of DMT (l50:7:1.5V/V) was used for the first stage. At a flow (0.65mM) in isopropanol leads to formation of four rate of 9.9 m//min, I11 was eluted after 2.8 min folisopropanol adducts as well as at least three of the lowed by a fraction containing I. 11, and IV after dimethylthymine cycolbutane dimers. The progress of 3.6 min. Unreacted DMT followed at 4.5 min. Cyclothe rcaction could be followed by GLC. The following butane dimers could be eluted from the column, after GLC temperature program was found to have suffi- a number of runs were made. by washing with methcient resolution to allow detection of all of the photo- anol. The fraction containing I, I1 and IV was evaporproducts in one run: 165 C for 8min followed by ated to dryness, taken up in isopropanol to a concentration of about 15mg/m/, and injected on the ;I temperature increase of 16 /min until 280C is reachcd. whereupon a constant temperature is main- column in 0.2 m/ portions. In this second stage heptaincd for 4 min. Under these conditions. using 250 C tane:isopropanol:methanol(150:2:0.3 V/V) was used inject ion and detector temperatures. DMT was eluted as the eluent at 9.9m//min. Product IV was eluted aftcr 2.15 min followed by I (3.1 rnin). I1 (3.6min). 111 after 7min while a broad doublet peak containing (4.4 min). IV (5.1 min). and the three cyclobutane 1 and I1 eluted during the time period between 10 and dimcrs (11.9. 13.7 and 14.3 min). respectively. [An 22 min. It was necessary to rechromatograph the fracauthentic sample of DMT cyclobutane dimer mixture tion containing IV. after evaporation. using the same was prepared according to Morrison et trl. (1968).] solvent in order to obtain a highly pure sample of For preparative purposes loom/ of the 0.65mM IV. The fraction containing I and I1 was evaporated solution was divided among eight 15 m/ quartz tubes to dryness, taken up in isopropanol to a concenand these tubes. after deoxygenation. were irradiated tration of approximately 15 mg/m/, and chromatofor 15 h on the Corex shielded Hanovia reactor. After graphed as above. using the same solvent and sample this period of time. about three-fourths of the DMT injections of 0.1 m/. Three fractions were collected: had disappeared as estimated by GLC. Using the fact the first contained 11, the second a mixture of 1 and that all four adducts resemble one another very 11. and the third I. The structures of photoproducts I. 11. 111, and IV closely in atomic composition and mol wt. the area under the peaks of the GLC trace at 135'C were used were established using UV spectroscopy, IR specto cstimate the relative amounts of the four adducts troscopy, NMR spectroscopy. and mass spectrometry. I -1V. The results are 1(37",,). 11(14nJ, 1I1(3On,,). Molecular weights were established using low resolulV( IY",,).These figures must be regarded as approxi- tion chemical ionization mass spectrometry with isomate iis there is some overlap between the peaks cor- butane as the reactant gas: molecular formulae were responding to 111 and IV and considerable overlap determined using high resolution chemical ionization mass spectrometry with methane as the reactant gas. for I and 11. In the case of photoproduct I. the mol wt was found to be 214 and the high resolution mass spectral data was consistent with molecular formula of C,,-,HI8N2O3 (predicted protonated peak mass: 215.1395, found: 215.1383). The FT-NMR in CDCI, I or il I or II gave the following peaks: 63.22, 3H and 63.09. 3H. singlets (N-CH3). 63.16, IH. doublet (6-CHI, a pair of almost coinciding doublets, one with comM e x N 5 t ' 0 ponents 61.275 and 61.224 and the other with com$, CH2-C-CH, N H ponents at 61.275 and 61.217. 9H (5-CH-CH3) and Me Me 6-C-C(CH3)20H. Although additional weak peaks 111 IV appeared in the region between 6 = 2.7 and 3.0. probably corresponding to 5-CH resonances. we cannot assign these with certainty: both the 5-methyl protons and the proton on the 6-carbon would be expected Me Me to split the resonance due to the 5-CH proton and V VI make it difficult to observe clearly. The IR spectrum in dry alcohol-free chloroform showed amide carAt this concentration and degree of conversion, the bony1 absorption at 1700cm-' and 1650cm-' with adducts I-IV account for about 80",, of the product the 1650cm-' peak significantly the stronger of the in the reaction mixture with cyclobutane dimer two. It also showed a strong broad absorption cen-
Photochemistry ofdimethylthymine in isopropanol tered at 3450cm-', corresponding to the O H stretch and absorption centered at 2950 cm -' corresponding to CH stretching. The I R spectrum also showed the 'isopropyl split' (Conley, 1972) centered at 1350 cmThe UV absorption spectrum showed no absorption maxima above 230 nm. Photoproduct I reacts readily with acetic anhydride in pyridine to give a compound with a different retention time on GLC. The product is presumably the acetate of I ; acetylations of products analogous to I in the y-ray irradiated system containing DMT and ethanol lead to acetates (Brown et d., 1966). All of the above data are consistent with I having one of the two structures shown above. Photoproduct I1 had a rnol wt of 214 and the high resolution mass spectrum pointed to a molecular 1 8 N 2 0 3(predicted protonated peak formula of C mass 215.1395, found: 215.1399). The following NMR peaks were obtained by FT-NMR in CDCI,: 63.17, 3H, singlet, and 63.10, 3H, singlet (N-CH3), overlapping doublets with peaks between 61.29 and 61.18, 9H (5-CH-CH3 and C(CHJ20H), 6 3.12, overlapped doublet, 1H (6-CH). Again the 5-CH absorption could not be assigned with certainty, although weak peaks appeared between 6 = 3.0 and 2.7 that probably correspond to this proton. The NMR spectrum of I1 in CDCI, on the Varian A-60 showed a broad peak at (52.0 which disappeared on shaking with D 2 0 : this is presumably due to the isopropyl hydroxy group. The IR spectrum of I1 in dry alcohol-free CHCl3 shows amide carbonyl absorption at 1705 cm-' and 1660cm-' with the 1660cm-' absorption being the stronger of the two. A broad OH stretching vibration is centered at 3450cm-': there is strong absorption centered at 2940 cm- corresponding to CH stretching with the 'isopropyl ,split' centered at 1335 cm-'. The UV spectrum has no maxima above 230nm. Photoproduct I1 readily reacts with acetic anhydride in pyridine to give a product with a different retention time on GLC: this product is presumably the acetate (see discussion above for photoproduct I). All of these data are consistent with I1 having one of the structures shown previously. The mol wt of photoproduct 111 was established as 212 and the high resolution mass spectral data was consistent with a molecular formula of C I 0 H l 6 N 2 O 3 (predicted protonated peak mass: 213.1239, found: 213.1253). The FT-NMR spectrum in CDCI, showed the following peaks: 63.19, singlet, 3H and 6 3.00, singlet, 3H (N-CH,), 6 1.44, singlet. 3H and 61.41, singlet, 3H (5-C-CH3) and (8-C-CH3), 63.20, singlet. 1H (5-CH), AB quartet with components centered at 62.39 and 61.87. 2H, J = 13 H, (7-CH-,). Morrison and Maleski (1972) have reported that the NMR of 2,4-diaza-2,4,6-trimethylbicyclo[4.2.0]octan-1,3-dione shows the 5-C-CH3 at 6 1.38 and that the center of the multiplet corresponding to the hydrogens at the 7 and 8 positions occurs at 62.04. The IR spectrum of I11 run in dry alcohol-free CHCl,, possesses amide carbonyl absorption at 1660cm-' and 1710cm-' with the 1660cm-'
'.
255
absorption being significantly stronger. A broad OH absorption is centered at 3450cm-' and a strong absorption centered at 2950 cm ' corresponds to CH stretching. The low resolution NMR, run on the Varian A-60. in CDCI,, showed a broad peak at 62.72 which disappeared on shaking with DzO, providing independent confirmation of the presence of the hydroxyl group. The UV absorption of 111 showed a maximum at 227.5 nm (6 = 2527) and a minimum at 215.5 nm (E = 2075). There is only slight tail absorption above 265 nm, but the absorbance rises sharply below 265 nm. having an E of 731 at 253.6 nm. Similar behavior is exhibited by the cyclobutane type dimers of thymine (Herbert et d.,1969). Irradiation of a 0.2 m M solution of I11 in isopropanol at 254 nm with a Hg germicidal lamp in a quartz spectrophotometer cuvette lead to rapid generation of the absorption spectrum of DMT. Within 30min almost quantitative conversion to DMT was accomplished, based on absorbance at i.= 269.0 nm. After evaporation of this photolyzed solution to dryness, the residue was taken up in 10 p/ of isopropanol and 1 p/ was injected into the GLC: a peak with the same retention time as DMT was eluted at three different temperatures. Coinjection with authentic DMT lead to the same conclusion. This behavior is, of course, that expected of cyclobutyl derivatives of pyrimidine bases. In summary, the spectral and photoreversal data are in complete agreement with that expected for structure 111. The mol wt of photoproduct IV was found to be 212 and the high resolution mass spectral data implied a molecular formula of C I o H l , N 2 0 , (predicted protonated peak mass: 213.1239, found: 213.1244). The FT-NMR spectra run in deuterated acetone (99.987;))displayed the following peaks: 6 3.03, singlet, 3H and 62.97, singlet. 3H (N-CH,). 62.16, singlet, 3H, (COCH,). 6 1.05, doublet, 3H, J = 7 H2 (5-CH-CH,), 63.93, slightly broadened quartet, 1H (6-CH on DMT ring), 62.70, complex multiplet, 2H. (CH-CH2-COCH,). In addition, a multiplet structure corresponding to the 5-CH on the DMT ring is overlapped by the N-CH, peaks. The FT-NMR spectrum in CDC13 showed this latter multiplet moved out partially from under the N-CH, peaks. taking the form of a IH broadened complex multiplet. The IR spectrum of I V showed no evidence of O H absorption at concentrations of up to 42, in dry alcohol-free CDCI,. There was strong absorption centered at 2920 cm-'. corresponding to CH stretching, as well as amide carbonyl absorption at 1660cm-' and 1710cm-'. However. in contrast to the IR spectra of I, 11. and 111, the 1710cm-' and 1660cm-' absorptions were of comparable intensity. This is attributed to the additional carbonyl contained in the acetone-like moiety of IV: acetone itself absorbs at 1710cm-'. The UV spectrum of IV shows absorption starting to rise at 340nm, having values of ~ ( 3 3 0= ) 8.7, ~ ( 3 0 0= ) 37.3 and ~ ( 2 7 0 = ) 89.7. The ~
256
MARTIND. SHETLAR
absorbance starts to rise sharply below 270 nm. having an E of 481 at 250nm. There is an absorption maximum at 217.5 nm (c = 4790) and an absorption minimum at 214nm ( E = 4740). The values of E between 330 nm and 270 nm are comparable to those for acetone. Treatment of IV with acetic anhydride in pyridine under very stringent conditions (100°C for I h) did not lead to reaction as evidenced by GLC and NMR. In summary. the spectral and chemical data are all in accord with that expected of structure 1V. Acetonc p/ioto.sriisiti=erl rrtrctions of
DM T nith isopro-
ptIlUJ/
The acetone photosensitized reaction of DMT with isopropanol at E. > 290 nm was carried out in deoxygenated 0.65 m M solution which was S',, by volume acetone. Within 6.5 h approximately 95",, of the DMT was converted to product. The GLC of the photoreaction mixture showed four adducts with the same retention times as I. 11, 111, and IV. The products were isolated and NMR confirmed the identity of the adducts formed in the direct photolysis and the acetone sensitized reaction. The relative distribution of yield of the four adducts in the acetone sensitized reaction and the unsensitized reaction was quite similar. as determined by GLC pattern of peak intensities. In addition to the photoadducts I-IV at least three cyclobutane type dimers were formed. along with a small amount of 5.6-dihydro-1,3-dimethyIthymine (VI) (less than 2",,). The ratio of total adduct to total dimer was estimated to be about 4:l by GLC. Thc, DMT-isoproptrnol Lidducts 1. 11. 111 trrrd prit iiorj. phot opt'oclitc.t s
Iv m?
Studies of the photochemistry of DMT in isopropanol. using GLC at 135'C as a monitor of reaction progress. were made at low percentage conversion of DMT to photoproducts to determine if I. 11. Ill and 1V were primary photoproducts. In the case of the unsensitized reaction at i. > 260 nm (0.65 mM in DMT). the products all appeared when less than 5",, of the DMT had been converted to product: furthermore. the pattern of relative intensities of the GLC peaks for 1, 11, 111. and IV was closely similar to that found after 75",, conversion. Progressive conversion to product percentages higher than 5",, maintained the same pattern of peak intensities. Thus. it may be concluded that I. 11. 111, and IV are all primary photoproducts. Similar results were obtained for the acetone sensitized reaction of DMT (0.65 mM) with isopropanol at i. > 290 nm: analogous conclusions can. therefore. be drawn for this situation as well. Concentrot ion qffecrs on the DM Tisopropanol photorruction
The photochemical reaction of DMT with isopropanol was run at a tenfold higher concentration of DMT than was normal in this study (6.5mM). After 42 h irradiation at i. > 260 nm approximately 900, of
the DMT was converted to product The DMT cyclobutane dimers were the predominant products. However, at least 1004, of the product consisted of adducts I, 11, 111 and IV. The distribution of photoadduct yields among the four adducts. as studied by GLC, was closely similar to that obtained for the same reaction in the 0.65 mM solutions described above. Thus. a tenfold concentration change in DMT appears to have no significant effect on the distribution of yields of adduct formed. In addition to the four adducts and cyclobutane dimers found in this more concentrated system, a small amount of V1 was detected by GLC. This is in contrast to the situation in the reaction mixture resulting from irradiation of DMT at 0.65 mM solution where VI was not detected. Free radical initiator induced chemistry in the DMTisopropanol system
DBP (di-t-butyl peroxide) is a free radical initiator which. upon photolysis with light of i. > 290nm. decomposes to form t-butoxy radicals. In isopropanol these radicals readily abstract hydrogen to form a-hydroxy isopropyl radicals. In an effort to determine the similarities and differences between the photochemistry of DMT in isopropanol and the chemistry induced by a-hydroxy isopropyl radical attack on DMT. we decomposed 10m/ of DBP in 190mf of isopropanol which was 6.5 mM in DMT, with light of i. > 290 nm. After 28 h the D M T was about 90:; converted to products. The reaction mixture. after evaporation, was taken up to a concentration of 30 mg/m/ in isopropanol and loads of 0.3 m/ were injected onto the Partasil PXS column using heptane:isopropanol: methanol (150:6:0.3) with the eluent pumped at a rate of 5m//min. Three peaks were detected by the UV monitor and collected. Fraction 1 contained photoadduct 11. identified by the identity of its NMR with that obtained from the DMT-isopropanol reaction mixture and by the fact that its retention time on GLC was the same as that of 11. Fraction 2 was identified as unreacted DMT containing some I1 as an impurity. Fraction 3 consisted of material whose structure was determined to be that described by structure V. Adduct V has been reported previously by Elad and co-workers (Leonov et ul.. 1973). but its spectral characteristics have not been recorded. We found that V has the following properties. The low resolution mass spectrum gives the expected mol wt of 212. The UV spectrum shows a maximum at 272.4nm ( E = 6595) and a minimum at 2380 ( E = 1450). The NMR spectrum, run on the Varian A-60 in CDC13 shows the following peaks: 63.35 and 63.38, both singlets and both 3H (N-CH,): 1.21 singlet. 6H (C(CH,),OH: 2.54: singlet, 2H (5-C-CH2-): 7.06 singlet, 1H (6-CH). The IR spectrum showed broad amide carbonyl absorption centered at 1650cm-' and a second amide carbonyl peak of lesser intensity at 1700cm-'. A broad peak, corresponding to the O H stretch was centered at
Photochemistry of dimethylthymine in isopropanol
3450 cm- ' while absorption corresponding to CH stretching occurred centered at 2990 cm- The 'isopropyl split' appeared in the spectrum centered at 1360cm-'. These data are all in accord with the structure V reported by Elad and co-workers for this compound. The relative amounts of I1 and V in the DBP initiated reaction mixture was determined by isolation and weighing in conjunction with NMR. In this experiment I1 and D M T were collected in one fraction and V in a second. Fraction 1 and 2 were weighed and the relative amounts of D M T and I1 in fraction 1 were determined by comparing integrated areas of peaks in the NMR spectrum of fraction 1. Under our reaction conditions the relative yields of the two products were 73% V and 27% 11. In the DBP initiated reactions which worked optimally (see below), there was no evidence of formation of I, I11 and IV. We also ran the same reaction at 0.65 mM in DMT-isopropanol systems using the same conditions. Again, I1 and V were the major products as evidenced by CLC and there was no evidence for formation of l, 111, and IV. The rate of reaction is much slower in these more dilute systems. In the absence of DBP, reaction did not occur. In some experimental runs on the DMT-DBP-isopropanol system, we found that the reaction product mixture changed with irradiation time. At low irradiation times (lOh), the reaction mixture was as described above. At longer times. photoproducts I, 111, and IV, as well as cyclobutane dimers, started to appear: after 28 h the reaction mixture strongly resembled that from a DMT-acetone-isopropanol reaction mixture with the amount of 11 exaggerated and, of course, V present. This is presumably due to the accumulation of acetone in the system due to the decomposition of t-butoxy radical to acetone and methyl radicals. Why this occurs in some runs and not in others is unclear.
'.
Possible occurrence of V irr the DMT-isopropanol sy.stern
Using the GLC at 180C. a search for the occurrence of V in irradiated DMT-isopropanol and DMT-acetone-isopropanol systems was carried out. At this temperature, V was eluted after 7.5 min. Injection of a l o p / sample of the reaction mixture (30 mg/m/) resulting from irradiation of D M T (6.5mM) in isopropanol indicated that V might be present in small amounts. The DMT-isopropanol reaction mixture was chromatographed by HPLC using the conditions established for the DMT-DBPisopropanol reaction mixture. The fraction eluting at the retention time of V showed slight absorbance according to the UV monitor and was collected. After evaporation, the collected material was taken up in isopropanol and injected onto the GLC: a peak with a retention time identical with an authentic sample of V was eluted from the column. This combination of identical retention times by both GLC and HPLC
251
is suggestive that a small amount of V may be formed in the photolyzed DMT-isopropanol system. However, because the amount is small compared to the amounts of the other photoproducts formed, it was not feasible to collect sufficient material to definitely establish that the product was indeed V. The DMT-acetone-isopropanol system (0.65mM) showed no evidence for formation of V. Chemical properties of I , 11, 111 and I V .
Using GLC, the stabilities of the photoadducts 1. 11, I l l and IV toward heat, acid, and base were determined. For these studies a reaction mixture containing I, 11, Ill and IV at a total photoadduct concentration of 20mg/m/ was used. Heating at 1 W C in distilled water for 15min had no effect on the total or relative amount of each adduct. Treatment with 1 N HCI at room temperature for 15min resulted in decomposition of I11 with an accompanying increase in amount of a compound with the same retention time as DMT. The photoadducts I, 11, and 1V appeared to be stable in this situation as well as to more extended treatment for 1.5 h. The GLC of the reaction mixture, when dissolved in 0.1 M NaOH and allowed to stand for 5 min, showed that 111 was again decomposed while a peak eluting with the same retention time as DMT appeared: the peak corresponding to IV was also decreased significantly in intensity. After 30min of this treatment, I and 11 appear to be stable. After 30min in 1 N NaOH. all photoadducts were decomposed. DlSCUSSlOY
Types of adducts formed Our results indicate that DMT shows an interesting set of photoaddition reactions in the presence of isopropanol. The photoaddition reactions of cx-methylz,B-cycloalkenones with alcohols do not appear to have previously been studied, although Dauben et al. (1968) have studied the cycloaddition (dimerization) reactions of 2-methylcyclohex-2-en-1-one.However, compounds 1 and 11 are the types of photoproduct which might be expected, based on the known photochemistry of other z,S-unsaturated cyclic ketones in alcoholic solution. For example, 2-cyclopentan-I-one adds isopropanol at the 3-position to form 3-(2-hydroxy-2-propy1)-cyclopentanone(Pfau rr ul., 1962) while 1,3-dimethyluracil adds isopropanol (Leonov rt a/., 1973). ethanol (Shetlar, 1974), and methanol (Shetlar, 1976) at the 6-carbon to form r-hydroxyalkyl adducts. Several studies of the free radical initiated reactions of DMT and related compounds in alcoholic solution have appeared in the literature. Brown rt a / . (1966) showed that cis and truns ethanol adducts, analogous to I and 11, were formed when DMT and thymine were irradiated with prays in aqueous solution containing ethanol. Zarebska and Shugar (1972) confirmed and extended these results. Elad and co-
258
MARTIND. SHETLAR
workcrs (Lconov ct d.. 1973: Elad. 1976) have re- from isopropanol to form the a-hydroxyisopropyl radical (Elad, 1976). This radical then attacks the ported that thc DBP initiated reaction of isopropanol with DMT and thymine leads to products of structure 6-carbon of a ground state DMT molecule to form type V : the acetone sensitized reaction of thymidine an adduct radical: the reaction is then completed by evidcntly leads to a similar type of adduct (Leonov abstraction of another H from an isopropanol to form 11. along with another a-hydroxyisopropyl radical. and Elad. 1974). Other types of products were not Thus, the potential for a free radical chain mechanism reported in these studies. More recently, Havron ef exists. (Note that excited states of DMT do not enter t r l . (1976) h a w found that at least one isomer of 5.6-dihydro-6-(2-hydroxy-2-propyl)-3’(5’)-thymidineinto this mechanism as DMT does not absorb signifimonophosphatc is formed from 3’(5’)-thymidine cant amounts of light under our reaction conditions monophosphatc when free radical reaction with iso- at 1 > 290 nm.) The experimental observations in the propanol is initiated with DBP. Adducts of structure DMT-isopropanol and DMT-DBP-isopropanol system are that I, rather than 11, is the major adduct type V werc not reported in this study. Reactions leading to the interesting compounds of of the 6-r-hydroxyalkyl type in the DMT-isopropanol system and that I. 111. and IV are not formed at all structure types 111 and IV appear to be unprecedented in reported studies of the photochemical reactions of in the DMT-DBP-isopropanol system. This rules out mechanisms for formation of I, I11 and IV that inr,/l-cycloalkenones with alcohols. volve attack on ground state DMT by r-hydroxyiso,M~,chtriisiic considerrit ions propyl radicals. Little can be said with certainty about the mechanAny mechanism proposed for formation of the photoadducts 1. 11. 111 and IV must take into account ism of formation of I1 in the DMT-isopropanol and DMT-acetone-isopropanol systems. The experiments the following observations. I . Adducts I, 11, 111. and IV are primary photo- in the DMT-DBP-isopropanol system certainly indicate that I1 can be formed by the chain type mechanproducts. both in the directly photolyzed DMT-isoism described above. but do not imply that this is propanol system and in the acetone sensitized reacthe only or even dominant pathway of formation. tion of DMT with isopropanol. With regard to possible alternative mechanisms of 2. The distributions of yields of photoadducts I. 11. I 1 1 and IV in the direct photolysis of DMT in isopro- formation of I and 11, it is interesting to note that panol and in the acetone sensitized formation of Fisher er cd. (1974) found that the photoaddition of cysteine to thymine proceeds by triplet state reaction ndducts in isopropanol display a close similarity. 3. Increase of the concentration of DMT in the di- which could be sensitized by acetone and that radical rectly photolyzed reaction from 0.65 mM// to combination reactions account for the main adduct. 6.5mM:/ does not have a significant effect on the rather than the type of mechanism involving attack of cysteinyl radicals on ground state thymine. pattern of relative photoadduct yields. A solid mechanistic proposal which incorporates the implications of the above observations must await further studies. One important type of mechanism can he ruled out. however. by considering the results from the DMT - isopropanol and DMT-DBP-isopropanol systems together. In the case of the DBP induced reaction of isopropanol with DMT to form 11, the probable sequence of reaction steps presumably starts with abstraction of H by the t-butoxy radical
Ackno,c/etfgenienrs-Research support from NIH Grant No. GM-23526 and from the Cancer Research Coordinating Committee of the University of California is gratefully acknowledged, Research support to the Magnetic Resonance Laboratory (Grant No. RR00892-01A1) of the University of California. San Francisco. from the NIH Division of Research Resources is also acknowledged. Helpful discussions of this work with Dr. Paul Ortiz de Montellano and Dr. Chi-Gen Lee are also gratefully acknowledged .
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