The Photoreactions of Thymine with Hypoxanthine and Imidazole*

Grazyna Wenska Faculty of Chemistry, A. Mickiewicz University, 6 Grunwaldzka str., 60—780 Poznan, Poland Z. Naturforsch. 40b, 108-114 (1985); received May 15, 1984 Photocycloaddition, Thymine, Azetidine, Cyclobutane Adducts Photochemical reactions of thymine linked to hypoxanthine or imidazole by a trimethylene chain were studied in aqueous solution. Irradiation (A = 254 nm) of thymine-hypoxanthine pair yielded two internal cycloadducts with azetidine and cyclobutane part structures. Sensitization and quenching experiments suggested that the excited singlet was the reactive state in the photocycloaddition reactions. Only cyclobutanes were isolated from irradiated (A = 290 nm) solutions of thymine-imidazole pairs. Photocycloadditions were reversible upon irradiation at A = 254 nm.

Introduction Results and Discussion (2+2) Photocycloaddition of thymine, a pyri- Thymine and hypoxanthine midine base of nucleic acids is well documented. Aqueous solutions of la, b were irradiated with Thymine photodimerization has been subjected to low-pressure mercury lamp (A = 254 nm) until no extensive studies as the dimers account for most of further changes in the absorption spectrum were ob- photobiological effects observed upon UV irradia- served (10 min, Fig. 1). tion of cells [1, 2], Dimers are not the only cyclobutane-type products of thymine. Simple alkenes, vinyl esters and ethers [1], alkynes [3], acetylenic esters [4], coumarins and psoralens [5] when undergo photoaddition to pyrimidines give cyclobutanes as well. For several years we have been studied the reactions of thymine irradiated in the presence of some compounds of biological importance. Bichromo- phoric systems in which thymine was linked to a respective compound by a trimethylene chain were used in these studies. Previously, we have described the photocycloaddition of thymine and adenine lead- -i 1 * 1 1 1 1 1 r 1 1 r"- 240 260 280 300 320 340 ing to a novel adduct with azetidine part structure A(nm] [6, 7], Fig. 1. UV spectra of la, ( ) before (1), ( ) after The investigation of the photoreactions of thy- irradiation at A = 254 nm for 3 min (2), 6 min (3), 9 min, mine-hypoxanthine pair was carried out as a conti- 30 min (4). nuation of the previous studies. Hypoxanthine is a minor base found in certain ribonucleic acids [8]. In this paper the photoreactions of thymine-imidazole Solvent evaporation (at —50 °C) followed by col- pairs are also described. These reactions may be re- umn chromatography (ambient temperature) gave levant to the interaction of proteins and nucleic acids photoproducts 2 and respective substrates 1 in the under irradiation. The naturally occuring aminoacid- ratio of ca. 1:9 (Scheme 1). Under those conditions histidine (a-amino-lH imidazole-4-propanoic acid) cyclobutanes 2 were the only stable photoproducts was shown to react with uracil under UV irradiation [10]. [9]. There is, however, no decisive information about However, the UV spectrum of 2 (Fig. 2) indicated the structure of the photoproduct(s). that the direct formation of this product under UV irradiation could not account for the absorbance changes presented in Fig. 1. Thus, some other unsta- * This work was supported within the project 09.7./.7. ble products must also be present in the reaction 0340-5087/85/0100-0108/$ 01.00/0 mixture. G. Wenska • The Photoreactions of Thymine 109

RNH on silica gel. The tentative assignment of the struc- OC H,C N^l "V K ture of 3 (Scheme 1) followed from the chemical and NR photochemical properties of the photoproduct. kXN N ) 0xJ N' < = i N 0 The compound 2a was shown (TLC, UV) to be NH H formed in the dark after 3 had been treated with .CHO water (Fig. 3). Nucleophilic addition of a water 2 molecule in the dark to the C=N bond and ring opening are well-established reactions for 5,6-di- a) R = CH3 b) R = H hydro-4-oxopyrimidines [11]. Moreover, the product 3 like azetidine 4, converted back to the substrate la CH3N after reirradiation at A = 254 nm (Fig. 3).

CH3N

SCHEME

Fig. 3. UV Spectra in Me0H-H20 (1:1), ( ) of 3 im- mediately after dissolution, ( ) after irradiation of 3 at A = 254 nm (6 min), (-•-•-•) after the solution of 3 was left in the dark for 3 h.

The photoreactions of la can be therefore described as follows (eq. (1)):

240 260 280 300 320 340 360 \(nm) hv(254 nm) la < — 4 Fig. 2. UV spectra of la ( ), 2a (-•-•-•) and 4 ( ) hv in H,0. (254 nm) (1) H O 2 -> 2a Photoreactions of la were studied in detail. Two major photoproducts: 3 (Rf 0.21, solvent system A, Similar scheme presumably describes the photo-

Experimental) and 4 (Rf 0.29, A) in addition to the reactions of lb. The composition of the photo- substrate la (^0.36, A) were detected by TLC im- equilibrium mixture la:3:4 was estimated from the mediately after irradiation. Some traces of 2a (Rf UV spectrum to be 7:1:2. The concentration of the 0.56, A) were observed only. product 3 was calculated using molar extinction coef-

Azetidine 4 (Scheme 1) was isolated from the ficients determined in H20—MeOH solution (AMAX reaction mixture when the temperature was carefully 227 nm, £max 11.000, Fig. 3). The photoproduct 2a controlled and kept below 10 °C throughout. Photo- was obtained with a higher yield (25%) under irradi- product 3 was found to undergo a dark reaction dur- ation of la with A>265 nm. ing workup to yield 2 a. Therefore 3 was obtained The quantum yield for la disappearance was esti- only in a small quantity by a column chromatography mated to be 0.05 (A = 254 nm) and is of the same 110 G. Wenska • The Photoreactions of Thymine 110

order as that found for intramolecular photodimeri- form (/NHa.CHbo =1-5 Hz). This form has been shown zation of trimethylenebisthymine [12]. The photo- to be favoured for N-monosubstituted aliphatic cleavage reactions: 4-^la (0 = 0.43 at 254 nm, amides including N-r-butylformamide [16]. Both ro-

A = 254 nm, determined in MeOH) proceeded with because two formyl (Hb) signals at d 8.18 and d 8.23 much higher efficiencies. Acetone could not sensitize have been observed. The dependence of the isomer the formation of the products 4 and 2a. The triplet ratio on the solvent has been observed previously energy of acetone is higher than that of nucleotide [17]. The *H NMR spectrum of 2 a also indicates that bases and it has been reported previously that triplet in CDC13 solution NH proton of the formamido acetone was quenched by oxopurines and thymine group is involved in intramolecular hydrogen bond- mainly via triplet-triplet energy transfer [13]. Addi- ing most likely with C=0 of the ortho substituent. tionally, oxygen, the well known triplet quencher This proton gives rise to the concentration [14] was found not to affect the rate of disappearance (0.90—0.025 M) independent signal shifted about of la. Thus, both sensitization and quenching experi- 2 ppm downfield as compared with the NH proton ments suggested that the excited singlet was the reac- signal of N-r-butyl aliphatic amides in nonpolar sol- tive state in photocycloaddition reactions of la. vent [16]. In more polar solvent-DMSO these in- The structures of the isolated photoproducts 2 a, tramolecular bonds are broken and intermolecular 2 b and 4 were established on the basis of spectral hydrogen bonds with solvent molecules are formed. data. The rigid, cyclobutane-type structure of the The NH signal appears at 6 9.16 (0.15 M, DMSO-d6) products 2 a and 2 b was evident from the NMR and its chemical shift is concentration dependent. spectra. The aromatic atom resonances typical for In the electron impact mass spectrum of 2 a and 2 b purine and pyrimidine rings [15] were not observed the major fragment is at m/e 320 and 292, respective- in the 13C NMR spectrum of 2a. The JH NMR spec- ly, indicating the loss of CO from molecular ion to be trum of 2a is presented in Fig. 4. The signals were the major fragmentation pattern. The peaks corres- assigned to respective protons by double resonance, ponding to molecular ions are the major peaks in the exchange with D20 and a comparison of the chemi- field-desorption mass spectra of 2a and 2b. cal shifts to those of compounds alike. The structure of the product 2 a has been further The spectrum indicates that formamido group of confirmed by thermal and photochemical transfor-

2 a exists in CDC13 solution predominantly in the syn mations. Heating 2a at 210 °C for 3 h gives the sub-

h d CH3NH j

0 = CH N- NCH 3 g A C H N HN H, 1 f I HC0

e H2C^ e CH,

NI N OAH b

slAI AAA. 1 Fig. 4. 'H NMR spectrum of I 1 1 — 1 i i — 10 9 8 7 6 5 4 3 2 1 5 (ppm) 2a in CDCl G. Wenska • The Photoreactions of Thymine 111 strate la. Irradiation with light of wavelength NL H3C X NR, 2 = 254 nm cleaves a cyclobutane ring (eq. (2)). 3 • I " Y hvl>290nm). /"^f K J

A N = - N 0 2a la (2) R, H hv (254 nm) 2a 5 7 The resulting product 5 (Scheme 2) was identified by a comparison (TLC, NMR, UV) with an indepen- dently synthesized sample. Similarly, the cleavage of 5,2a R, = C-NHCH3 a cyclobutane ring of 2 b occured since the irradiation R2=NHC-H resulted in increasing of absorbance at A — 250 nm. The quantum yield for the appearance of 5 was esti- R3=CH3 mated to be 0.50 at A = 254 nm. It should be pointed 6,7 R^, . R3 - H out that the initially formed compound 5 undergoes further photochemical reaction under prolonged irradiation (see below). The remaining photoproduct, azetidine 4, is ther- mally unstable. It converts to the starting la by bonylation leading to 8 competed with photocycload- heating at 50 °C with t1/2 of 100 min. The photo- dition (Scheme 2). The product distribution (2 a and product 4 is also unstable in acidic and basic solu- 8) was dependent on irradiation wavelength. Cyclo- tions. These properties of the product 4 are identi- butane 2a was the major product (90% yield based cal with those of the previously described azetidine on 5 reacted) accompanied by the traces of 8 when 5 derivatives [6, 7]. The *H NMR spectrum of 4 con- was irradiated with A>290 nm. Photoproduct 8 firmed the suggested structure. Cycloadduct 4 is 4,5- could be obtained in quantity by irradiation of 5 at diaminopyrimidine derivative and therefore it A = 254 nm. In the latter case, initially formed cyclo- shows, in the UV spectrum, the long wavelengths butane 2 a was converted back to 5, and since 8 was absorption band (Fig. 2) [18]. stable to irradiation, it was the eventual product (see Unfortunately, the spectral data are insufficient Experimental). for the definite assignment of the stereochemistry of The structures of all the photoproducts were con- the cycloadducts. Careful inspection of Dreiding firmed spectroscopically. The NMR coupling con- models suggests cis-syn geometry for 2 and 4 due to stants (see Experimental) and inspection of Dreiding the shortness of the trimethylene chain [19]. models suggest that the cycloadduct 7 is cis-syn Photocycloadditions of carbon-nitrogen double isomer [19, 23, 24], bonds are relatively rare [20]. Photoreactions involv- The photocycloaddition of imidazole derivatives ing the "inner" C=C bond of oxopurines have been has been reported previously. The imidazole C=C reported recently [21, 22]. bond has been suggested to be involved in the formation of oxetanes in Paterno Büchi reaction of alipha- Thymine and imidazole tic and aromatic ketones [25] and of dioxetanes in dye sensitized photooxidation [26], From the study In the case of thymine-imidazole pairs one would described here it seems likely that photocycloaddi- expect the competitive addition of thymine C=C tion may contribute to a covalent linking of histidine bond to C=C and C=N bonds of imidazole moiety. and pyrimidine bases. However, irradiation of 5 and 6 through Pyrex (A >290 nm) gave only cyclobutanes 2a and 7, re- spectively (Scheme 2). Experimental The photocycloaddition was a photoreversible Melting points were uncorrected. Ultraviolet process; substrates were obtained upon irradiation of spectra were recorded on a Zeiss UV-VIS spec- cyclobutanes 2a and 7 at A = 254 nm. In the case of trophotometer. Twice distilled water was used as a the compound 5 a slower, irreversible photodecar- solvent. IR spectra were obtained on a Perkin 112 G. Wenska • The Photoreactions of Thymine 112

Elmer 580 spectrophotometer by using KBr pellets. FD-MS: 348(100). 13 13 *H NMR, C NMR spectra were measured on C NMR (CDC13) Ö: 170.01, 168.93, 161.39, Varian A 60 and Jeol FX 90 Q spectrometers with 159.56, 151.21 (C=0 and C=N), 79.10, 78.56, TMS as the internal standard. Electron impact 75.74, 63.66, 48.11, 46.54, 43.39, 28.66, 27.57, 25.68, mass spectra (MS) were determined at 70 eV with 21.50. Jeol JMS-D-100 and field-desorption mass spectra UV (Fig. 2), !H NMR (Fig. 4). (FD-MS) with Varian MAT 311 A spectrometers. Analysis for C H N O Microanalyses were made on Carlo Erba or Perkin 15 20 6 4 Calcd C 51.70 H 5.79 N 24.13, 240 Elemental Analysers. Serva silica gel Found C 51.72 H 5.76 N 24.23. 200—300 mesh was used for column chromatography, Merck silica gel plates 60 F254 for preparative 4: In a separate experiment azetidine 4 was iso- thick layer chromatography (2 mm thickness) and lated from the CHC13 extract by preparative thick for TLC analysis (0.20 mm thickness) Solvent sys- layer chromatography (A). The plates were de- tems: veloped twice. Temperature was kept below 10 °C during the workup. A. EtOAc—MeOH (1:1), ] B. EtOAc-MeOH (5:1), H NMR (DMSO-d6) <5: 7.80 (s, 1, HC=N), 5.77 and 4.27 (2 d, / = 5 Hz, 2, azacyclobutane H), C. CH2Cl2-MeOH-HCOOH (7:2:1), D. CHCI3—MeOH (5:1), 4.10-3.50 and 2.30 and 1.50 (m, 6, CH2CH2CH2), E. CHCl -MeOH (30:1), 3.40 and 2.96 (2 s. 6. N-CH3), 1.71 (s, 3, CCH3). 3 UV (Fig. 2). F. CHCl3-MeOH (3:1), 9-[3-(Thym-l-yl)propyl] hypoxanthine lb was syn- thetized as described previously [27]. The compound Analysis for Ci5H18N603 la was prepared from lb by methylation (CH3I, Calcd C 54.54 H 5.49 N 25.45, NaH, DMF). Mild alkaline hydrolysis of la Found C 54.28 H 5.62 N 25.12. (0.125 M NaOH in H20-Et0H, room temp., 4 h) gave 5. The compound 6 was synthesized by alkyla- Irradiation of lb: The irradiated (A = 254 nm) sol- tion of imidazole with l-(3-bromopropyl)thymine. ution was concentrated. The precipitate of unreacted All new compounds la, 5, 6 exhibited consistent lb was filtered off and the filtrate applied on a silica spectra (NMR, UV, IR, MS) and elemental analyses. gel plate. After drying under vacuum silica gel was placed on the top of a chromatographic column. Elu- Preparative irradiations tion (D) gave 2 b which crystallized from the concentrated eluate (10% yield). Starting material lb was The preparative irradiations were carried out in a recovered in 80% yield. cylindrical reactor using Original Hanau immersion 2b: m.p. 305-306 °C (dec). mercury lamps: low-pressure TNN 15/32 (A = 254 nm) UV Amax 223 nm (e 5, 340), (0,1 N KOH) Amax and high-pressure TQ 150 provided with a cylindrical 239 nm (£ 8, 170). Pyrex filter (A >290 nm). A solution filter (0.5 cm IR cm"1: 3400, 3280, 3190 (NH), 1720, 1700, 1670 layer) of potassium iodide in water (1%, w:v) and (C=0). high-pressure lamp was used for A >265 nm irradia- >H NMR (DMSO-d6) <3:10.32 and 9.21 (2 s, 2, tions. Irradiations were performed on 0.50—0.30 g N-H), 8.25 and 8.21 (overlapping d, total 1, CHO), scale using 1 mM aqueous solutions. All chemical 7.36 (s, 1, HC=N), 6.48 (br, s, 2, CONH2), yields reported are the yields of isolated compounds. 4.14-2.08 (m, 7, cyclobutyl H, CH2CH2CH2 over- Irradiation of la: The photolyzed solution of la lapping with solvent), 1.49 (s, 3, CH3). (A = 254 nm) was concentrated in vacuo. The precipi- MS mle (rel. int.): 292(100), 275(27), 167(54), tate of unreacted la was filtered off and the filtrate 154(6), 150(27), 140(51), 136(16), 126(16), 123(68), extracted with CHC13. 109(16). 2 a: The CHC13 extract was chromatographed on a FD-MS mle (rel. int.): 320(100). column (E). Cyclobutane 2a (10% yield) was crystal- Analysis for C13Hi6N604 lized from CHCl3-isopentane. Starting material la was recovered in 85% yield. 2a was isolated with Calcd C 48.73 H 5.04 N 26.24, 25% yield from A>265 nm irradiated la; m.p. Found C 48.41 H 4.96 N 26.02. 220-223 °C. Irradiation of 5: The irradiated (Pyrex) solution of 1 IR cm" : 3420, 3240 (NH), 1700, 1670 (C=0). 5 was concentrated and extracted with CHC13. Col- MS mle (rel. int.): 348(2), 320(100), 289(8), umn chromatography (B) of the CHC13 extract gave 181(57), 167(5), 164(9), 154(47), 153(9), 140(9). the photoproduct 2 a (47% yield) followed by the G. Wenska • The Photoreactions of Thymine 113 starting material 5 (48% yield). Traces of 8 were Quantum yields, sensitization and detected. quenching experiments When 5 (0.5 mmol) was irradiated at A = 254 nm Irradiations were carried out on an optical bench the reaction was followed by TLC (C) and carried to using low-pressure mercury lamp (A = 254 nm) or ca. 50% conversion of 5. The isolation procedure as high pressure mercury lamp HBO 200 and a combi- described above. 2a (ca. 3% yield) was eluted first nation of interference (Zeiss) and glass BC-4 (Mash- from the column, then the major photoproduct 8 priborintog, USSR) filters (A = 313 nm). Uranyl oxa- (52% yield) was obtained, followed by the starting late was used as an actinometer [28]. Aqueous solu- material 5 (42% yield). Prolonged irradiation gave 8 tions of la (0.2 mM) 4 and 2a (1 mM) were deoxy- with 73% yield and 5 (20% yield). genated and irradiated in a cuvette. In the case of 3 a 8: UV AMAX 265 nm (e 16,400); m.p. 179-180 °C. -1 solution in MeOH (0.3 mM) was used. In quenching IR cm : 3430, 3320, 3140 (NH), 1700, 1670 experiment oxygen was bubbled through the solution (C=0). of la. The amount of the material that underwent NMR (CDC13) 7.04 (s, 1, HC=N), 6.97 (s, 1, the reaction was determined from UV measure- HC=C), 6.73 (br, s, 1, NHCH3), 5.00 (br, s, 2, ments. The irradiations were carried out to a low NH2), 3.89 (t, 2, N-CH2), 3.77 (t, 2, N-CH2), 3.33 conversion of the starting materials (<10%) and the (s, 3, N-CH3), 2.91 (d, 3, NHCH3), 2.46-2.10 (m, calculated quantum yields were extrapolated to zero 2, CCH2C), 1.90 (s, 3, CCH3). irradiation time. MS m/e (rel. int.): 320(100), 289(4), 181(54), In the sensitization experiment the solution of la 167(4), 154(55), 153(13), 150(10), 140(10), 136(6), (1 mM) in water-acetone mixture (1:1) was deoxyge- 123(40). nated and irradiated at A = 313 nm. Under these con- Precise mass for C14H20N6O3: calcd 320.1596, ditions acetone absorbs 99% of the incident light. found 320.1594. The solvent was evaporated and a residue subjected Irradiation of 6: The irradiated (Pyrex) solution of to TLC analysis. 6 was evaporated to dryness. The residue was dis- solved (D) and chromatographed on a column (F). Thermolysis of 2 a The substrate 6 (42% yield) was obtained followed by the product 7 (58% yield). A sample of 2 a in a NMR tube was heated at 7: m.p. 232-235 °C (dec). 210 °C (oil bath) for 3 h. Then, DMSO-d6 was added and NMR spectrum recorded. NMR spectrum UV AMAX 225 nm (e 6,200). IR cm"1: 3220 (NH), 1710 (C=0). and TLC of the reaction mixture indicated the pre- l sence of la. H NMR (DMSO-d6 + CF3COOH) (5: 8.91 (s, 1, CH=N), 5.07 (part A of AMX, JAX = 5.1, 7AM = 5 8.2 Hz, 1, Imid C H), 4.58 (part M of AMX, /Mx = Photocleavage of the cycloadducts 4 2.9 Hz, 1, Imid C H), 4.01 (part X of AMX, 1, Thy Solutions (1 mM) of 2 a, 4 and 7 were deoxygen- 6 C H), 4.40-3.80, 3.52-3.17, 2.95-2.63, 1.86-1.70 ated and irradiated in the reactor at A = 254 nm (2 a, (4 m, 6, CH2CH2CH2), 1.55 (s, 3, CH3). 4, 7) or through Pyrex (4). The reactions were followed by UV spectroscopy. Water was evaporated Analysis for CnH14N402 Calcd C 56.38 H 6.02 N 23.92, off and the reaction mixtures were analyzed by TLC Found C 55.95 H 5.86 N 23.65. and 'H NMR spectroscopy.

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