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Isomerization and new specific synthesis of thymine glycol
ISOMERIZATION AND NEW SPECIFIC SYNTHESIS OF THYMINE GLYCOL J. CADET,* J. ULRICH’and R. TEOULE* Laboratoirede Radiobiologie.Departementde RechercheFondamentale.CEA-CEN/G,BP85Centredetri, 38041 Grenoble.France (Receivedin the UK 4 Fehruury 1975: Ac~~pptedforplthlication Abnirect-The
mechanism of the cpimcrization
of 5,6dihydroxy-S&dihydrothymine
IO April 1975) was studied using ‘“0 labeled
compounds(positions 5 or 6) prepared by specificsyntheses. Results provided by mass spectrometrywere in agreement with a mechanism involving a N,-C, glycol into its (-) trans form was observed.
ring chain tautomerism.
Cis and trans $6 - dihydroxy - 5,6 - dihydrothymine derivatives probably occur on DNA after in situ gamma irradiation in uitro and in uiuo ’ and may play an important role in mutagenesis.* Trans 5,6 - dihydroxy - 5,6 dihydrothymine,*’ among the various thymine radiolysis products was prepared6 a yield greater than 10% by the isomerization of the cis isomer in aqueous solution. This reaction is reported in this paper using C-2 labeled compounds. It appeared to be very specific and no substances other than thymine glycol isomers could be detected by sensitive autoradiography in efficient chromatographic solvent systems. We describe two synthetic methods to prepare these important biological compounds specifically labeled with (‘*O)OHin positions 5 or 6 of the pyrimidine ring. Their structures were unambiguously demonstrated by mass spectrometry. Using these labeled glycols, and particularly their enantiomeric forms, a base catalyzed mechanism” involving a ring chain tautomerism is proposed. RESULTS
Synthesis of thymine dials labeled either in position 5 or 6 The labeling of an OH substituant in the pyrimidine ring
was carried out trans 5 - 6 - hydroxy - 5,6 dihydrothymine or 6 - hydroxy - 5 - iodo - 5,6 dihydrothymine by adapting the method described previously.9 (a) Cis and trans 5,6 - dihydroxy - 5,6 - dihydrothymine “O-Sexo. Warming these halohydrins in a water-pyridine mixture for one hour at 60°C gave mostly cis 5,6dihydrothymine with a little of the trans isomer. The assignment of the cis configuration was based on the comparison with an authentic sample prepared by the permanganate oxidation of thymine?” The position (5) of the heavy oxygen was determined by mass spectrometry
Specific isomerization
of (+) cis thymine
A quantitative shift of ‘*OH from C-6 to C-5 occurred during the substitution of the halogen by the hydroxyl ion. A similar anionotropic rearrangement has been observed with these halohydrins by the action of hydrogen peroxide which gave mainly cis 6 - hydroperoxy - 5 - hydroxy - 5,6 dihydrothymine in the presence of silver oxide.” In both cases, the loss of the halogen atom and the hydroxyl intramolecular attack probably took place according to a concerted process, involving a bridged ion during the transition state. The rearrangement has an anti character, the migrating group being on the opposite side of the ring with respect to the leaving group.‘* As a result an inversion of configuration at the “migrating terminus” C-5 took place. This process was SNIlike; the hydroxyl function assisted the removal of halogen. Nucleophilic attack preferentially took place with retention of configuration during the third step of the reaction and gave mostly a cis derivative. (b) Cis and trans 5,6 - dihydroxy - 5,6 - dihydrothymine ‘*0&x0. The solvolysis of trans 6 - hydroxy - 5 - iodo 5.6 - dihydrothymine “0&x0 in a methanol-hydrogen peroxide mixture gave mostly trans thymine glycol “0-6exo with a little of the cis isomer ‘“0-6exo. The position of the heavy oxygen label was based on mass spectrometry studies (aide infra). The formation of “0-6exo trans and cis glycols resulted from the hydrolytic decomposition of the hydroxy hyperoxide” generated according to a S,+ substitution. In our experimental conditions more stable thymine bromohydrins did not react. Determination of the position of “0 in these compounds bj mass spectrometry The fragmentation pattern provided by electron impact
of 5,6 - dihydroxy - 5,6 - dihydrothymine has been studied in an earlier report.” There was no significant difference between the cis and the trans isomers. 0 The study of the mass spectra allowed us to determine unambiguously the nature of fragments m/e = 46.89, 115 and 72 which were of interest in order to localize the labeled “0 hydroxyl. The fragmentation of the molecular ion was mainly directed by the initial cleavage of 5,6 C-C bond and gave two types of fragments: (a) fragments in which the Fig. 1. positive charge was IocaIized on oxygen 4 or 5 (m/e = 72.0211; GRO,; m/e = 115.0269; C,H5N0,). The peak m/e 72 was of low intensity and it could be ‘Laboratoire de SpectromCtriede Masse, DCUDCAICAGI EAPC, C.E.A./CENde Grenoble. somewhat difficult to distinguish the ion m/e 74
(uide infra).
2057
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J.
CADET, J.
ULRICH and R. TEOULE
Fig. 3. (C&‘b’6O) from m/e 74 (C&.OZ). (b) protonated fragments which bore the positive charge located on oxygen4 or nitrogen-l (m/e = 46.0293; CH,NO; *NH, = CHOH; m le = WO351; C2HIN202; NH,-CO~H=CHOH). The assignment of the “OH labeling in position 5 was baaed on the presence of an isotopic ion C,HJN’602’*0 (m/e = 117) associated with the ion GHJN’~O, m/e =
115.0269. No isotopic cluster appeared with the ion CH,N’% m/e = 46.0293. The assignment of the labeling in position 6 for thymine glycol resulted from the observation of the isotopic cluster CH,N’*O and CH.N’“O m/e = 46.0293 and m le = 48. The complementary fragment CSI3N’%, m/e = 115.0269did not exhibit the peak m/e = 117 related to isotopic ion, The major peak C2HJN202m/e = 89.0351appeared as a doublet m/e = 89, 91 in the compound labeled “0-6exo. The periodate oxidation of cis 5,6 - dihydroxy - 5,6 dihydrothymine “0-5exo gave N’-formyl N-pyruvyl urea” which was in turn transformed into labeled 5hydroxyJ-methyl hydantoin. 0 -
IO,-
0-C’*O-CH, ‘i NH CO-NH-CHO
kH0 Fig. 6.
L
The intensity of molecular ion for 5-hydroxy-S-methyl hydantoin was low; however, the peak m/e = 102~0429 C,H&O, (M” CO) was very valuable for this purpose. In the labeled compound it appeared as a cluster m le = 104, 102. 150
Isomerization of 5,6 - dihydroxy - 5,6 - dihydrothymine The warming of cis 5,6 - dihydroxy - 5,6 - dihydrothymine “C-2 in an aqueous solution at WC for 4 hr gave only the trans isomer as could be shown by autoradiography of bidimensional chromatographic plates. The purity of both substances obtained was tested by spectroscopic means and the yield of the reaction was about 10% in agreement with preceding reports.“’ The relative &tensities in 1the isotopic clusters were not modified after the isomerizat ion of the cis glycol “Odexo 0
0
m/u
Fig. 5.
Fig. 7.
J. CADET,J.
ULWCH and
into trans glycol “0-5exo. A reduction of the label close to fifty percent was observed in both isomers when dials ‘*0&x0 were warmed for 4 hr at 90°C. This loss in the label could be explained by a ring chain tautomerism (Fig. 7). The warming in water of the enantiomeric (+) form of cis thymine diol preparedby nitric acid hydrolysis of cis (+) 5,6dihydroxy-SJGdihydrothymidinegave specifically the trans (-) epimer. Likewise the cis (-) gave the trans (+) epimer.
R. TE~ULE
2059
95.0); 91 (NHCO-NH=CHOH, 13.0); 89 (NHCQ-NH=CHOH, 8.7); 48 (N&=CH”QH, 100);46 (NH#ZHOH, 67.0). (Found: C, 2686; N, 1244; H, 3.19; Br, 35.53; Calc. for GH,N,O,Br: C, 2692; N, 12.56; H, 3.16; Br, 3582%). Cis 5,6 - dulydrvxy - 5,6 - dihydrothymhe “0-5~0. Trans 5 bromo - 6 - hydroxy - 5,6 - dihydrothymine “0&x0 (I I5 mg) or 6 hydroxy - 5 - iodo - 5,6 - dihydrothymine ‘%-6exo (135mg) were dissolved in a pyridine-water (1: 1 v/v) mixture (20ml). The solution was warmed for one hour at 60°C. The mixture was evaporated to dryness. The resulting yellow syrup was applied to five plates of silicagel (20x 2Ocm) which were developed in solvent B. Two compounds were located by their weak UV absorption and by the NaOH-AgNO, reagent spray. The major General procedures. IR spectra were registred on a Perkin bond (Rf, 0.46)was extracted with methanol (4 x 4 ml).The solvent Elmer, Model 257 instrument using the KBr pellet micro method. was evaporated to a syrup and crystallized from water to yield NMR spectra were recorded on a Varian Associates, Model T 60 52 mg (65%) of 5,6 - dihydroxy - 5;6 - dihydrothymine as a white instrument (6OMHz) at 35” using dimethyl sulfoxide-ds as a solid. m.o. 218-m fLit.“.“~ 213.214-215.215.22O’l:IR (cm-‘) solvent and tetramethylsilane as internal standard. High resolution 3350;3425(OH); 3221(NH); 1734,’1700,1668(C=o);‘i229; 1172; mass spectra were obtained on an GEC AEI MS 9 mass 1088, 1053; NMR. (DMSO 6) 8 I.26 (s, 3, S-CH,); 4.36 (dd, I, spectrometer with sample introduction by direct probe; maas H-6); 5.22 (s, I, S-OH);5.% (d, I, J,,, = 4.5 Hz, 6-OH); 8.09 (d, I. measurements were made by computer. Optical rotations were Jti.6= 4 Hz. I-NH): 999 Is. broad. 1. 3-NH). Mass snectrum m/e registered with a Roussel and Jouan “Quick polarimeter”. CD (rel intensity) 145~(1~8); 144(1.4); i 17(M’ -NH=CHGH, 47.7); I i5 spectra were obtained on a Roussel and Jouan, Model 1960 (M* -NH=CHOH, 27.3); 101(5.5); 100(2.7); 99 (2.3); 90 (3.3); 89 instrument. Melting points were taken on a Reicher hot stage (NHICO-NH=CHOH, 100);76 (5); 74 (9.1); 73 (3.2); 72 (4.5); 71 apparatus without correction. Elementary analyses were per- (2.3); 70 (4.1); 61 (5.5); 60 (3.2); 46 (NHXHOH, 90.9); 45 (34.1); formedby C.N.R.S. laboratory, Thiais. All solvents were removed 44 (22.7); 43 (CH,CO, 17.3). These data are identical with those in a Buchi rotatory evaporator under reduced pressure, unless reoorted meviouslv.‘J~‘o(Found: C. 37.45:H. 5.03: N. 1760: Calc. otherwise indicated. Thin layer chromatography” (TLC) was for C,H$lN,O.(160.1):Ci37.50; H,‘5Gl; N, l750%): Workup of performed on Macherey Nagel MN-N-HR/UV 254 silica gel the faster running band (Rf,, 0.61) yielded 4 mg (5%) of tram 5,6 (0.25mm thickness) using chloroform-methanol-water (4: 2: I) dihydroxy - 5,6 - dihydrothymine “O-Sexo. Mass spectrum m/e (v/v/v) lower phase added with 5% methanol (solvent A) and ethyl (rel intensity) 117(M’ -NH=CHOH, 43.4); 1IS (M’ -NH-CHOH. acetate-2-propanol-water (75: 16:9) (solvent B) as the developing 23.5); 89 (NHXO-NH=CHOH, 100);46 (NHKHOH, 87.3). solvents. Trans 5.6 - dihydroxy - 5,6 - dihydtuthymine ‘Wk.xo. To 50 ml of a mixture H,02 30% methanol (1: 1 v/v) was added trans 6 Trans 6 - hydroxy - 5 - iodo - 5,6 - dihydrothymine “Odexo. To thymine-‘%- (126mg) in anhydrous dimethyl sulfoxide (100ml) hydroxy - iodo - 5,6 - dihydrothymine “0-6exo (135mg). The were added N-Iodosuccinimide (600mg) followed by 100111of solution was stirred at 25” for IShr. The transformation of the trifluoroacetic acid and 80 4 of HZ’% (Yeda, Reovoth, label rate iodohydrin was complete as shown by the presence of three new 60%). After IS hr at room temp. and in the dark the solution was compounds in the bidimensiomralchromatographic analysis of the evaporated to a SyNp. The material was applied to five plates mixture. These compounds were separated by preparative (20 X20 cm) and the plates were developed with solvent B. One chromatography on 5 plates of silicagel (20x 20 cm) in solvent B. band was detected by its weak UV absorption. This zone The major band (Rfs = 0.61) was excised and the product was (Rfs = 0.91) was carefully scraped off and the absorbent was extracted from the absorbent with methanol (4 x4 ml). The extracted with methanol (4 x 4 ml). ‘Ihe solvent was evaporated to solvent was evaporated to a syrup and crystallized from 95% dryness and the residue was crystallized in water to give a white ethanol to yield 43 ma (54%) of tram 5.6 - dihvdroxv - 5.6 solid (172mg) in 64% yield m.p. 16&16pC; IR (cm-‘) 3420 (NH); dihydrothymine “0-6exd as a white solid, m.p. 219-220”(Lit. 3061(NH); 1712(GO); 1105,1023;NMR (DMSO 6) 8 198 (s, 3, 218-219”).IR (cm-‘) 3418,3352(OH); 3202(NH); 1745,1714.1695 S-CH,); 4.85 (dd, I, Ja,u = 4 Hz 6-QH); 8.49 (d, I, J,, = 4.5 I-NH (GO); 1279, 1163, 1096.NMR. (DMSO 6) 8 1.26 (s, I, S-CH,); Hz) and IO.35(s, broad, 3-NH). Mass spectrum m/e (rel intensity) 4.35 (dd, I, 6-H); 5.78 (s, I, S-OH); 6.09 (d, I, J&o” = 4.5 Hz, 272 (M’, 7,6); 270(M’, 6.3); 225(M’ -NH=CHOH, 7.8); 183(4.4); 6-OH); 7.96 (d, I, J,+=4Hz, l-NH); 9.85 (s, I, 3-NH). Mass spectrum m/e (rel intensity) 116(4.1); 1IS (M’-NH=CHOH, 100); 100);145(M’ -1,15.3); 143(M’-1.12.5); 126 100(2.7); 99 (4.6); 92 (2.4); 91 (NH,-CO-NH=CH’aOH, 94.6); 90 (M’ -IOH. 58.0):(Found: C, 22.13; H. 2.69; N, 10.45%.Calc. for (2.2); 89 (NHCO-NH=CHOH, 59.5); 76 (8.1); 75 (2.2); 74 (5.4); C,H,N,O,I (270): C. 22.24: H. 2.61: N. 10.37%). 73 (2.2); 72 (23.0); 71 (7.3); 70 (4.9); 48 (NH#ZH“OH, 81.8); 46 Trans 5 - biomb - 6’ - hydroiy - 5.6 -’ dihydrothymine (NHPCHOH. 55.4); 45 (7.3); 44 (67.6); 43 (67.0). These data are “0-6exo. Thymine (126mg) was treated with N- identical with those reported previously.* (Found: C, 37.47; H, bromosuccinimide (460mg) in anhydrous dimethyl sulfoxide 497; N, 17.69;Calc. for C,H,N20. (160.1):C, 37.50; H, 5.04; N, containing 100~1 of trifluoroacetic acid and 80 d of “0 labeled 17.50%). water (label rate 60%). After 15hr at room temp., in the dark, the The extract from the slower zone yielded cis 5,6 - dihydroxy disappearence of thymine was complete as shown by bidimen- 5.6 - dihydrothymine “0+x0; yield 7 mg (P?G),mass spectrum sional chromatogranhic - _ analysis (solvent A and B):The solution m/e (rel intensity) 115 (M’ -NH=CHOH, 100); 91 (NH,-COwas evaporated to dryness. The residue was applied to five plates NH=CH”OH, 90.1); 89 (NH&O-NHXHOH, 56.1); 48 (NH, = of silica gel (20 x 20 cm) which were develoued with solvent B. The CH”OH, 84.2); 46 (NHKHOH, 53.4). zone which was absorbing weakly in UV iight was excised from The upper band (Rfa = 066) was extracted with methanol to the plates and the product was extracted from the silica gel with give 7 mg (6%) of thymine. methanol (4 X4 ml). Evaporation of the solvent produced a dry N-jorrnyl N’-pyrrtuyl urea ‘9. Cis 5,6 - dihydroxy - 5,6 syrup which was crystallized from water to aflord 132mg (59%)of dihydrothymine “O&no (30mg) was dissolved in a solution of trans 5 - bromo - 6 - hydroxy - 5.6 - dihydrothymine as white sodium metaperiodate (I.6 g) in water (50ml) and the mixture was needles.“~‘OM.p. 165-168”(dec); IR (cm-‘) 3423(OH); 3263,3062 kept in the dark for 1.5hr at room temp. The solution was (NH); 1711 (GO); 1110, 1044; NMR (DMSO 6) 6 180 (s. 3, evaporated to dryness. The resulting solid residue was extracted SCH,); 480 (d, I, J,., = 4.7 bH. Hz); 6.41 (s, broad, I. 6-OH); 850 with methanol (4 x 4 ml). Evaporation of the extract gave a clear (d, J,.6 = 4.7 Hz, I-NH); IO.45(s, I, 3-NH). Mass spectrum m/e (rel amorphous material which was resolved on 2 plates of silica gel intensity) 226 (M+. 1.5); 244 (M’, 2.5); 222 (M’, 1.0); 179 (M’ (20 x 20 cm) with solvent A as a developer. The single radioactive -NH=CHC?H,9.8); 177(M* -NH=CHOH, 9.5); 145(M’ -Br, 6.2); zone (Rf, &59) was excised from the plates and the product was 143 (M’ - Br. 4.0); 136 (CO=CBrCH,, 94.6); 134 (CO-CBrCH, extracted from the silica gel with methanol (3 x 3 ml). Evaporation
Isomerization and new specitic synthesis of tbyminc glyeol Phys. Physicochim. Bio/. 56, 1046(l%l); D. Borszcz, Z Tramer and D. Shugaer, Acfa Biochim Polon. IO,9 (1963). ‘S. Iida and H. Hayatsu, Biachem. Biophys. Acto 213, 1 (1970); ibid. 213, I (1971). *L. R. Subbaraman, J. Subbaraman and E. J. Bebrman, J. Org. Chem. 38, 1499(1973). a. Lipkin and J. A. Rabi, J. Am. Chem. Sot. 93, 3309 (1971). ‘“M. H. BCM, B. Chatanua and A. S. Jones, J. Chem. Sot. 1014 (1969). “J. Cadet and R. Teoule, C. R Acad. Sci Paris, 276 C, 1743 (1973). “J. Matbieu and R. Panico, Mkhanismcs RLactionnelsen Chimie Oganique, p. 502 Hero~ann,Paris, (1972);J. March, Adwnced OrganicChemistry: Reaction Mechanisms and Structure,p. 781. McGraw-Hill, New York (1968); W. MaeCrae, Basic Organic Reactions. Heydeo, London (1973). “J. Cadet and R. Teoule, Tetrahedron Letters, 4245 (1973). ‘J. Ulrich, R. Teoule, R. Massot and A. Comu, Org. Mass Spectrum. 2, II83 (1969).
2%1
‘a. Teouk, J. Cadet and J. Ulrich, C. R. Acad Sci.. Paris, 270C, 362 (1970). ‘7. Cadet and R. Teouk, T&ah&m Letters 3329 (1972). “R. Dusebinsky, T. Gabriel, W. Tautz, M. Hoffer, E. Grunberg, J. H. Burebenal and J. J. Fox. J. Med. Chem. 10.47 (1%71. “C. Nofre. La radiolyse de la thymine et 3es hnplications radiophysiologiques,Thesis, University of Lyon No. 4% (1968). ‘J. Cadet and R. Teouk. 1. Chrumaron. 76. 407 (1973). “0. Baudisch and D. DaGdsoo, J. Bioj. Chum.G. 23j (1925). “M. Cbabre, D. Gagnaire and C. Nofre, Bull Sot. Chim. Fr. IO8 (1966). =S. Murahashi. K. S. Yuki and F. Doura, Bull Chem. Sot. Japan 39, I559 (l966). -J. Cadet and R. Teoule, Carbohydrate Res. 29. 345 (1973). “J. G. Buchanan, Natroe 168, 1091(1951). -P. Howgate, A. S. Jones and J. R. Titteosor, J. Chum.Sot. C 275 (I%@. “S. lida and H. Hayatsu, Biochem. Biophys. Acta 228, I (1971).
mechanism of the cpimcrization
of 5,6dihydroxy-S&dihydrothymine
IO April 1975) was studied using ‘“0 labeled
compounds(positions 5 or 6) prepared by specificsyntheses. Results provided by mass spectrometrywere in agreement with a mechanism involving a N,-C, glycol into its (-) trans form was observed.
ring chain tautomerism.
Cis and trans $6 - dihydroxy - 5,6 - dihydrothymine derivatives probably occur on DNA after in situ gamma irradiation in uitro and in uiuo ’ and may play an important role in mutagenesis.* Trans 5,6 - dihydroxy - 5,6 dihydrothymine,*’ among the various thymine radiolysis products was prepared6 a yield greater than 10% by the isomerization of the cis isomer in aqueous solution. This reaction is reported in this paper using C-2 labeled compounds. It appeared to be very specific and no substances other than thymine glycol isomers could be detected by sensitive autoradiography in efficient chromatographic solvent systems. We describe two synthetic methods to prepare these important biological compounds specifically labeled with (‘*O)OHin positions 5 or 6 of the pyrimidine ring. Their structures were unambiguously demonstrated by mass spectrometry. Using these labeled glycols, and particularly their enantiomeric forms, a base catalyzed mechanism” involving a ring chain tautomerism is proposed. RESULTS
Synthesis of thymine dials labeled either in position 5 or 6 The labeling of an OH substituant in the pyrimidine ring
was carried out trans 5 - 6 - hydroxy - 5,6 dihydrothymine or 6 - hydroxy - 5 - iodo - 5,6 dihydrothymine by adapting the method described previously.9 (a) Cis and trans 5,6 - dihydroxy - 5,6 - dihydrothymine “O-Sexo. Warming these halohydrins in a water-pyridine mixture for one hour at 60°C gave mostly cis 5,6dihydrothymine with a little of the trans isomer. The assignment of the cis configuration was based on the comparison with an authentic sample prepared by the permanganate oxidation of thymine?” The position (5) of the heavy oxygen was determined by mass spectrometry
Specific isomerization
of (+) cis thymine
A quantitative shift of ‘*OH from C-6 to C-5 occurred during the substitution of the halogen by the hydroxyl ion. A similar anionotropic rearrangement has been observed with these halohydrins by the action of hydrogen peroxide which gave mainly cis 6 - hydroperoxy - 5 - hydroxy - 5,6 dihydrothymine in the presence of silver oxide.” In both cases, the loss of the halogen atom and the hydroxyl intramolecular attack probably took place according to a concerted process, involving a bridged ion during the transition state. The rearrangement has an anti character, the migrating group being on the opposite side of the ring with respect to the leaving group.‘* As a result an inversion of configuration at the “migrating terminus” C-5 took place. This process was SNIlike; the hydroxyl function assisted the removal of halogen. Nucleophilic attack preferentially took place with retention of configuration during the third step of the reaction and gave mostly a cis derivative. (b) Cis and trans 5,6 - dihydroxy - 5,6 - dihydrothymine ‘*0&x0. The solvolysis of trans 6 - hydroxy - 5 - iodo 5.6 - dihydrothymine “0&x0 in a methanol-hydrogen peroxide mixture gave mostly trans thymine glycol “0-6exo with a little of the cis isomer ‘“0-6exo. The position of the heavy oxygen label was based on mass spectrometry studies (aide infra). The formation of “0-6exo trans and cis glycols resulted from the hydrolytic decomposition of the hydroxy hyperoxide” generated according to a S,+ substitution. In our experimental conditions more stable thymine bromohydrins did not react. Determination of the position of “0 in these compounds bj mass spectrometry The fragmentation pattern provided by electron impact
of 5,6 - dihydroxy - 5,6 - dihydrothymine has been studied in an earlier report.” There was no significant difference between the cis and the trans isomers. 0 The study of the mass spectra allowed us to determine unambiguously the nature of fragments m/e = 46.89, 115 and 72 which were of interest in order to localize the labeled “0 hydroxyl. The fragmentation of the molecular ion was mainly directed by the initial cleavage of 5,6 C-C bond and gave two types of fragments: (a) fragments in which the Fig. 1. positive charge was IocaIized on oxygen 4 or 5 (m/e = 72.0211; GRO,; m/e = 115.0269; C,H5N0,). The peak m/e 72 was of low intensity and it could be ‘Laboratoire de SpectromCtriede Masse, DCUDCAICAGI EAPC, C.E.A./CENde Grenoble. somewhat difficult to distinguish the ion m/e 74
(uide infra).
2057
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CADET, J.
ULRICH and R. TEOULE
Fig. 3. (C&‘b’6O) from m/e 74 (C&.OZ). (b) protonated fragments which bore the positive charge located on oxygen4 or nitrogen-l (m/e = 46.0293; CH,NO; *NH, = CHOH; m le = WO351; C2HIN202; NH,-CO~H=CHOH). The assignment of the “OH labeling in position 5 was baaed on the presence of an isotopic ion C,HJN’602’*0 (m/e = 117) associated with the ion GHJN’~O, m/e =
115.0269. No isotopic cluster appeared with the ion CH,N’% m/e = 46.0293. The assignment of the labeling in position 6 for thymine glycol resulted from the observation of the isotopic cluster CH,N’*O and CH.N’“O m/e = 46.0293 and m le = 48. The complementary fragment CSI3N’%, m/e = 115.0269did not exhibit the peak m/e = 117 related to isotopic ion, The major peak C2HJN202m/e = 89.0351appeared as a doublet m/e = 89, 91 in the compound labeled “0-6exo. The periodate oxidation of cis 5,6 - dihydroxy - 5,6 dihydrothymine “0-5exo gave N’-formyl N-pyruvyl urea” which was in turn transformed into labeled 5hydroxyJ-methyl hydantoin. 0 -
IO,-
0-C’*O-CH, ‘i NH CO-NH-CHO
kH0 Fig. 6.
L
The intensity of molecular ion for 5-hydroxy-S-methyl hydantoin was low; however, the peak m/e = 102~0429 C,H&O, (M” CO) was very valuable for this purpose. In the labeled compound it appeared as a cluster m le = 104, 102. 150
Isomerization of 5,6 - dihydroxy - 5,6 - dihydrothymine The warming of cis 5,6 - dihydroxy - 5,6 - dihydrothymine “C-2 in an aqueous solution at WC for 4 hr gave only the trans isomer as could be shown by autoradiography of bidimensional chromatographic plates. The purity of both substances obtained was tested by spectroscopic means and the yield of the reaction was about 10% in agreement with preceding reports.“’ The relative &tensities in 1the isotopic clusters were not modified after the isomerizat ion of the cis glycol “Odexo 0
0
m/u
Fig. 5.
Fig. 7.
J. CADET,J.
ULWCH and
into trans glycol “0-5exo. A reduction of the label close to fifty percent was observed in both isomers when dials ‘*0&x0 were warmed for 4 hr at 90°C. This loss in the label could be explained by a ring chain tautomerism (Fig. 7). The warming in water of the enantiomeric (+) form of cis thymine diol preparedby nitric acid hydrolysis of cis (+) 5,6dihydroxy-SJGdihydrothymidinegave specifically the trans (-) epimer. Likewise the cis (-) gave the trans (+) epimer.
R. TE~ULE
2059
95.0); 91 (NHCO-NH=CHOH, 13.0); 89 (NHCQ-NH=CHOH, 8.7); 48 (N&=CH”QH, 100);46 (NH#ZHOH, 67.0). (Found: C, 2686; N, 1244; H, 3.19; Br, 35.53; Calc. for GH,N,O,Br: C, 2692; N, 12.56; H, 3.16; Br, 3582%). Cis 5,6 - dulydrvxy - 5,6 - dihydrothymhe “0-5~0. Trans 5 bromo - 6 - hydroxy - 5,6 - dihydrothymine “0&x0 (I I5 mg) or 6 hydroxy - 5 - iodo - 5,6 - dihydrothymine ‘%-6exo (135mg) were dissolved in a pyridine-water (1: 1 v/v) mixture (20ml). The solution was warmed for one hour at 60°C. The mixture was evaporated to dryness. The resulting yellow syrup was applied to five plates of silicagel (20x 2Ocm) which were developed in solvent B. Two compounds were located by their weak UV absorption and by the NaOH-AgNO, reagent spray. The major General procedures. IR spectra were registred on a Perkin bond (Rf, 0.46)was extracted with methanol (4 x 4 ml).The solvent Elmer, Model 257 instrument using the KBr pellet micro method. was evaporated to a syrup and crystallized from water to yield NMR spectra were recorded on a Varian Associates, Model T 60 52 mg (65%) of 5,6 - dihydroxy - 5;6 - dihydrothymine as a white instrument (6OMHz) at 35” using dimethyl sulfoxide-ds as a solid. m.o. 218-m fLit.“.“~ 213.214-215.215.22O’l:IR (cm-‘) solvent and tetramethylsilane as internal standard. High resolution 3350;3425(OH); 3221(NH); 1734,’1700,1668(C=o);‘i229; 1172; mass spectra were obtained on an GEC AEI MS 9 mass 1088, 1053; NMR. (DMSO 6) 8 I.26 (s, 3, S-CH,); 4.36 (dd, I, spectrometer with sample introduction by direct probe; maas H-6); 5.22 (s, I, S-OH);5.% (d, I, J,,, = 4.5 Hz, 6-OH); 8.09 (d, I. measurements were made by computer. Optical rotations were Jti.6= 4 Hz. I-NH): 999 Is. broad. 1. 3-NH). Mass snectrum m/e registered with a Roussel and Jouan “Quick polarimeter”. CD (rel intensity) 145~(1~8); 144(1.4); i 17(M’ -NH=CHGH, 47.7); I i5 spectra were obtained on a Roussel and Jouan, Model 1960 (M* -NH=CHOH, 27.3); 101(5.5); 100(2.7); 99 (2.3); 90 (3.3); 89 instrument. Melting points were taken on a Reicher hot stage (NHICO-NH=CHOH, 100);76 (5); 74 (9.1); 73 (3.2); 72 (4.5); 71 apparatus without correction. Elementary analyses were per- (2.3); 70 (4.1); 61 (5.5); 60 (3.2); 46 (NHXHOH, 90.9); 45 (34.1); formedby C.N.R.S. laboratory, Thiais. All solvents were removed 44 (22.7); 43 (CH,CO, 17.3). These data are identical with those in a Buchi rotatory evaporator under reduced pressure, unless reoorted meviouslv.‘J~‘o(Found: C. 37.45:H. 5.03: N. 1760: Calc. otherwise indicated. Thin layer chromatography” (TLC) was for C,H$lN,O.(160.1):Ci37.50; H,‘5Gl; N, l750%): Workup of performed on Macherey Nagel MN-N-HR/UV 254 silica gel the faster running band (Rf,, 0.61) yielded 4 mg (5%) of tram 5,6 (0.25mm thickness) using chloroform-methanol-water (4: 2: I) dihydroxy - 5,6 - dihydrothymine “O-Sexo. Mass spectrum m/e (v/v/v) lower phase added with 5% methanol (solvent A) and ethyl (rel intensity) 117(M’ -NH=CHOH, 43.4); 1IS (M’ -NH-CHOH. acetate-2-propanol-water (75: 16:9) (solvent B) as the developing 23.5); 89 (NHXO-NH=CHOH, 100);46 (NHKHOH, 87.3). solvents. Trans 5.6 - dihydroxy - 5,6 - dihydtuthymine ‘Wk.xo. To 50 ml of a mixture H,02 30% methanol (1: 1 v/v) was added trans 6 Trans 6 - hydroxy - 5 - iodo - 5,6 - dihydrothymine “Odexo. To thymine-‘%- (126mg) in anhydrous dimethyl sulfoxide (100ml) hydroxy - iodo - 5,6 - dihydrothymine “0-6exo (135mg). The were added N-Iodosuccinimide (600mg) followed by 100111of solution was stirred at 25” for IShr. The transformation of the trifluoroacetic acid and 80 4 of HZ’% (Yeda, Reovoth, label rate iodohydrin was complete as shown by the presence of three new 60%). After IS hr at room temp. and in the dark the solution was compounds in the bidimensiomralchromatographic analysis of the evaporated to a SyNp. The material was applied to five plates mixture. These compounds were separated by preparative (20 X20 cm) and the plates were developed with solvent B. One chromatography on 5 plates of silicagel (20x 20 cm) in solvent B. band was detected by its weak UV absorption. This zone The major band (Rfs = 0.61) was excised and the product was (Rfs = 0.91) was carefully scraped off and the absorbent was extracted from the absorbent with methanol (4 x4 ml). The extracted with methanol (4 x 4 ml). ‘Ihe solvent was evaporated to solvent was evaporated to a syrup and crystallized from 95% dryness and the residue was crystallized in water to give a white ethanol to yield 43 ma (54%) of tram 5.6 - dihvdroxv - 5.6 solid (172mg) in 64% yield m.p. 16&16pC; IR (cm-‘) 3420 (NH); dihydrothymine “0-6exd as a white solid, m.p. 219-220”(Lit. 3061(NH); 1712(GO); 1105,1023;NMR (DMSO 6) 8 198 (s, 3, 218-219”).IR (cm-‘) 3418,3352(OH); 3202(NH); 1745,1714.1695 S-CH,); 4.85 (dd, I, Ja,u = 4 Hz 6-QH); 8.49 (d, I, J,, = 4.5 I-NH (GO); 1279, 1163, 1096.NMR. (DMSO 6) 8 1.26 (s, I, S-CH,); Hz) and IO.35(s, broad, 3-NH). Mass spectrum m/e (rel intensity) 4.35 (dd, I, 6-H); 5.78 (s, I, S-OH); 6.09 (d, I, J&o” = 4.5 Hz, 272 (M’, 7,6); 270(M’, 6.3); 225(M’ -NH=CHOH, 7.8); 183(4.4); 6-OH); 7.96 (d, I, J,+=4Hz, l-NH); 9.85 (s, I, 3-NH). Mass spectrum m/e (rel intensity) 116(4.1); 1IS (M’-NH=CHOH, 100); 100);145(M’ -1,15.3); 143(M’-1.12.5); 126 100(2.7); 99 (4.6); 92 (2.4); 91 (NH,-CO-NH=CH’aOH, 94.6); 90 (M’ -IOH. 58.0):(Found: C, 22.13; H. 2.69; N, 10.45%.Calc. for (2.2); 89 (NHCO-NH=CHOH, 59.5); 76 (8.1); 75 (2.2); 74 (5.4); C,H,N,O,I (270): C. 22.24: H. 2.61: N. 10.37%). 73 (2.2); 72 (23.0); 71 (7.3); 70 (4.9); 48 (NH#ZH“OH, 81.8); 46 Trans 5 - biomb - 6’ - hydroiy - 5.6 -’ dihydrothymine (NHPCHOH. 55.4); 45 (7.3); 44 (67.6); 43 (67.0). These data are “0-6exo. Thymine (126mg) was treated with N- identical with those reported previously.* (Found: C, 37.47; H, bromosuccinimide (460mg) in anhydrous dimethyl sulfoxide 497; N, 17.69;Calc. for C,H,N20. (160.1):C, 37.50; H, 5.04; N, containing 100~1 of trifluoroacetic acid and 80 d of “0 labeled 17.50%). water (label rate 60%). After 15hr at room temp., in the dark, the The extract from the slower zone yielded cis 5,6 - dihydroxy disappearence of thymine was complete as shown by bidimen- 5.6 - dihydrothymine “0+x0; yield 7 mg (P?G),mass spectrum sional chromatogranhic - _ analysis (solvent A and B):The solution m/e (rel intensity) 115 (M’ -NH=CHOH, 100); 91 (NH,-COwas evaporated to dryness. The residue was applied to five plates NH=CH”OH, 90.1); 89 (NH&O-NHXHOH, 56.1); 48 (NH, = of silica gel (20 x 20 cm) which were develoued with solvent B. The CH”OH, 84.2); 46 (NHKHOH, 53.4). zone which was absorbing weakly in UV iight was excised from The upper band (Rfa = 066) was extracted with methanol to the plates and the product was extracted from the silica gel with give 7 mg (6%) of thymine. methanol (4 X4 ml). Evaporation of the solvent produced a dry N-jorrnyl N’-pyrrtuyl urea ‘9. Cis 5,6 - dihydroxy - 5,6 syrup which was crystallized from water to aflord 132mg (59%)of dihydrothymine “O&no (30mg) was dissolved in a solution of trans 5 - bromo - 6 - hydroxy - 5.6 - dihydrothymine as white sodium metaperiodate (I.6 g) in water (50ml) and the mixture was needles.“~‘OM.p. 165-168”(dec); IR (cm-‘) 3423(OH); 3263,3062 kept in the dark for 1.5hr at room temp. The solution was (NH); 1711 (GO); 1110, 1044; NMR (DMSO 6) 6 180 (s. 3, evaporated to dryness. The resulting solid residue was extracted SCH,); 480 (d, I, J,., = 4.7 bH. Hz); 6.41 (s, broad, I. 6-OH); 850 with methanol (4 x 4 ml). Evaporation of the extract gave a clear (d, J,.6 = 4.7 Hz, I-NH); IO.45(s, I, 3-NH). Mass spectrum m/e (rel amorphous material which was resolved on 2 plates of silica gel intensity) 226 (M+. 1.5); 244 (M’, 2.5); 222 (M’, 1.0); 179 (M’ (20 x 20 cm) with solvent A as a developer. The single radioactive -NH=CHC?H,9.8); 177(M* -NH=CHOH, 9.5); 145(M’ -Br, 6.2); zone (Rf, &59) was excised from the plates and the product was 143 (M’ - Br. 4.0); 136 (CO=CBrCH,, 94.6); 134 (CO-CBrCH, extracted from the silica gel with methanol (3 x 3 ml). Evaporation
Isomerization and new specitic synthesis of tbyminc glyeol Phys. Physicochim. Bio/. 56, 1046(l%l); D. Borszcz, Z Tramer and D. Shugaer, Acfa Biochim Polon. IO,9 (1963). ‘S. Iida and H. Hayatsu, Biachem. Biophys. Acto 213, 1 (1970); ibid. 213, I (1971). *L. R. Subbaraman, J. Subbaraman and E. J. Bebrman, J. Org. Chem. 38, 1499(1973). a. Lipkin and J. A. Rabi, J. Am. Chem. Sot. 93, 3309 (1971). ‘“M. H. BCM, B. Chatanua and A. S. Jones, J. Chem. Sot. 1014 (1969). “J. Cadet and R. Teoule, C. R Acad. Sci Paris, 276 C, 1743 (1973). “J. Matbieu and R. Panico, Mkhanismcs RLactionnelsen Chimie Oganique, p. 502 Hero~ann,Paris, (1972);J. March, Adwnced OrganicChemistry: Reaction Mechanisms and Structure,p. 781. McGraw-Hill, New York (1968); W. MaeCrae, Basic Organic Reactions. Heydeo, London (1973). “J. Cadet and R. Teoule, Tetrahedron Letters, 4245 (1973). ‘J. Ulrich, R. Teoule, R. Massot and A. Comu, Org. Mass Spectrum. 2, II83 (1969).
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‘a. Teouk, J. Cadet and J. Ulrich, C. R. Acad Sci.. Paris, 270C, 362 (1970). ‘7. Cadet and R. Teouk, T&ah&m Letters 3329 (1972). “R. Dusebinsky, T. Gabriel, W. Tautz, M. Hoffer, E. Grunberg, J. H. Burebenal and J. J. Fox. J. Med. Chem. 10.47 (1%71. “C. Nofre. La radiolyse de la thymine et 3es hnplications radiophysiologiques,Thesis, University of Lyon No. 4% (1968). ‘J. Cadet and R. Teouk. 1. Chrumaron. 76. 407 (1973). “0. Baudisch and D. DaGdsoo, J. Bioj. Chum.G. 23j (1925). “M. Cbabre, D. Gagnaire and C. Nofre, Bull Sot. Chim. Fr. IO8 (1966). =S. Murahashi. K. S. Yuki and F. Doura, Bull Chem. Sot. Japan 39, I559 (l966). -J. Cadet and R. Teoule, Carbohydrate Res. 29. 345 (1973). “J. G. Buchanan, Natroe 168, 1091(1951). -P. Howgate, A. S. Jones and J. R. Titteosor, J. Chum.Sot. C 275 (I%@. “S. lida and H. Hayatsu, Biochem. Biophys. Acta 228, I (1971).