Photochemistry of 4-Thiothymine Derivatives in the Presence of N-9-Substituted-Adenine Derivatives: Formation of N-6-Formamidopyrimidines

Article abstract of DOI:10.1021/jo971162p

UV irradiation of aqueous solutions containing either 4-thiothymin-1-ylacetic acid (1b) and adenosine (2a), 4-thiothymidine (1a) and adenin-9-ylacetic acid (2b), or 1b and 2b led to 4,5-diamino-6-formamidopyrimidine (N-6-Fapy-Ade) derivatives as observed after irradiation of a mixture of 1a and 2a (J. Am. Chem. Soc. 1996, 118, 8142-8143). These new observations demonstrate that the replacement of one or both nucleoside sugar residues by a carboxymethyl group does not affect the regioselective course of the photochemical reaction. The thermal decomposition of 3a that resulted from irradiation of 1a in the presence of 2a, was examined along with its behavior under mild alkaline conditions. Finally, irradiation of N-3-methyl-4-thiothymidine (6a) in the presence of adenosine gave the N-3-methylcytidine derivative 7.

Full text of DOI:10.1021/jo971162p

J . Org. Chem. 1997, 62, 8125-8130
8125
P h otoch em istr y of 4-Th ioth ym in e Der iva tives in th e P r esen ce of
N-9-Su bstitu ted -Ad en in e Der iva tives: F or m a tion of
N-6-F or m a m id op yr im id in es
Carole Saintom e´ ,†,‡ Pascale Clivio,* Alain Favre, and J ean-Louis Fourrey*
,†
‡
,†
Institut de Chimie des Substances Naturelles, CNRS,
1198 Gif-sur-Yvette Cedex, France, and Institut J acques Monod,
9
CNRS-Universit e´ Paris VII, 2 Place J ussieu, 75251 Paris Cedex 05, France
Received J une 26, 1997X
UV irradiation of aqueous solutions containing either 4-thiothymin-1-ylacetic acid (1b) and
adenosine (2a ), 4-thiothymidine (1a ) and adenin-9-ylacetic acid (2b), or 1b and 2b led to 4,5-diamino-
6
-formamidopyrimidine (N-6-Fapy-Ade) derivatives as observed after irradiation of a mixture of
1
a and 2a (J . Am. Chem. Soc. 1996, 118, 8142-8143). These new observations demonstrate that
the replacement of one or both nucleoside sugar residues by a carboxymethyl group does not affect
the regioselective course of the photochemical reaction. The thermal decomposition of 3a that
resulted from irradiation of 1a in the presence of 2a , was examined along with its behavior under
mild alkaline conditions. Finally, irradiation of N-3-methyl-4-thiothymidine (6a ) in the presence
of adenosine gave the N-3-methylcytidine derivative 7.
In tr od u ction
glycosylase (fpg) protein.11 Later, during our examina-
tion of the light-induced reaction between 4-thiothymi-
dine and adenosine, we observed the formation of a
unique adduct (3a ) having a formamidopyrimidine-
adenine (Fapy-Ade) structure.12 It occurred to us that
such an easy access to this type of compound might be
very helpful in the field of DNA lesion studies. Indeed,
in regard to the strategy of site specific incorporation of
lesions in oligonucleotides, successfully developed for
Cyclobutane pyrimidine dimers and (6-4) pyrimidine
pyrimidone photoproducts are recognized as the major
deleterious adducts generated by UV irradiation of DNA.1
A great number of model studies have been designed to
examine their structural effect at the molecular level in
nucleic acids as well as their formation and repair
mechanisms.1
,2
In this context, the remarkable photo-
1
3
cyclobutane pyrimidine dimers and (6-4) pyrimidine
chemical properties of the sulfur analogues of nucleic acid
bases (4-thiouracil, 4-thiothymine) have successfully been
utilized to study in detail the mechanism of formation
1
4
photoproducts that allowed a number of biophysical and
biological studies,1
a,15
formamidopyrimidine-modified oli-
gonucleotides could be used to examine, at the molecular
level, either DNA repair mechanisms or to determine the
factors responsible for the toxicity and mutagenicity of
this type of adducts.
Herein, in view of elucidating the respective role of
each sugar residue on the course of the reaction between
3
,4
and photoreversal of (6-4) adducts.
Purine bases are
also suspected to lead, under UV radiation, to potent toxic
and mutagenic lesions.1
b,5
Among the purine photoprod-
ucts, formamidopyrimidine (Fapy) derivatives have been
identified recently.6 This latter type of DNA damage,
also induced under oxidative conditions (including ion-
izing radiations), alkylating agents, or alkaline condi-
tions,10 can be repaired by the formamidopyrimidine
4
-thiothymidine (1a ) and adenosine (2a ) to give 3a , we
7
,8
9
report the photochemical behavior of 4-thiothymin-1-
ylacetic acid (1b) in the presence of adenosine (2a ), as
well as that of 4-thiothymidine (1a ) in the presence of
adenin-9-ylacetic acid of (2b ) and also of 1b in the
†
Institut de Chimie des Substances Naturelles.
Institut J acques Monod.
Abstract published in Advance ACS Abstracts, November 1, 1997.
‡
X
(7) (a) Hems, G. Nature 1958, 181, 1721-1722. (b) Hems, G.
Radiation Res. 1960, 13, 777-787. (c) Teoule R. Int. J . Radiat. Biol.
1987, 51, 573-589. (d) Steenken, S. Chem. Rev. (Washington, D.C.)
1989, 89, 503-520. (e) Boiteux, S.; Gajewski, E.; Laval, J . Dizdaroglu,
M. Biochemistry 1992, 31, 106-110.
(
1) (a) Taylor, J .-S. Pure Appl. Chem. 1995, 67, 183-190. (b) Davies,
R. J . H. Biochem. Soc. Trans. 1995, 23, 407-418. (c) Carell, T. Chimia
995, 49, 365-373.
2) For selected examples see: (a) Cadet, J .; Vigny, P. In Bioorganic
Photochemistry: The Photochemistry of Nucleic Acids; Morrison, H.,
Ed.; Wiley and Sons: New York, 1990; Vol. 1, pp 1-272. (b) Begley, T.
P. Acc. Chem. Res. 1994, 27, 394-401. (c) Fenick, D. J .; Carr, H. S.;
Falvey, D. E. J . Org. Chem. 1995, 60, 624-631. (d) Carell, T.; Epple,
R.; Gramlich, V. Angew. Chem., Int. Ed. Engl. 1996, 35, 620-623. (e)
Prakash, G.; Falvey, D. E. J . Am. Chem. Soc. 1995, 117, 11375-11376.
3) (a) Fourrey, J .-L.; Gasche, J .; Fontaine, C.; Guittet, E.; Favre,
A. J . Chem. Soc., Chem. Commun. 1989, 1334-1336. (b) Clivio, P.;
Fourrey, J .-L.; Gasche, J .; Favre, A. J . Am. Chem. Soc., 1991, 113,
1
(
(8) Dizdaroglu, M. Mutat. Res. 1992, 275, 331-342.
(9) (a) Tomasz, M.; Lipman, R.; Verdine, G. L.; Nakanishi, K. J . Am.
Chem. Soc. 1985, 107, 6120-6121. (b) Tomasz, M.; Lipman, R.; Lee,
M. S.; Verdine, G. L.; Nakanishi, K. Biochemistry 1987, 26, 2010-
2027. (c) Hertzog, P. J .; Lindsay Smith, J . R.; Garner, R. C. Carcino-
genesis 1982, 3, 723-725. (d) Wood, M. L.; Lindsay Smith, J . R.;
Garner, R. C. Cancer Res. 1988, 48, 5391-5396. (e) Chetsanga, C. J .;
Polidori, G.; Mainwaring, M. Cancer Res. 1982, 42, 2616-2621.
(10) L o¨ nnberg, H.; Lehikoinen, P. J . Org. Chem. 1984, 49, 4964-
4969 and references cited herein.
(
5
481-5483. (c) Clivio, P.; Fourrey, J .-L.; Gasche, J .; Favre, A.
Tetrahedron Lett. 1992, 33, 1615-1618. (d) Clivio, P.; Fourrey, J .-L.;
(11) (a) Boiteux, S. J . Photochem. Photobiol. B: Biol. 1993, 19, 87-
96. (b) Demple, B.; Harrison, L. Annu. Rev. Biochem. 1994, 63, 915-
948. (c) Bhagwat, M.; Gerlt, J . A. Biochemistry 1996, 35, 659-665.
(12) Saintom e´ , C.; Clivio, P.; Favre, A.; Fourrey, J .-L.; Riche, C. J .
Am. Chem. Soc. 1996, 118, 8142-8143.
Szabo, T.; Stawinski, J . J . Org. Chem. 1994, 59, 7273-7283.
(
4) (a) Liu, J .; Taylor, J .-S. J . Am. Chem. Soc. 1996, 118, 3287-
3
2
288. (b) Clivio, P.; Fourrey, J .-L. J . Chem. Soc., Chem. Commun. 1996,
203-2204.
(
5) (a) Duker, N. J .; Gallagher, P. E. Photochem. Photobiol. 1988,
(13) Taylor, J .-S.; Brockie, I. R.; O’Day, C. L. J . Am. Chem. Soc. 1987,
109, 6735-6742.
4
1
2
8, 35-39. (b) Gallagher, P. E.; Duker, N. J . Photochem. Photobiol.
989, 49, 599-605. (c) Zhao, X.; Taylor, J .-S. Nucleic Acids Res. 1996,
4, 1561-1565.
(14) Iwai, S.; Shimizu, M.; Kamiya, H.; Ohtsuka, E. J . Am. Chem.
Soc. 1996, 118, 6742-6743.
(
6) Doetsch, P. W.; Zastawny, T. H.; Martin, A. M.; Dizdaroglu, M.
(15) (a) Taylor, J .-S. Acc. Chem. Res. 1994, 27, 76-82. (b) Fujiwara,
Y.; Iwai, S. Biochemistry 1997, 36, 1544-1550.
Biochemistry 1995, 34, 737-742.
S0022-3263(97)01162-6 CCC: $14.00 © 1997 American Chemical Society

8
126 J . Org. Chem., Vol. 62, No. 23, 1997
Saintom e´ et al.
Sch em e 1
presence of 2b. In addition, we have examined the
temperature, the H-8 proton signal of 3a ,b appeared as
a very broad signal coalescing around 70 °C. This
indicated that 3a and 3b consisted of a slow equilibrium
mixture of various species, with respect to the NMR time
1
2
mechanism of the thermal degradation of 3a . Further-
more, in an attempt to get insight into the mechanism
of formation of 3a , we have studied the photochemical
reaction between 6a , the N-3-methyl derivative of 1a , and
adenosine.
scale, which could be caused by the restricted rotation of
the formamido group.9
b,17
Moreover, since the signals of
protons H-8 of 3c and 3d appeared as well-resolved
singlets, it could be suggested that the ribose moiety was
implicated in this restricted rotation.
Resu lts a n d Discu ssion
1
3
Isola tion of P h otop r od u cts 3a -d a n d 7. Photo-
chemical reactions were monitored by the disappearance,
on the UV spectrum, of the thiocarbonyl absorption
maximum (ca. 335 nm) of 1a ,b and 6a . Photoproducts
C NMR data of 3b-d supported the proposed struc-
tures. The corresponding spectra were analyzed by
analogy with those of 3a and its peracetyl derivative 5
(Table 1) for which unambiguous HMBC correlations
were observed in methanol-d4. Confirmation of the
opening of the adenosine imidazole ring to give a N-6-
formyl-4,5,6-triaminopyrimidine was established by the
3
a -d were obtained as unique adducts by irradiation of
a 1:1 mixture of 1a and 2a (aqueous solution), and of
mixtures of 1b and 2a , 1a and 2b, and 1b and 2b,
1
2
respectively, as inferred from the H NMR analysis of
presence of a characteristic C-8 sp carbon around 166
the crude irradiation products. For these three latter
irradiation experiments, the concentration of the adenine
derivatives (2a ,b) was twice that of the 4-thiothymine
derivatives (1a ,b) in order to accelerate photoproduct
formation. In the same concentration conditions, pho-
pm that correlates with H-1′ of ribose on the HMBC
spectrum of 5. Furthermore, chemical shifts of C-4, C-5,
and C-6 of the pyrimidine nucleus of 3b-d were in very
good agreement with those of 3a and of other 5-methyl-
cytosine structures.18 The 6 ppm deshielding of the
signals due to C-6 of the thymine base in 3b and 3d
compared to 3a and 3c was attributed to the replacement
of the deoxyribose residue by a carboxymethyl group.
Signal broadening was noticed at positions corresponding
to C-4, C-5, and C-1′ of the adenosine part of 3a and 3b,
and these observations corroborated the hypothesis of a
restricted rotation of the formamido group.
tolysis of 6a in the presence of 2a led to a 50:50 mixture
1
H NMR) of photoproducts 7 and 6b.16
(
Photoproducts 3a and 7 were purified by medium-
pressure reverse phase (RP 18) chromatography using a
gradient of acetonitrile in water. Photoproducts 3b-d
were purified by reverse phase HPLC eluting with a
gradient of acetonitrile in an aqueous solution of triethy-
lammonium acetate.
Formation of photoproduct 3a was by far the most
efficient.12 Photoadducts 3b-d and 7 were isolated after
purification in 20-27% yield.
Mech a n ism of F or m a tion of 4 by Th er m a l Deg-
r a d a tion of 3a . Since the formation of N-4-(4-amino-
6-formamidopyrimidin-5-yl)-5-methylcytosine between a
4-thionopyrimidine and a purine base appeared to be
quite general, 3b-d being obtained as unique products,
we decided to investigate more precisely the previously
observed thermal degradation of 3a 12 that was found to
undergo both deformylation and cleavage of the N-
ribosidic bond in neutral aqueous solution to lead to the
4,5,6-triaminopyrimidine derivative 4. As thermally
induced decomposition of purine derivatives in alkaline
conditions is well documented,10 we examined first the
Ch a r a cter iza tion of P h otop r od u cts 3b-d . The UV
spectrum of 3b-d (λmax ca. 295 nm) was similar to that
of 3a , immediately suggesting a 5-methylcytosine struc-
ture for these adducts. The positive FAB mass spectra
of the three compounds 3b-d displayed a quasimolecular
+
ion at m/z 474, 458, and 400 [(M + Na) ], respectively,
indicating that all of them were formed by means of a
condensation reaction between the 4-thiothymine and
adenine derivatives implicating the replacement of the
sulfur atom by an oxygen atom. From the comparison
1
stability of 3a in alkaline solution by H NMR spectros-
copy. Thus, treatment of an aqueous solution of 3a , at
room temperature, with K CO (pD ca. 10) led within 2
2 3
h to (1) a decrease of the H-2 and H-8 signals (overlapped
singlets) which was accompanied by the appearance of a
signal at 8.1 ppm (attributed to H-2 of 8) and (2) a
1
of the H NMR spectra of 3b-d with that of 3a , it was
clear that 3b-d contained the same N-4-(4-amino-6-
formamidopyrimidin-5-yl)-5-methylcytosine motif. In-
1
deed, the H NMR spectra of 3a -d displayed three
singlets in the downfield region corresponding to H-6,
H-2, and H-8 (see Experimental Section). At room
(
17) (a) Humphreys, W. G.; Guengerich, F. P. Chem. Res. Toxicol.
1
991, 4, 632-636. (b) Raoul, S.; Bardet, M.; Cadet, J . Chem. Res.
Toxicol. 1995, 8, 924-933.
(16) Clivio, P.; Fourrey, J .-L.; Gasche, J .; Favre, A. J . Chem. Soc.,
(18) Shaw, A. A.; Sheltlar, M. D. J . Am. Chem. Soc. 1990, 112, 7736-
Perkin Trans. 1 1992, 2383-2388.
7742.

Formation of N-6-Formamidopyrimidines
J . Org. Chem., Vol. 62, No. 23, 1997 8127
1
3
Ta ble 1.
C NMR Ch em ica l Sh ifts (62.90 MHz)* of 3a -d , 7 (in D2O r ela tive to d ioxa n e 67.8 p p m ), 5 (in CD3OD set a t
4
9.0 p p m ), a n d 4 (in DMSO-d 6 set a t 39.7 p p m )
3
a
5**
4
7
3b
146.7
159.0
163.9a
106.1
13.4
3c
3d
C6T
140.6
139.5
157.6
164.2
105.6
13.5
87.8
38.8
75.8
89.7
138.4
156.1
163.7
104.7
13.9
85.6
40.7
70.9
87.4
136.3
152.3
154.4
108.9
16.6
140.7
158.0
163.3a
106.6
13.6
146.8
159.0
163.3
106.0
13.3
C2T
C4T
157.9
164.0
106.5
13.6
87.3
40.6
71.6
87.9
62.5
157.9
152.9, 155.6
113.0, 115.0
164.0
166.0
91.5, 93.9
73.1
70.7
84.6
62.4
a
C5T
CH3
C1′T
C2′T
C3′T
C4′T
C5′T
C2A
87.0a
39.9
87.9b
40.6
71.5b
87.7
71.6
87.3b
62.3
a
62.3c
153.2
150.9
126.1
159.2
143.9
89.7
65.0
61.8
157.6
156.6
111.1
164.2
164.2
90.8
74.0
71.1
80.1
64.0
155.3
159.8
97.1
157.8
154.5 br
114.6 br
164.3a
166.2
91.6, 94.0
73.3
70.8
84.8
62.2
157.5
156.6
110.6
164.4a
165.5
157.4
156.5
110.9
164.6
165.6
C4A
C5A
C6A
C8A
C1′A
C2′A
C3′A
C4′A
C5′A
CH2T
CH2A
COOH
NCH3
a
159.8
b
b
74.9
71.8
b
a
87.2
a
62.3c
54.2
54.1
48.8
176.0
48.7
175.6
175.9
32.4
*
For convenience, numbering of the parent adenosine is maintained. ** Acetate signals: 20.4; 20.5; 20.7; 20.9 (×2) and 172.2 (×2);
a-c
1
72.1; 171.3; 171.2 ppm.
Interchangeable attributions within a column.
complete disappearance of the anomeric H-1′ signal of
the ribose part, concomitantly with the apparition of two
anomeric doublets at 5.86 ppm (J ) 4.3 Hz) and 5.64 ppm
mixture of 8a and 8b in D
2
O (pD ca. 7, after careful
addition of K CO ) was heated for 20 min at 90 °C. The
2
3
1
only result of this treatment, as observed by H NMR
spectroscopy, was the partial conversion of 8a and 8b to
the pyranosyl forms 8c and 8d and not the release of
D-ribose; however, this experiment did establish that 8a
and 8b were thermally stable under neutral conditions
and ruled out pathway B.
(
J ) 5.5 Hz) (integrating for one proton) corresponding
to R- and â-ribofuranosyl derivatives 8a and 8b, respec-
tively.10 The LSIMS spectra of both compounds displayed
+
the expected quasimolecular peaks at m/z 504 (M + Na )
+
and m/z 520 (M + K ). After 28 h, two additional
anomeric doublets characteristic of the two R- and
â-pyranosyl derivatives 8c and 8d were present at 5.48
ppm (J ) 3.5 Hz) and 5.35 ppm (J ) 9.2 Hz).1 After 7
days, only the signals of 8c and 8d remained (quasimo-
lecular ions at m/z 504 (M + Na ) and 520 (M + K ) on
LSIMS spectra) demonstrating that deformylation had
occurred first.
The easy and clean transformation of 3a into 4 is
reminiscent of the release of Fapy-G from ionizing
0
20
radiation-treated DNA upon warming at pH 7. This
underlines the interest of this reaction pathway in view
+
+
of mapping to eventual N7-C8 purine cross-linked sites
2
1
in photolabeling experiments and for designing artificial
specific endonucleases.22
We then studied the thermally induced decomposition
of 3a 12 at neutral pD using variable temperature H NMR
spectroscopy. Initial deformylation should lead to the
above mentioned intermediates 8a -d . Conversely, ini-
tial loss of the ribose residue should be accompanied by
the formation of intermediate 9.
Ch a r a cter iza tion of P h otop r od u ct 7. In an at-
tempt to confirm the postulated mechanism leading to
3a ,12 we have studied the photochemistry of N-3-methyl-
4-thiothymidine (6a ) in the presence of adenosine (2a ),
reasoning that the suggested thiaazetidine intermediate
should be trapped as was the thietane intermediate in
1
4
3c,d
the case of Tps T.
Unexpectedly, the compound which
Heating a solution of 3a (10 mg in 500 µL of D
0 °C for 4 h 15 min led (1) to the decrease of the signal
2
O) at
was formed in this reaction was attributed structure 7.
9
-
1
-1
The UV spectrum of 7 (λmax 304 nm, ꢀ 14817 mol cm
at 8.4 ppm (overlapped H-2/H-8 singlets) which was
accompanied by the progressive appearance of a singlet
at 8.1 ppm and (2) to the disappearance of the anomeric
H-1′ of ribose associated with the progressive apparition
of a set of four anomeric proton doublets at δ 5.3 (J ) 4
Hz), 5.2 (J ) 2 Hz), 4.9 (J ) 6 Hz), and 4.8 (J ) 2 Hz)
3
2
dm , H O, pH 7) indicated the loss of the thiocarbonyl
chromophore. Exact mass measurement of its pseudo-
+
molecular ion (m/z 506.1975, (M + H) ) in the positive
mode FAB mass spectrum indicated that 7 resulted from
2
the condensation of 6a and 2a followed by H S elimina-
tion. Inspection of the 13C NMR data (Table 1) provided
key structural informations. Assignment of all the
carbons were made through the analysis of the 2D one
ppm, corresponding to the release of free D-ribose as its
R- and â-ribofuranosyl and pyranosyl isomers.19 Accord-
ingly, the absence of any of the intermediates 8 during
the thermal decomposition of 3a was in favor of pathway
A. However, we could not exclude pathway B due to the
known very fast thermal hydrolysis of N-arylglycosamines
analogues that proceeds via a Schiff base.10 Thus, a
(20) Hems, G. Nature 1960, 186, 710-712.
(21) Favre, A. In Bioorganic Photochemistry: Photochemistry and
the Nucleic Acids; Morrison, H., Ed.; J . Wiley & Sons: New York, 1990;
Vol. 1, pp 317-378. (b) Sontheimer, E. J . Mol. Biol. Rep. 1994, 20,
3
3
5-44. (c) Favre, A., Fourrey, J .-L. Acc. Chem. Res. 1995, 28, 375-
82.
(
19) Observed anomeric proton chemical shifts and coupling con-
(22) (a) Sigman, D. S.; Chen, C.-H. Annu. Rev. Biochem. 1990, 59,
207-236. (b) Sigman, D. S.; Mazumder, A.; Perrin, D. M. Chem. Rev.
1993, 93, 2295-2316.
stants were identical with those obtained with an authentic sample of
D-ribose.

8
128 J . Org. Chem., Vol. 62, No. 23, 1997
Saintom e´ et al.
Sch em e 2
Sch em e 3
bond and multiple bond 13C- H spectra. The signals of
the anomeric H-1′ of each residue allowed attribution of
the signals of carbons C-2 (δC 152.3 ppm) and C-6 (δC
1
that subsequently undergoes hydrogen sulfide elimina-
tion. Since the C-4 position of 4-mercaptopyrimidine
bases is susceptible to undergo nucleophilic displacement
by amines, we have confirmed that the formation of 7
was not the result of a thermal reaction (no reaction
occurred in the absence of light). This postulated mode
of formation of 7 might suggest another pathway, differ-
ent from the previously proposed one,12 to account for the
formation of 3a . As with 7, 3a could have resulted from
a nucleophilic attack of the N-7 nitrogen of 2a at C-4 of
the excited thiocarbonyl group of 1a leading to an
1
36.3 ppm) of the pyrimidine nucleus and those of C-4
δC 150.9 ppm) and C-8 (δC 143.8 ppm) of the adenine
nucleus ( J ). Carbons C-4 of the pyrimidine base (δC
54.4 ppm) and C-5 of the purine base (δC 126.1 ppm)
were assigned ( J correlations) from protons H-6 and H-8,
respectively. Confirmation of the attribution of C-4 of
the pyrimidine base was obtained from long range (LR)
J ) correlations with the two methyl group protons. The
remaining aromatic proton H-2 allowed attribution of C-6
(
3
1
3
3
(
3
2
unstable adduct that, after elimination of H S, would
undergo imidazole ring opening as observed with N-7-
alkylated purines.9
(
δC 159.2 ppm) from its J coupling. Bonding between
the 6-amino group of the adenine with position C-4 of
the pyrimidine was deduced from the carbon chemical
shifts of 7. Compared to 2a , the chemical shifts of carbon
C-6 (∆δ ) 8.7 ppm), C-5 (∆δ ) 7.6 ppm), C-2 (∆δ ) 5.3
ppm), and C-4 (∆δ ) 5.8 ppm) of the adenine part were
deshielded (no significant variation was observed at the
C-8 position) as expected for a substitution of the 6-amino
group.23 Finally, the high shielding of the signal due to
C-4 of 6a (192.4 ppm), now observed at 154.4 ppm (see
above) in 7, is in agreement with the proposed structure.
Moreover, an authentic sample of 3,5-dimethyl-2′-deoxy-
cytidine 6c, prepared by treatment of 6a with liquid
ammonia, exhibited very close 13C chemical shift data.
From a mechanistic point of view, we propose that
photoproduct 7 results from an attack of the 6-amino
group of 2a at the electrophilic C-4 position of the excited
thiocarbonyl group leading to an unstable intermediate
Con clu sion
In summary, we have demonstrated that the formation
of N-4-(4-amino-6-formamidopyrimidin-5-yl)-5-methylcy-
tosine adducts by irradiation of N-1-substituted-4-thio-
thymine and N-9-substituted-adenine is a general reac-
tion. In this process an imidazole ring fission reaction
could be observed. It might be related to the action
mechanism of biologically important DNA-modifying
agents known to induce the formation of N-7-alkyl-Fapy
sites.9 It is probable that the preferred N-7;C-8 imidazole
ring fission of adenosine leading to N-9-formyl derivatives
be governed by the electron deficient nature of the N-7-
substituent. Indeed, this pathway is reminiscent of the
analogous ring opening of N-9-adenines derivatives upon
(
23) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; Ebel,
H. F., Ed.; VCH: Weinheim, 1987.

Formation of N-6-Formamidopyrimidines
J . Org. Chem., Vol. 62, No. 23, 1997 8129
N-7-acylation.24 In connection with this regioselective
ring fission between N-7 and C-8, it is important to note
that, although the majority of N-7-alkylated purine
derivatives led to N-5-formamidopyrimidines, N-7-ad-
Gen er a l P u r ifica tion P r oced u r e. Photoproducts 3a and
were purified on a medium pressure (400 mbar) RP-18
7
column (40-63 µm; 14 × 3 cm i.d. for 3a and 12 × 3 cm i.d.
for 7). Fractions (4 mL) were collected. The eluant system
for 3a was water (80 mL) then water-acetonitrile 1% (400
mL); 2% (500 mL); 5% (100 mL); 10% (200 mL). Fractions
containing 1a (229/246), 2a (96/228), and 3a (37/88) were
concentrated then lyophilized leading to 3a (60% yield), 2a
(33%), and 1a (24%). The eluant system for 7 was water (100
mL) and then water-acetonitrile 1% (50 mL); 4% (100 mL);
ducts of aflatoxin B1 can lead also to N-6-forma-
midopyrimidines.9
c,d
Interestingly, structurally related
DNA adducts, involving the N-7 position of purines, give
rise to divergent biological responses.25 Consequently,
it can be suggested that derivatives of type 3 could be
useful for studying the structure-activity relationship
of such adducts together with the enzymatic excision
mechanism of the Fpg protein11 and the enzymatic
reclosure of opened imidazole rings of purine.2
6
% (100 mL); 8% (100 mL), 12% (100 mL), 14% (100 mL).
Fractions (4 mL) containing 7 (134/141) were concentrated and
then lyophilized (22% yield).
The other photoproducts 3b-d were purified by HPLC using
a C18 (6 µm, 60 Å) cartridge (25 × 100 mm) with a flow rate
of 8 mL/min. A photodiode array detector was employed.
Fractions containing the products were lyophilized or concen-
trated under high vacuum below 20 °C and then desalted on
a medium pressure (400 mbar) RP-18 column (40-63 µm; 9 ×
2.5 cm i.d. for 3b,c and 9 × 1.5 cm i.d. for 3d ). Fractions (2
6
Exp er im en ta l Section
Ch em ica ls. 4-Thiothymidine (1a ), 4-thiothymin-1-ylacetic
acid (1b), and N-3-methyl-4-thiothymidine (6a ) were prepared
2
7,28
according to literature procedures.
(
Adenin-9-ylacetic acid
2b) was prepared by saponification of its ethyl ester.30
NMR chemical shifts (δ) are reported relative to residual
solvent traces (CD OD: 3.3 ppm, DMSO-d : 2.6 ppm D O: 4.8
ppm). C chemical shifts (δ) are reported relative to solvent
DMSO-d 39.7 ppm, CD OD: 49.0 ppm) or for spectra
recorded in D O to external dioxane (67.8 ppm). HMBC
experiments were optimized to suppress J C-H coupling of
00 Hz and recorded at 276 K for 3a , 253 K for 5 and 300 K
2
9
1
H
mL) were collected, and elution was performed with H O from
2
fractions 1 to 17 and then 1% CH CN for 3b,c and H O for
3
2
3
6
2
3d . Fractions 18/39 contained 3b, fractions 16/42 3c and 1/4
3d .
Photoproduct 3b. A 40 min linear gradient of 0-12%
acetonitrile in 0.05 M triethylammonium acetate (pH 6.0) was
used. Fractions containing 3b were concentrated and desalted
(27% yield).
1
3
(
6
:
3
2
1
2
for 7. FAB HRMS were performed by the Service Central
d’Analyze du CNRS (Lyon, France).
Ir r a d ia tion Con d ition s. Irradiation experiments were
performed under continuous nitrogen bubbling in aqueous
solution. To obtain 3a , an equimolecular solution (140 mL)
of adenosine and 4-thiothymidine (1.2 mmol) in a Pyrex tube
Photoproducts 3c and 3d . An isochratic 0.01 M triethyl-
ammonium acetate (pH 6.0) solution was used for 30 min, and
then a 10 min (3c) or 30 min (3d ) linear gradient of 0-12%
acetonitrile in 0.01 M triethylammonium acetate (pH 6.0) was
used. The fractions containing the photoproducts were con-
centrated and desalted leading to 3c (24% yield) and 3d (20%
yield).
(
17 × 3.5 cm i.d.) was kept at 5 °C and irradiated for 234 h
using an Original Hanau Quarzlampen Fluotest-Forte ref
261. To obtain 3b, a 50 mL aqueous solution of adenosine
0.26 mmol) and 4-thiothymin-1-ylacetic acid (0.13 mmol) was
1
Photoproduct 3a : UV (H O) see ref 12; H NMR (300 MHz;
2
5
(
D O) δ 8.40 (brs, 1H, H-8), 8.38 (s, 1H, H-2), 7.81 (s, 1H, H-6),
2
6.26 (t, 1H, J ) 6.5 Hz, H-3′ T), 5.50 (brs, 1H, H-2′ A), 4.45
(m, 2H, H-2′ A, H-3′ T), 4.06 (m, 2H, H-3′ A, H-4′ T), 4.00-
3.40 (m, 5H, H-5′/H-5′′ A, H-5′/H-5′′ T, H-4′ A), 2.37 (m, 2H,
placed in a cylindrical Pyrex flask (2.7 × 6 cm i.d.) and
irradiated for 20 to 26 h at a 4.5 cm distance from a super-
pressure 350 W lamp equipped with a filter to cutoff wave-
lengths below 310 nm. The system was thermostated at 4 °C.
To obtain 3c: A 50 mL aqueous solution of adenin-9-ylacetic
acid (2b) (0.25 mmol) and 4-thiothymidine (0.12 mmol) (ad-
1
3
H-2′/H-2′′ A), 2.09 (s, 3H, CH ); C NMR (62.90 MHz; D O)
3
2
+
see Table 1; HRMS (FAB) calcd for C H N O Na (M + Na)
2
0
27
7
9
532.1770, found 532.1790. Anal. Calcd for C H N O ‚2
2
0
27
7
9
H O: C, 44.03; H, 5.73; N, 17.97. Found: C, 43.56; H, 5.55;
2
dition of an aliquot of Na
2
CO
3
allowed dissolution of 2b) was
N, 17.57.
Photoproduct 3b: UV (H O) λ
irradiated for 14 to 20 h using the conditions reported for 3b.
To obtain 3d a 50 mL aqueous solution of adenin-9-ylacetic
acid (2b) (0.25 mmol) and 4-thiothymin-1-ylacetic acid (1b)
294 nm; H NMR (300
1
2
max
MHz; D O) δ 8.49 (br s, 1H, H-8), 8.34 (s, 1H, H-2), 7.47 (s,
2
1H, H-6), 5.52 (d, 1H, J ) 4.2 Hz, H-1′), 4.40 (br m, 1H, H-2′),
(0.12 mmol) (addition of an aliquot of Na
2
CO
3
allowed dissolu-
4.30 (m, 2H, N-1 CH ), 4.06 and 3.90 (br m, 2H, H-3′ and H-4′),
2
tion of 2b) was irradiated for 12-18 h using the conditions
reported for 3b. To obtain 7, a 50 mL aqueous solution of
adenosine (0.49 mmol) and N-3-methyl-4-thiothymidine (6)
3.42-3.80 (m, 2H, H-5′/H-5′′), 2.04 (s, 3H, CH ); FAB-MS
3
+
+
]; 13C NMR
(positive mode) m/z 452 [(M + H)
], 474 [(M + Na)
(62.90 MHz; D O) see Table 1.
2
(0.24 mmol) was irradiated for 106 h using the conditions
O) λmax 295 nm; 1H NMR (250
Photoproduct 3c: UV (H
MHz; D O) δ 8.37 (br s, 1H, H-8*), 8.33 (s, 1H, H-2*), 7.83 (s,
H, H-6), 6.28 (t, 1H, J ) 6.3 Hz, H-1′), 4.47 (m, 1H, H-3′),
.25 (s, 2H, N-9 CH ), 4.07 (m, 1H, H-4′) 3.94-3.73 (m, 2H,
) *:
interchangeable attributions; FAB-MS (positive mode) m/z 458
2
reported for 3b.
2
Progress of these reactions was monitored by UV (disap-
pearance of the thiocarbonyl absorption at ca. 335 nm, ap-
1
4
2
pearance of a new maximun at ca. 295 nm for 3a -d ) or 1
H
H-5′/H-5′′), 2.57-2.25 (m, 2H, H-2′/H-2′′), 2.10 (s, 3H, CH
3
NMR spectroscopy. After completion of the reaction, the
irradiated solutions were lyophilized or concentrated under
high vacuum below 20 °C and stored at -18 °C prior purifica-
tion.
+
+
13
[
D
(M + Na) ], 480 [(M - H + 2Na) ]; C NMR (62.90 MHz;
2
O) see Table 1.
Photoproduct 3d : UV (H
O) λmax 298 nm; 1H NMR (250
2
MHz; D
.25 (2s, 4H, N-1-CH
interchangeable attributions; FAB-MS (positive mode) m/z 400
2
O) δ 8.37 (s, 1H, H-8*), 8.31 (s, 1H, H-2*), 4.36 and
(
24) (a) Altman, J .; Ben-Ishai, D. J . Heterocycl. Chem. 1968, 5, 679-
82. (b) Leonard, N. J .; McDonald, J . J .; Henderson, R. E. L.;
Reichmann, M. E. Biochemistry 1971, 10, 3335-3342.
25) Baertschi, S. W.; Raney, K. D.; Shimada, T.; Harris, T. M.;
Guengerich, F. P. Chem. Res. Toxicol. 1989, 2, 114-122.
26) Chetsanga, C.; Grigorian, C. Proc. Natl. Acad. Sci., U.S.A. 1985,
2, 633-637.
27) Fox, J . J .; Van Praag, D.; Wempen, I.; Doerr, I. L.; Cheong, L.;
4
2
and N-9-CH ), 2.07 (s, 3H, CH ) *:
2
3
6
+
13
(
[(M + Na) ]; C NMR (62.90 MHz; D
Th er m ic Degr a d a tion of P h otop r od u ct 3a . An aqueous
solution of 3a (88 mg, 0.173 mmol) in 1.15 mL of H O was
2
O) see Table 1.
(
2
8
heated to 90 °C for 4 h 40 min. The solution was concentrated
to dryness and then purified by RP HPLC on a column
identical with that used to purify photoproducts 3b-d . An
isochratic 0.01 M triethylammonium acetate (pH 6.0) solution
was used for 20 min, and then a 5 min linear gradient of
(
Knoll, J . E.; Eidinoff, M. L.; Bendich, A.; Brown, G. B. J . Am. Chem.
Soc. 1959, 81, 178-183.
(28) Saintom e´ , C.; Clivio, P.; Fourrey, J .-L.; Woisard, A.; Favre, A.
Tetrahedron Lett. 1994, 35, 873-876.
29) J ones, A. S.; Lewis, P.; Withers, S. F. Tetrahedron 1973, 29,
293-2296.
30) Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.;
(
0
6
-12% acetonitrile in 0.01 M triethylammonium acetate (pH
.0) was used. Fractions containing 4 were concentrated and
2
(
desalted leading to 35 mg of product (58% yield). UV (H
λmax 284 nm, ꢀ 8942 mol cm dm ; H NMR (400 MHz;
2
O)
Hansen, H. F.; Vulpius, T.; Peterson, K. H.; Berg, R. H.; Nielsen, P.
E.; Buchardt, O. J . Org. Chem. 1994, 59, 5767-5773.
-
1
-1
3
1

8
130 J . Org. Chem., Vol. 62, No. 23, 1997
Saintom e´ et al.
1
DMSO-d
6
) δ 7.92 (s, 1H, N-7-H), 7.85 (s, 1H, H-2), 7.76 (s, 1H,
), 4.32
m, 1H, H-3′), 3.84 (m, 1H, H-4′), 3.66 (m, 2H, H-5′/H5′′), 2.15
m, 1H, H-2′), 2.09 (s, 3H, CH ), 2.03 (m, 1H, H-2′′); FAB MS
positive mode) m/z 350 [(M + H) ]; C NMR (62.90 MHz;
) see Table 1.
(5 to 40%). UV (H
2
O, pH 7) λmax (nm) 268, ꢀ 1430 mol- cm-1
3
1
H-6), 6.26 (t, 1H, J ) 6.7 Hz, H-1′), 6.06 (br s, 2H, NH
2
2
dm ; H NMR (300 MHz; D O) δ 7.69 (s, 1H, H-6), 6.30 (t, 1H,
(
(
(
J ) 6.4 Hz, H-1′), 4.48 (m, 1H, H-3′), 4.08 (m, 1H, H-4′), 3.85
3
(m, 2H, H-5′/H-5′′), 3.48 (s, 3H, NCH ), 2.42 (m, 2H, H-2′/H-
3
+
13
13
2′′), 2.06 (s, 3H, CH ); C NMR (75.47 MHz; D O) δ 161.1 (C4),
150.9 (C2), 136.3 (C6), 106.7 (C5), 88.0 (C1′*), 87.8 (C4′*), 71.4
(C3′), 62.2 (C5′), 40.1 (C2′), 31.4 (NCH ), 14.1 (CH ) *: inter-
3
2
DMSO-d
6
Acetyla tion of 3a . Photoproduct 3a (260 mg, 0.51 mmol)
was dissolved in anhydrous pyridine (9 mL), and acetic
anhydride (2.5 mL) was added. The solution was stirred
overnight at rt with exclusion of moisture. The solution was
chilled to 0 °C, and methanol (2.5 mL) was added. The solution
was then concentrated to dryness below 20 °C under high
vacuum and purified by silica gel chromatography using a
3
3
changeable attributions; HRMS (CI, CH ) calcd for C H N O
4
4
11 18
3
+
(M + H) 256.1262, found 256.1258.
Ad en in -9-yla cetic Acid (2b). Ethyl adenin-9-yl acetate30
994 mg, 4.5 mmmol) was dissolved in MeOH (25 mL) and
(
cooled to 0 °C. A 2 N aqueous NaOH solution (25 mL) was
added. After stirring 30 min at 0 °C, the aqueous solution
was brought to pH 1 by addition of a 4 N aqueous HCl solution.
After filtration, washing with water, and drying, 822 mg of
gradient of methanol in CH
2
Cl
2
affording 127 mg of the penta-
OH) λmax (nm) 291;
OD) δ 8.42 (br s, 1H, H-8), 8.25 (s,
O-acetyl product 5 (34.6% yield). UV (CH
3
1
H NMR (300 MHz; CD
3
2
b was obtained (95% yield). For 1H NMR data see ref 29.
1
5
H, H-2), 7.65 (s, 1H, H-6), 6.17 (t, 1H, J ) 6.8 Hz, H-1′ T),
1
3
C NMR (62.90, D
2
O) δ 174.9 (COOH), 155.9 (C6), 153.0 (C2),
.67-5.36 (m, 3H, H-1′ A, H-2′ A, H-3′ A), 5.23 (m, 1H, H-3′
1
49.4 (C4), 143.5 (C8), 118.5 (C5), 47.6 (CH
2
); CI-MS m/z 194
T), 4.47-3.98 (m, 6H, H-5′/H-5′′ A, H-5′/H-5′′ T, H-4′ A, H-4′
+
[(M + H) ].
T), 2.52 (m, 1H, H-2′ T), 2.24 (m, 1H, H-2′′ T), 2.10-2.08-
2
.02-2.00 (4s, 18H, 5 OAc, CH
3
); FAB MS (positive mode) m/z
OD) see Table 1.
O, pH 7) λmax 304 nm, ꢀ 14817
mol cm dm ; H NMR (250 MHz; D O) δ 8.53 (s, 1H, H-2),
.47 (s, 1H, H-8), 7.41 (s, 1H, H-6), 6.32 (t, 1H, J ) 6.4 Hz,
+
13
7
42 [(M + Na) ]; C NMR (62.90 MHz; CD
P h otop r od u ct 7: UV (H
3
Ack n ow led gm en t. Thanks are due to M.-T. Martin
2
for recording the HMBC spectra and M.-T. Adeline and
J . Hamon for their assistance in HPLC purifications.
We also acknowledge C. Girard, P. Varenne and O.
Lapr e´ vote for recording or interpreting some mass
spectra. We thank D. Guillaume for recording variable
-
1
-1
3 1
2
8
H-1′ T), 6.12 (d, 1H, J ) 6.0 Hz, H-1′ A), 4.83 (m, 1H, H-2′ A),
4
1
.45 (m, 2H, H-3′ T, H-3′ A), 4.29 (m, 1H, H-4′ A), 4.02 (m,
H, H-4′ T), 3.96-3.69 (m, 4H, H-5′/H-5′′ A, H-5′/H-5′′ T), 3.49
1
temperature H NMR experiments and for unvaluable
(
s, 3H, NCH
3
), 2.39 (m, 2H, H-2′/H-2′′ T), 1.34 (s, 3H, CH
3
);
1
3
discussions.
C NMR (62.90 MHz; D
2
+
O) see Table 1; HRMS (FAB) calcd
for C21
28 7 8
H N O (M + H) 506.1999, found 506.1975.
N-3,5-Dim et h yl-2′-d eoxycyt id in e (6c). N-3-Methyl-4-
thiothymidine (6a ) (50 mg, 0.18 mmol) was dissolved in liquid
ammonia (1 mL), and the solution was heated to 60 °C for 39
h in a steel container. After evaporation of ammonia, the
residue was dissolved in methanol and concentrated to dryness
leading to 24 mg of 6c (51% yield). An analytical sample was
obtained after purification by chromatography on a silica gel
column eluted with a gradient of methanol in dichloromethane
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
and C NMR spectra of compounds 3a -d , 4, 5, 6c, and 7 (17
pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
13
J O971162P