Synthesis and photochemical behavior of peptide nucleic acid dimers and analogues containing 4-thiothymine: Unprecedented (5-4) photoadduct reversion

Article abstract of DOI:10.1021/ja971983b

Pna dimers 1, 5, containing either 4-thiothymine or N³-methyl- 4-thiothymine, were prepared, and the crystal structure of compound 3 was established. With regard to their photochemistry none of these PNA analogues were able to fully mimic the p

Full text of DOI:10.1021/ja971983b

J. Am. Chem. Soc. 1998, 120, 1157-1166
1157
Synthesis and Photochemical Behavior of Peptide Nucleic Acid
Dimers and Analogues Containing 4-Thiothymine: Unprecedented
(5-4) Photoadduct Reversion
Pascale Clivio,*,† Dominique Guillaume,‡ Marie-The´re`se Adeline,† Jeanine Hamon,†
Claude Riche,† and Jean-Louis Fourrey*,†
Contribution from the Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-YVette Cedex,
France, and the Laboratoire de Chimie The´rapeutique, Faculte´ de Pharmacie, UniVersite´ Rene´ Descartes,
4 AVenue de l’ObserVatoire, 75006 Paris, France
ReceiVed June 15, 1997
Abstract: PNA dimers 1-5, containing either 4-thiothymine or N3-methyl-4-thiothymine, were prepared, and
the crystal structure of compound 3 was established. With regard to their photochemistry, none of these PNA
analogues were able to fully mimic the photochemical behavior observed in the dinucleotide series. Whereas
1 and 2 displayed a sequence dependent photochemistry to give mainly 4-(R-thyminyl) adducts, their rearranged
isomers 3 and 4 yielded mostly (5-4) photoadducts. Photoproducts originating from 3 and 4 were shown to
undergo spontaneous reversal to their dithyminyl parent derivatives under acid conditions. Moreover, as a
result of this photochemical study, it can be suggested that in solution, even at the dimer stage, PNAs adopt
a conformation reminiscent of A-type DNA.
Introduction
targeted to mixed ssDNA, RNA, and PNA5 as well as the
formation of helical duplexes,5,6 might play a key role in these
exceptional binding properties.
Peptide nucleic acids1 (PNAs), which exhibit outstanding
hybridization properties, have attracted considerable attention
due to the potential use of these new molecules either in
antisense or antigene strategies for modulating gene expression
or as diagnostic and molecular biology tools.2 PNAs are achiral
and neutral oligonucleotide analogues in which the nucleobases
are attached, via a methylenecarbonyl linker, to the secondary
amine of a poly-N-(2-aminoethyl)glycine backbone (Figure 1).
These polyamide oligomers have demonstrated a remarkable
ability to bind sequencesspecifically to single-stranded DNA
(ssDNA), RNA and double-stranded DNA (dsDNA).2 Whereas
binding to mixed ssDNA or RNA occurs via duplex formation,
binding to homo ssDNA or RNA is achieved via (PNA)2-DNA
(or -RNA) triplex formation. Upon targeting dsDNA with
pyrimidine PNA oligomers, a unique strand displacement mode2
takes place forming a (PNA)2-DNA triplex with a D loop that
was recently found to be a part of a four-stranded PNA-DNA
bundle.3 All these properties cannot be fully rationalized since
PNAs physicochemical behavior is not yet fully elucidated. Base
pairing, stacking, and lack of a charged backbone are not
sufficient enough to justify the high affinity of PNAs toward
nucleic acids. In fact, the poorly understood backbone struc-
turation, possibly mediated by interresidue hydrogen bonding4
and suggested by the PNA preferred binding orientation when
Some years ago, we demonstrated that the sequence-depend-
ent photochemistry of dideoxynucleotides incorporating a
4-mercaptopyrimidine at the 3′- or 5′-end was controlled by the
intrinsic 2-deoxyribose phosphate backbone conformation.7 In
particular, biologically important (6-4) pyrimidine-pyrimi-
done-type photoproducts8a could mainly be obtained when the
modified base was introduced at the 3′-end of the dimer.
Conversely, although no conformational modification was to
be expected, dimers having the 4-mercaptopyrimidine base at
the 5′-end yielded R-thymidinyl adducts upon irradiation.
Recently, our results were fully validated at the ssDNA level.8b
To get further insight into the PNA backbone conformation,
we are currently comparing the photochemical behavior of
ssPNA and their analogues, incorporating a 4-mercaptopyrimi-
dine at their C- or N-end, with regard to the one observed in
the ssDNA series. In this respect, we have recently reported
that 4-thiothymine-containing PNA dimers manifest a sequence
dependent photochemistry that leads exclusively to R-thyminyl
adducts9 in contrast to what is observed in the dinucleotide
phosphate series.7 In a continuation of this study, we herein
describe, the synthesis of 1 and 2 and the characterization of
(5) (a) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier,
S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E.
Nature 1993, 365, 566-568. (b) Wittung, P.; Nielsen, P. E.; Buchardt, O.;
Egholm, M.; Norden, B. Nature 1994, 368, 561-563.
(6) Wittung, P.; Eriksson, M.; Lyng, R.; Nielsen, P. E.; Norden, B. J.
Am. Chem. Soc. 1995, 117, 10167-10173.
(7) (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, 5481-5483 and
unpublished results from this laboratory.
† Institut de Chimie des Substances Naturelles.
‡ Laboratoire de Chimie The´rapeutique.
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
254, 1497-1500.
(2) For a recent review on PNA properties see: Hyrup, B.; Nielsen, P.
E. Bioorg. Med. Chem. 1996, 4, 5-23.
(3) Footer, M.; Egholm, M.; Kron, S.; Coull, J. M.; Matsudaira, P.
Biochemistry 1996, 35, 10673-10679.
(4) (a) Almarsson, O.; Bruice, T. C.; Kerr, J.; Zuckermann, R. N. Proc.
Natl. Acad. Sci. U.S.A. 1993, 90, 7518-7522. (b) Almarsson, O.; Bruice,
T. C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9542-9546. (c) Torres, R.
A.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 649-653.
(8) (a) Taylor, J.-S. Pure Appl. Chem. 1995, 67, 183-190. (b) Liu, J.;
Taylor, J.-S. J. Am. Chem. Soc. 1996, 118, 3287-3288.
(9) Clivio, P.; Guillaume, D.; Adeline, M.-T.; Fourrey, J.-L. J. Am. Chem.
Soc. 1997, 119, 5255-5256.
S0002-7863(97)01983-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/03/1998

1158 J. Am. Chem. Soc., Vol. 120, No. 6, 1998
CliVio et al.
Figure 1.
their photoadducts together with the preparation and the
photochemical behavior of three novel PNA analogues (3-5)
(Figure 1) which were designed to elucidate the constraints
applied, by the peptide backbone, on the PNA conformation.
otides.7 This unambiguously indicated that both 1 and 2 are
conformationally constrained in a form different from the
B-DNA form that is necessary for (6-4) photoproduct forma-
tion.
It is noticeable that 5,6-dihydro-5-(R-thyminyl)thymine pho-
toproduct, also known as spore photoproduct (SP), is the major
adduct produced by irradiation of A-like form DNA.14 Although
SP and adducts 24, 25, and 27 are structurally distinct, they
very likely derive from a similar mechanistic pathway which
might originally involve hydrogen abstraction from the methyl
of the adjacent thymine by a vicinal excited species. The
proposed mechanism of formation of SP is outlined in Figure
2. In the case of 1 and 2, radical binding, with the common
methylene radical, occurred at either C-4 or C-6 positions of
s4T, depending of the structure of the final product, to give 24,
25, and 27.
Results and Discussion
Synthesis of Dimers 1 and 2. Dimers 1 and 2 (Scheme 1)
were obtained in millimole preparative scale using liquid-phase
synthesis employing tert-butyloxycarbonyl (Boc)-protected mono-
mers and carbodiimide coupling activation as previously
described.10 Synthesis of 1 and 2 primarily required the
preparation of two units, namely N-[2-(Boc-amino)ethyl]-N-
(thymin-1-ylacetyl)glycine methyl ester 13 and its thiolated
analogue [N-[2-(Boc-amino)ethyl]-N-(4-thiothymin-1-ylacetyl)-
glycine methyl ester] 14. Coupling N1-carboxymethylated bases
6a (thymin-1-ylacetic acid)11 and 7b (4-thiothymin-1-ylacetic
acid) with 12 was accomplished by means of their pentafluo-
rophenyl esters, which were prepared in situ, to afford 13 and
14, respectively (86% yield). 4-Thiothymin-1-ylacetic acid was
prepared in three steps from 6a,12 whereas 12 was obtained by
methyl bromoacetate monoalkylation of N-Boc-1,2-aminoethane
(11)13 of ethylenediamine (10) (62% yield, two steps).
The carbamate function of 13 and 14 was cleaved using 20%
trifluoroacetic acid (TFA) in anhydrous CH2Cl2 to give quan-
titatively trifluoroacetates 16 and 17, respectively, whereas
saponification of the ester function of 13 and 14 gave in
quantitative yields, after acidification, 19 and 20, respectively.
After DCC/pentafluorophenol activation 19 and 20 were con-
densed with 17 and 16, respectively, to yield dimers 21 and
22. The latter were subsequently saponified, as described for
the preparation of 19 and 20, to yield the corresponding acids.
Removal of the Boc protecting group of these carboxylic acid
intermediates furnished 1 and 2, respectively. After purification
using reverse-phase HPLC, the overall yields (two-step depro-
tection) were 53% for 1 and 55% for 2.
As in the dinucleotide series, sulfur substitution in 1 and 2
should have very little effect on their conformation compared
to their parent molecules. Since, only R-thyminyl adducts could
be obtained by irradiation of 1 and 2, it might be concluded
that in solution, both dimers exhibit a conformational state in
which the nucleobase stacking is the one adopted by the A-like
form of DNA observed in spores. The differences which were
noticed, in terms of yields and of photoproduct structures,
reflected the expected distance variations between the methyl
group and atoms C-4 and C-6 of the thiolated base.
Interestingly, a P-form, where base stacking is reminiscent
of the one found in A-form DNA duplexes, has recently been
proposed to depict the conformation of a dsPNA in the solid
state.15a Moreover, the P-form seems generally to be adopted
by PNAs since it was also observed, in the solid state, for a
PNA2/DNA triplex15b and in solution for a PNA/DNA duplex.15c
From our study, it can also be reasonably proposed that, even
at the single strand and dimer level, PNAs adopt in solution a
conformation with base stacking corresponding to the same A-
(or related A-) form.
A number of chemical modifications of the PNA backbone16
has been made in an effort to understand their binding properties.
Among them, analogues having more than 11 atoms between
the nucleobases have been proven to retain base pairing.16a,b
We therefore took advantage of the known easy N-acyl
migration reaction,17 to prepare dimers 3 and 4 that contain 15
bonds between the nucleobases. Accordingly, we explored their
Interestingly, photolysis of 1 and 2 led to R-thyminyl
photoproducts 24-27, exclusively whose formation resulted
from an hydrogen abstraction mechanism (Scheme 2).9 Since
(6-4) photoproducts could never be obtained, starting from
compounds 1 and 2, the latter appeared unable to give rise to
the photochemistry which was observed with dideoxynucle-
(10) (a) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am.
Chem. Soc. 1992, 114, 1895-1897. (b) Dueholm, K. L.; Egholm, M.;
Behrens, C.; Christensen, L.; Hansen, H. F.; Vulpius, T.; Petersen, K. H.;
Berg.; R. H.; Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59, 5767-
5773.
(11) Jones, A. S.; Lewis, P.; Withers, S. F. Tetrahedron 1973, 29, 2293-
2296.
(12) (a) Saintome´, C.; Clivio, P.; Fourrey, J.-L.; Woisard, A.; Favre, A.
Tetrahedron Lett. 1994, 35, 873-876. (b) Saintome´, C.; Clivio, P.; Fourrey,
J.-L.; Laugaˆa, P.; Favre, A. To be published.
(14) (a) Varghese, A. J. Biochem. Res. Commun. 1970, 38, 484-490.
(b) Varghese, A. J. Biochemistry 1970, 24, 4781-4787. (c) Setlow, P. Mol.
Microbiol. 1992, 6, 563-567.
(15) (a) Rasmussen, H.; Kastrup, J. S.; Nielsen, J. N.; Nielsen, J. M.;
Nielsen, P. E. Nat. Struct. Biol. 1997, 4, 98-101. (b) Betts, L.; Josey, J.
A.; Veal, J. M.; Jordan, S. R. Science 1995, 270, 1838-1841. (c) Eriksson,
M.; Nielsen, P. E. Nat. Struct. Biol. 1997, 3, 410-413.
(13) Krapcho, A. P.; Kuell, C. S. Synth. Commun. 1990, 20, 2559-2564.

Unprecedented (5-4) Photoadduct ReVersion
J. Am. Chem. Soc., Vol. 120, No. 6, 1998 1159
Scheme 1a
a (i) Thionyl Chloride, MeOH; (i) P2S5; (ii) 2 N aqueous NaOH, H3O+; (iv) MeI, CO3K2; (v) C6F5OH, DCC, DMF; (vi) TEA; (vii) TFA, CH2Cl2.
photochemical behavior in order to determine if they possibly
could give rise to (6-4) photoproducts.
Synthesis of Dimers 3 and 4. These dimers were prepared
from intermediates 21 and 22 (Scheme 1) and the synthetic route
is almost identical with that followed for the preparation of 1
and 2. It only differed by the inversion of the last two
deprotection steps. Hence, reaction of 21 and 22 with 20% TFA
in CH2Cl2 induced the cleavage of the carbamate while
subsequent alkaline treatment concomitantly led to ester sa-
ponification and migration of the acyl group to the N-terminal
position. Rearranged dimers 3 and 4 were finally purified by
reverse-phase HPLC and isolated in 36.5% and 35% yield from
21 and 22, respectively. The N-acyl rearrangement was
ascertained from the simplification of the NMR spectra (1H and
13C) due to the removal of one tertiary amide. It was definitively
established by X-ray crystallographic analysis of 3 (Figure 3).
The molecule shown in Figure 3 is in a zwitterionic form
where an intramolecular hydrogen bond links the charged
nitrogen N4′B-H to the peptidic oxygen O7′B. The peptide
chain is folded to approach the bases at ∼4 Å. Moreover, by
translation along the b axis, all bases are stacked in the crystal
in a such way that the 4-thiothymine is sandwiched between
two thymines. Two water molecules of crystallization were
(16) (a) Hyrup, B.; Egholm, M.; Rolland, M.; Nielsen, P. E.; Berg, R.
H.; Buchardt, O. J. Chem. Soc., Chem. Commun. 1993, 518-519. (b) Hyrup,
B.; Egholm, M.; Nielsen, P. E.; Wittung, P.; Norden, B.; Buchardt, O. J.
Am. Chem. Soc. 1994, 116, 7964-7970. (c) Dueholm, K. L.; Petersen, K.
H.; Jensen, D. K.; Egholm, M.; Nielsen, P. E.; Buchardt, O. Bioorg. Med.
Chem. Lett. 1994, 4, 1077-1080. (d) Lagriffoul, P.-H.; Egholm, M.; Nielsen,
P. E.; Berg, R. H.; Buchardt, O. Bioorg. Med. Chem. Lett. 1994, 4, 1081-
1082. (e) Kosynkina, L.; Wang, W.; Liang, T. C. Tetrahedron Lett. 1994,
35, 5173-5176. (f) Krotz, A. H.; Buchardt, O.; Nielsen, P. E. Tetrahedron
Lett. 1995, 36, 6937-6940. (g) Krotz, A. H.; Buchardt, O.; Nielsen, P. E.
Tetrahedron Lett. 1995, 36, 6941-6944. (h) Haaima, G.; Lohse, A.;
Buchardt, O.; Nielsen, P. E. Angew. Chem., Int. Ed. Engl. 1996, 35, 1939-
1942.
(17) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen,
H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.; Coull, J.; Berg,
R. H. J. Peptide Science 1995, 3, 175-183.

1160 J. Am. Chem. Soc., Vol. 120, No. 6, 1998
CliVio et al.
which we tried to circumvent by transforming 28 in a more
stable derivative. Presuming the presence of a thiol group, and
in the light of previous observations on s5(6-4) bipyrimidine
chemistry,7b we attempted to obtain 29 by adding methyl
methanethiosulfonate (CH3SO2SCH3) to the crude irradiation
mixture just after photolysis. Indeed, addition of this reagent
to the solution of 28 induced instantaneously the apparition, in
1
the H NMR spectrum of the reaction mixture, of two new
methyl signals corresponding to CH3SO2H and SSCH3 (2.50
and 2.43 ppm), attesting to its transformation into 29 in
agreement with the presence of a thiol group within 28.
After the mixture stood at room temperature, 29 slowly gave
rise to 30 that could finally be isolated in pure form by reverse-
phase HPLC in 32% yield (from 3). The UV spectrum of 30
(λmax 318 nm) suggested the presence of either a (6-4) or (5-
4) pyrimidinone chromophore.19,20a Its FAB mass spectrum
(positive mode) exhibited a peak at m/z 551 (M + H)+,
suggesting the replacement of the sulfur atom by an oxygen
atom. Inspection of the 1H NMR spectrum (D2O/TFA) revealed
two singlets at 7.50 and 5.27 ppm, supporting a pyrimidinone
and a 5,6-dihydrothymine structure, respectively. In addition
to the carbon signals of the pyrimidinone, the 13C NMR
spectrum of 30 displayed a quaternary (55.7 ppm) and a methine
(84.3 ppm) sp3 carbon. This latter resonance was unambigu-
Figure 2.
3
ously attributed to C-6 of the saturated thymine from its J
correlations, in the HMBC spectrum, with N1CH2 and CH3
protons within the same base. In the same experiment, the
corresponding H-6 proton 3J-correlated with carbons C-2, C-4,
N1CH2, and CH3. Chemical shifts of carbons C-5 and C-6 (55.7
and 84.3 ppm, respectively), being typical of (5-4) adducts20
considering substituent differences, definitively ruled out a (6-
4) structure7b,20 for this photoadduct. Consequently, we pro-
posed the mixed-disulfide structure for 29 and the s6(5-4)
bipyrimidine structure for 28 that should result from opening
of a thietane intermediate generated by [2 + 2] photocycload-
dition between the two bases in a respective antiparallel
orientation as already suggested20a,21 (Scheme 3). Difficulties
encountered in isolating 28, and obtention of 30 instead, are
not surprising since spontaneous transformation of s6(5-4)
adducts into o6(5-4) derivatives has already been observed in
a bimolecular (Thy; s4Urd) model reaction.20a It is noteworthy
(18) Salient features of the molecule. Torsion angles: ø3: C2-N1-
C8′-C7′) 87.8°, C2B-N1B-C8′B-C7′B ) -84.8°; ø2: N1-C8′-C7′-
N4′ ) 165.8°, N1B-C8′B-C7′B-N1′B ) 159.6°; ø1: C8′-C7′-N4′-
C3′) -171.3°, C8′B-C7′B-N1′B-C2′B ) 167.9°, C5′-N4′-C3′-C2′
) -111.1°, C7′-N4′-C3′-C2′ ) 70.2; N4′-C3′-C2′-N1′ ) 54.2°, C3′-
C2′-N1′-C6′B ) -88.4°, C2′-N1′-C6′B-C5′B ) 174.9°, N1′-C6′B-
C5′B-N4′B′ ) 175.2°; C6′B-C5′B-N4′B-C3′B ) 173.2°; C5′B-N4′B-
C3′B-C2′B ) -84.8°; N4′B-C3′B-C2′B-N1′B ) -75.0°; C3′B-
C2′B-N1′B-C7′B ) 75.4°. Each molecule of compound 3 crystallizes
with two molecules of water (W40 and W41), hydrogen bonded to 3.
Intramolecular hydrogen bonds: N4′BsHx‚‚‚O7′B, 2.733 (3); W40sH‚‚‚-
O2, 2.721 (4); W41sH‚‚‚O6′′, 2.776 (4). Intermolecular hydrogen bonds:
N3sH‚‚‚W41, (1/2 + X, -1/2 - Y, 1/2 + Z), 2.767 (5); N1′sH‚‚‚O4B (-1/2
+X, Y - 1/2 Z), 2.803 (4); N4′BsHy‚‚‚O6′ (-1/2 + X, -1/2 - Y, -1/2 + Z)
2.679 (3); N1′BsH‚‚‚O6′ (1/2 + X, -1/2 - Y, -1/2 + Z), 2.876 (3).
Figure 3. X-ray structure of compound 3.
found in the asymmetric unit. All possible N-H or O-H
hydrogen bonds are present in the crystal.18
Photolysis of 3 and 4. While photolysis of 1 and 2 was
completed within 30 min, in the same irradiation conditions
the photolysis of 3 was roughly 80% completed. In contrast,
that of 4 was less than half completed after 6 h (estimated from
the 1H NMR spectra of the crude irradiation mixtures) of
irradiation. Altogether this indicated a strong difference of
reactivity within the two series. Irradiation of an aqueous
solution of 3 led to the formation of a major (28) and a minor
1
N3BsH‚‚‚W40 (X, -Y, -1/2 + Z) 2.799 (4) W40sH‚‚‚S4 (X, -Y, /2 +
1
Z), 3.383 (3); W41sH‚‚‚O2B (1/2 + X, -1/2 - Y, /2 + Z), 2.891 (5).
(19) Rycyna, R. E.; Alderfer, J. L. Nucleic Acids Res. 1985, 13, 5949-
5963.
(20) (a) Blazek, E. R.; Alderfer, J. L.; Tabaczynski, W. A.; Stamoudis,
V. C.; Churchill, M. E.; Peak, J. G.; Peak, M. Photochem. Photobiol. 1993,
57, 255-265. (b) Alderfer, J. L.; Soni, S.-D.; Arakali, A. V.; Wallace, J.
C. Photochem. Photobiol. 1993, 57, 770-776.
(21) (a) Leonard, N. J.; Bergstrom, D. E.; Tolman, G. L. Biochem.
Biophys. Res. Commun. 1971, 44, 1524-1530. (b) Bergstrom, D. E.;
Leonard, N. J. Biochemistry 1972, 11, 1-9. (c) Thomas, G.; Fourrey, J.-
L.; Favre, A. Biochemistry 1978, 17, 4500-4508.
1
(32) photoproduct (Scheme 3) as indicated by the H NMR
spectrum of the crude irradiation mixture recorded immediately
after photolysis.
Instability of compound 28 in aqueous solution was mani-
1
fested by a slow broadening of its H NMR spectrum signals

Unprecedented (5-4) Photoadduct ReVersion
J. Am. Chem. Soc., Vol. 120, No. 6, 1998 1161
Scheme 29
Scheme 3a
Scheme 4
bimolecular reactions only.21,23 This is the first report of such
a photochemical pathway occurring via an intramolecular
photochemical pathway.
Under acid conditions (pH 1-3), compound 30 rearranged
slowly into the two thymine-containing derivative 31 which
could also be prepared in quantitative yield by treatment of 3
with CH3SO2SCH3 (Scheme 3). To the best of our knowledge,
such a rearrangement has never been observed and a plausible
mechanism is suggested in Scheme 4.
Compound 32, isolated in 4% yield, was easily identified as
a 4-(R-thyminyl) adduct by comparison of its spectral data with
those of similar photoproducts obtained in dinucleotide7,24 and
PNA9 series. Its FAB mass spectrum (positive mode) exhibited
a peak at m/z 533 (M + 1)+ in accordance with H2S elimination.
Its UV spectrum (λmax at 269 and 315 nm) was also typical of
a (i) MeSO2SMe; (ii) H2O.
that (5-4) adducts are relevant of nucleic acids photobiology22,23
and that model (5-4) adducts have so far been obtained in
1
such an adduct. The high field part of its H NMR spectrum
displayed only one methyl group signal (s, 2.10 ppm). The
13C NMR spectrum evidenced the appearance of a new
methylene (40.5 ppm) and the replacement of the thiocarbonyl
group (193.0 ppm) by a C-4 carbon (180.8 ppm) in accordance
with a 4-(R-thyminyl) adduct.7,9
(22) (a) Favre, A.; Yaniv, M.; Michelson, A. M. Biochem. Biophys. Res.
Commun. 1969, 37, 266-271. (b) Yaniv, M.; Favre, A.; Barrell B. G. Nature
1969, 223, 1331-1333. (c) Favre, A.; Michelson, A. M.; Yaniv, M. J. Mol.
Biol. 1971, 58, 367-379. (d) Favre, A.; Roques, B.; Fourrey, J.-L. FEBS
Lett. 1972, 24, 209-214.
(23) (a) Rhoades, D. F.; Wang, S. Y. J. Am. Chem. Soc. 1971, 93, 3779-
3781. (b) Rhoades, D. F.; Wang, S. Y. Biochemistry 1971, 10, 4603-4611.
(24) Clivio, P.; Favre, A.; Fontaine, C.; Fourrey, J.-L.; Gasche, J.; Guittet,
E.; Laugaˆa, P. Tetrahedron 1992, 48, 1605-1616.

1162 J. Am. Chem. Soc., Vol. 120, No. 6, 1998
CliVio et al.
Scheme 5a
a (i) MeSO2SMe; (ii) H2O.
Photolysis of the other analogue 4 led, after the same reaction
cascade as depicted for 3, to the (5-4) adduct 35 (13.7%) which,
under acidic conditions, was transformed into the dithymine
dimer 31 (Scheme 5), confirming the generality of the spontane-
ous reversal of (5-4) photoadducts in acid conditions. No trace
of the product corresponding to hydrogen abstraction pathway
was observed in this case.
Since formation of (5-4) adducts is thought to proceed
through a four-membered heterocyclic ring intermediate,21,23 we
attempted to isolate the putative corresponding thietane inter-
mediate using the strategy successfully applied in the s5(6-4)
series.25 Thus, we prepared the N3-methyl-4-thiothymine-
containing dimer 5 by closely following the route used in the
cases of 3 and 4 (Scheme 1). The N3-methyl-4-thiothymine
derivative 9b was prepared from methyl thymin-1-ylacetate 6b
that was regioselectively N3-methylated (8) and thiolated to give
methyl (N3-methyl-4-thiothymin-1-yl)acetate 9a. Subsequent
alkaline saponification and acidification afforded N3-methyl-4-
thiothymin-1-ylacetic acid 9b. The latter was condensed with
12, providing 15. Deprotection of the primary amine afforded
18 which was condensed with 19, leading to the diprotected
dimer 23. Successive 20% TFA/CH2Cl2 and 2 N NaOH
treatments provided 5 which after purification by HPLC was
isolated in 27% yield (from 23).
Photolysis of 5 did not afford the expected thietane but,
instead, yielded one major 6-(R-thyminyl) photoadduct 36 (21%)
resulting from an hydrogen abstraction pathway (Scheme 5) as
deduced from (1) its FAB mass spectrum (positive mode) that
displayed a peak at m/z 581 (M + 1)+, (2) its UV spectrum
(λmax 277 nm),7,24 and (3) its 1H NMR spectrum that displayed
signals for only one methyl group [doublet at δ, 1.35 ppm (major
component) and 1.31 ppm (minor component)] in the high-
field region and one olefinic H-6 proton in the downfield
region [δ 7.41 ppm (minor component), 7.25 ppm (major
component)]; these results were definitively confirmed by the
13C NMR data which particularly showed the presence of a
methylene at 25.6 ppm, two sp3 methine carbons at 59.0 and
45.5 ppm (major component)7,9,26 and finally a characteristic
CdS at 210 ppm.7,9 Interestingly, this is the first report of a
6-(R-thyminyl) adduct formation from a N3-methyl-4-thiothym-
ine although it was previously found to lead to 4-(R-thyminyl)
adducts.25
Conclusion
In the present work we have established that incorporation
of thio-substituted nucleobases in PNA requires no particular
precaution. As it is well-known that such nucleobases exhibit
remarkable photochemical properties which are currently ex-
ploited in photolabeling studies of systems containing nucleic
acids,27 the introduction of such modifications in PNA could
receive many applications. For example, since oligonucleotides
incorporating a 4-thiouridine at its 5′ end exhibit 100% cross-
linking capacity with the residue located at the 3′ end of its
complement,28 thio-substituted nucleobase containing PNAs
could be used to determine unambiguously the binding orienta-
tion of a stretch of PNA relative to its target.
Moreover, the comparison of the photochemistry of 4-thio-
thymine-containing PNA dimers 1 and 2 with that of their
corresponding N-acyl rearranged analogues 3 and 4, has shown
a remarkable behavioral difference. Under irradiation, 1 and 2
undergo an hydrogen abstraction reaction that, compared to the
dinucleotide series, is typical of the dimers in A-DNA form. In
contrast, dimers 3 and 4 gave rise to a major [2 + 2]
cycloaddition photoreaction as observed with dinucleotides
containing the thionucleobase at their 3′ end. However, whereas
in the dinucleotide series, photocycloaddition led to (6-4)
adducts, in the latter case (5-4) adducts were formed. These
adducts are analogues of those previously observed in tRNA
and other model studies.20-23 Interestingly, in contrast to that
of 1 and 2,9 the photoreactivity of dimers 3 and 4 did not exhibit
any sequence effects. The less constrained backbone structure
of 3 and 4 could account for these observations. In this case,
the antiparallel orientation of the bases, a prerequisite for (5-
4) adduct formation, is permitted due to the presence of three
supplementary atoms in the chain linking the two pyrimidines.
Indeed, the structural constraints exercised in model dimers
containing 11 atoms between the two bases (PNA or nucleotides)
preclude the formation of these adducts.
(25) (a) Clivio, P.; Fourrey, J.-L.; Gasche, J.; Favre, A. Tetrahedron Lett.
1992, 33, 1615-1618. (b) Clivio, P.; Fourrey, J.-L.; Szabo, T.; Stawinski,
J. J. Org. Chem. 1994, 59, 7273-7283.
(26) Shaw, A. A.; Cadet, J. J. Chem. Soc., Perkin Trans. 2 1990, 2063-
2070.
(27) (a) Sontheimer, E. J. Mol. Biol. Rep. 1994, 20, 35-44. (b) Favre,
A.; Fourrey, J.-L. Acc. Chem. Res. 1995, 28, 375-382.
(28) Saintome´, C.; Clivio, P.; Favre, A.; Fourrey, J.-L.; Laugaˆa, P. J.
Chem. Soc., Chem. Commun. 1997, 167-168.

Unprecedented (5-4) Photoadduct ReVersion
J. Am. Chem. Soc., Vol. 120, No. 6, 1998 1163
Nova-Pak HR C18 (6µm 60 Å) PrepPak cartridge (25 × 100 mm). A
photodiode array detector (Waters 991) was used.
Finally, it is noteworthy that most of all biological photoad-
ducts identified so far29 (cyclobutane pyrimidine dimers, (6-
4) photoproducts, and spore photoproduct) can be physiologi-
cally repaired by their respective specific (photo)lyases.30-32
Excepted for these latter, studies with model compounds have
demonstrated their possible reversion to their parent bases, under
relatively common physical conditions.8b,33,34 Interestingly, in
this respect we have found that (5-4) adducts, which are
naturally occurring nucleic acid photoproducts,22,23 can also
undergo a spontaneous repair process under mild acidic pH
conditions. Since these adducts could be substrates for a
photoreactivating enzyme,23b the biological significance of this
reversal pathway remains to be assessed.
Photolyzed dimers 1 and 2 were dissolved in H2O (20 mg/200 µL)
and injected, then a 30 min, 8 mL/min linear gradient of 0-9.6% CH3-
CN for 1 and 0-12% CH3CN for 2 in 0.05 M aqueous ammonium
acetate (pH 4.7) was used. Capacity factor (k′): k′ 24 ) 2.5; k′ 25 )
6.3; k′ 26 ) 5.8; k′ 27 ) 1.4; k′ 31 ) 4.4.
Photolyzed dimers 3 and 4 were dissolved in H2O (20 mg/200 µL)
and injected. A 30 min, 8 mL/min linear gradient of 0-12% CH3CN
in 0.05 M aqueous ammonium acetate followed by a 10 min plateau
was used. Capacity factor: k′ 30 ) 2.0; k′ 31 ) 4.4; k′ 32 ) 3.2; k′
35 ) 3.1.
Photolyzed dimer 5 was dissolved in H2O (10 mg/100 µL) and
injected. A 20 min, 7 mL/min gradient (curve 4, System Controller
Waters 600E) composed of solvents A (0.05 M aqueous ammonium
acetate, pH 4.8) and B (0.05 M aqueous ammonium acetate (pH 4.8)/
CH3CN 88/12) 0-80% B, followed by a 30 min linear gradient of 80
to 100% B and a 10 min plateau, were used. Capacity factor: k′ 36 )
7.9.
Fractions containing photoproducts were concentrated, desalted by
RP-18 column chromatography with water used as eluent for 10 min
then a linear gradient of methanol in water (50/50) 0-100% B in 20
min with a flow rate of 8 mL/min.
A. Synthesis. 1. General Procedures. Boc-Deprotection: Pro-
cedure A. Carbamate derivatives were stirred for 3 h at room
temperature with 20% TFA in CH2Cl2. The solvent was removed under
reduced pressure and the residue was repeatedly diluted with CH2Cl2
and concentrated until obtention of a foam that was rapidly used for
the next step. Procedure B. Carbamate derivatives were stirred for 1
h at room temperature with 50% TFA in CH2Cl2. The solvent was
removed under reduced pressure, and the residue was repeatedly diluted
with CH2Cl2 and concentrated until obtention of a foam that was purified
by HPLC.
Experimental Section
General Remarks. Reagents and solvents were obtained from
commercial sources and used without further purification unless
indicated. CH2Cl2 and triethylamine were dried by heating, under
reflux, with calcium hydride. Dry DMF was obtained by azeotropic
distillation with toluene then redistilled over barium oxide. Amine (as
their trifluoroacetate salts) and acids were dried at room temperature
in a desiccator over P2O5 under high vacuum overnight prior to
condensation.
Spectroscopic Measurements. UV spectra were recorded in reverse
osmosed water (pH 6) on a Perkin-Elmer Lambda 5 spectrophotometer.
1H and 13C NMR spectra were recorded on Bruker AC250, AM300, or
AC300P instruments. 1H chemical shifts (δ) are reported in parts per
million relative to residual solvent peak (HOD or HOD/TFA δ 4.80,
DMSO δ 2.60, MeOD δ 3.30, CHCl3 δ 7.27). 13C chemical shifts are
reported in parts per million relative to solvent peak (CDCl3 δ 77.7,
CD3OD δ 49.0, DMSO δ 39.7). Spectra recorded in D2O were
calibrated relative to an external capillary standard of dioxane (δ 67.8).
At room temperature in D2O, compounds 1 and 2 were present as a
mixture of four rotamers due to restricted rotation around tertiary amide
Hydrolysis of the Methyl Ester Group: Procedure C.
A
suspension of methyl ester derivative in 2 N aqueous NaOH was stirred
for 15 to 60 min at room temperature. The aqueous solution was cooled
at 0 °C and the pH was adjusted to 2 by dropwise addition of 4 N
HCl. The aqueous phase was either extracted three times with EtOAc
and the joined organic phases were dried over sodium sulfate and
concentrated to dryness (preparation of 19 and 20) or carefully
concentrated and either used for the next step (saponification of 21
and 22) or purified by HPLC (preparation of 3-5).
Coupling Reaction: Procedure D. To a solution of the free acid
in dry DMF, pentafluorophenol (1.1 equiv) was added in a dry
atmosphere. The solution was cooled to 0 °C and DCC (1 equiv) was
added. The ice bath was removed after 1 h and the stirring was
continued for 5 h at room temperature. The suspension was filtered.
A freshly prepared solution of ammonium salt (1 equiv), and triethy-
lamine (1 equiv) in dry DMF was poured dropwise to the filtrate. The
solution was stirred for 20 h and then the solvent was removed in vacuo
at room temperature. The residue was purified by silica gel chroma-
tography.
2. Monomer Synthesis. Methyl (N3-Methylthymin-1-yl)acetate
(8). To a suspension of 6b (1.48 g, 7.47 mM) and K2CO3 (2.43 g,
17.61 mM) in acetone (40 mL) was added CH3I (2.4 mL, 38.28 mM).
The reaction was stirred overnight at room temperature then filtrated.
The filtrate was concentrated and chromatographed on silica gel using
EtOAc/heptane 80:20 as eluent, affording 8 in 96% yield. 1H NMR
(300 MHz, CDCl3): δ 6.94 (s, 1H), 4.45 (s, 2H), 3.78 (s, 3H), 3.35 (s,
3H); 1.94 (s, 3H). 13C NMR (75.5 MHz, CDCl3): δ 168.8, 164.4,
152.2, 138.7, 110.8, 53.3, 50.2, 28.5, 13.5. HRMS (CI) (M + H)+:
calcd for C9H13N2O4 213.0881, found 213.0881. Anal. Calcd for
C9H12N2O4: C, 50.94; H, 5.70; N, 13.20. Found: C, 51.03; H, 5.78;
N, 13.11.
Methyl (N3-Methyl-4-thiothymin-1-yl)acetate (9a). To a boiling
solution of 8 (2.35 g, 11.1 mM) in dioxane (65 mL) was added P2S5
(3.17 g, 14.28 mM). The solution was stirred for 2 h and then filtered
on Celite, and the filtrate was concentrated to dryness. Purification of
the residue by silica gel chromatography using a gradient of EtOAc in
heptane (0f20%) afforded 2.53 g (90.7%) of 9a as a yellow powder.
1H NMR (250 MHz, DMSO-d6): δ 7.86 (s, 1H), 4.73 (s, 2H), 3.81 (s,
1
bonds and consequently displayed a complex H NMR pattern. 1H
NMR spectra of these compounds are given in the Supporting
Information. Compounds 3-5 were present as mixtures of two
rotamers (60:40, 55:45, and 60:40, respectively), consequently some
1
signals in the H NMR are described as major (ma.) and minor (mi.)
component. For compounds 25, 26, 35, and 36 only base moiety
characteristic NMR signals are provided. EIMS were carried out using
an AEI MS 50 spectrometer. FABMS and HRMS (EI or CI (CH4))
were carried out using a Kratos MS 80 spectrometer. FABHRMS were
performed by the Service Central d′Analyse du CNRS (Lyon, France).
Melting points (mp) were determined with a Reichert apparatus and
are uncorrected.
HPLC Purifications. Dimers 1-5 were purified on a Delta-Pak
C18 (15 µm 100 Å) PrepPak Cartridge (47 mm × 30 cm) using a
linear gradient composed of A (H2O/CH3CN/TFA 95/5/0.1) and B
(H2O/CH3CN/TFA 50/50/0.1): time 0, 0%B; time 60 min, 80%B with
a flow rate of 25 mL/mn and the effluent monitored at 260 nm; retention
time (RT) 1, 42 min; RT 2, 44 min; RT 3, 38 min; RT 4, 45 min; RT
5, 51 min. Products 24-27, 30-32, and 35 were purified on a Prep
(29) Cadet, J.; Vigny, P. In The Photochemistry of Nucleic Acids;
Morrison, H., Ed.; Wiley and Sons: New York, 1990; Vol. 1, pp 1-272.
(30) (a) Sancar, A. Biochemistry 1994, 33, 2-9. (b) Carell, T. Angew.
Chem., Int. Ed. Engl. 1995, 34, 2491-2494. (c) Heelis, P. F.; Hartman, R.
F.; Rose, S. D. Chem. Soc. ReV. 1995, 289-297.
(31) (a) Todo, T.; Takemori, H.; Ryo, H.; Ihara, M.; Matsunaga, T.;
Nikaido, O.; Sato, K.; Nomura, T. Nature 1993, 361, 371-374.(b) Chen,
J.-J.; Mitchell, D. L.; Britt, A. B. Plant Cell 1994, 6, 1311-1317. (c) Kim,
S.-T.; Malhotra, K.; Taylor, J.-S.; Sancar, A. Photochem. Photobiol. 1996,
63, 292-295. (d) Kim, S.-T.; Malhotra, K.; Smith, C. A.; Taylor, J.-S.;
Sancar, A. J. Biol. Chem. 1994, 269, 8535-8540.
(32) (a) Van Wang, T.-C.; Rupert, C. S. Photochem. Photobiol. 1977,
25, 123-127. (b) Fajardo-Cavazos, P.; Salazar, C.; Nicholson, W. L. J.
Bacteriol. 1993, 175, 1735-1744.
(33) (a) Begley, T. P. Acc. Chem. Res 1994, 27, 394-401. (b) Fenick,
D. J.; Carr, H. S.; Falvey, D. E. J. Org. Chem. 1995, 60, 624-631.
(34) Clivio, P.; Fourrey, J.-L. J. Chem. Soc., Chem. Commun. 1996,
2203-2204.

1164 J. Am. Chem. Soc., Vol. 120, No. 6, 1998
CliVio et al.
3H), 3.76 (s, 3H); 2.14 (s, 3H). 13C NMR (62.9 MHz, DMSO-d6): δ
191.1, 168.3, 149.4, 136.7, 117.7, 52.7, 50.6, 35.4, 18.5. HRMS (CI)
(M + H)+: calcd for C9H13N2O3S 229.0647, found 229.0645. Anal.
Calcd for C9H12N2O3S: C, 47.37; H, 5.30; N, 12.28; S, 14.02. Found:
C, 47.37; H, 5.19; N, 12.07; S, 14.13.
N3-Methyl-4-thiothymin-1-ylacetic Acid (9b). A suspension of 9a
(1.5 g, 6.58 mM) in 2 N NaOH (aqueous, 12 mL) was sonicated for
10 min. The reaction was cooled to 0 °C and acidified to pH 1 with
concentrated HCl. The formed precipitate was filtrated, washed with
cold water, and dried, yielding 1.3 g (93%) of 9b as a slightly yellow
powder. 1H NMR (250 MHz, DMSO-d6): δ 13.38 (br s, 1H), 7.86 (s,
1H), 4.62 (s, 2H), 3.76 (s, 3H), 2.13 (s, 3H). 13C NMR (75.5 MHz,
DMSO-d6): δ 191.1, 169.2, 149.5, 137.0, 117.6, 50.7, 35.5, 18.6.
HRMS (CI) (M + H)+: calcd for C8H11N2O3S 215.0490, found
215.0473. Anal. Calcd for C8H10N2O3S: C, 44.86; H, 4.71; N, 13.08.
Found: C, 45.24; H, 4.88; N, 13.11.
Methyl N-[2-(Boc-amino)ethyl]glycinate (12). To a solution of
N-Boc-1,2-aminoethane (11, 2.2 g, 13.75 mM) and triethylamine (2.19
mL, 15.61 mM) in anhydrous CH2Cl2 (25 mL) was added dropwise
methyl bromoacetate (1.27 mL, 13.70 mmol). The solution was stirred
overnight in a dry atmosphere at room temperature, then diluted with
CH2Cl2 (25 mL), and washed with brine. The organic phase was dried
over sodium sulfate and concentrated. The residue was chromato-
graphed on silica gel using a gradient of EtOAc in heptane (25f50%),
and 12 was isolated as an oil (2.19 g, 68.6%). 1H NMR (250 MHz,
CDCl3): δ 5.20 (br t, 1H), 3.66 (s, 3H); 3.35 (s, 2H), 3.14 (q, 1H, J )
5.8 Hz), 2.67 (t, 1H, J ) 5.8 Hz), 1.89 (br s, 1H), 1.37 (s, 9H). 13C
NMR (62.9 MHz, CDCl3): δ 173.5, 156.6, 79.6, 52.3, 50.8, 49.3, 40.7,
28.9. MS (EI): m/z 232 (M)+.
Methyl N-[2-(Boc-amino)ethyl]-N-(thymin-1-ylacetyl)glycinate
(13). This compound was prepared, according to general procedure
D, from 6a (1.19 g, 6.46 mM) and using 22 mL of dry DMF. The
alkaline solution was prepared using 10 mL of DMF. The residue was
purified by silica gel chromatography using a gradient of methanol in
CH2Cl2 (0f10%) to afford 13 as a white foam (2.2 g, 85.6%). 1H
NMR (300 MHz, CDCl3): δ 9.74 (br s, 1H), 7.03 (mi.) and 6.97 (ma.)
(s, 1H), 5.72 (ma.) and 5.19 (mi.) (br t, 1H), 4.57 (ma.) and 4.42 (mi.)
(s, 2H), 4.22 (mi.) and 4.05 (ma.) (s, 2H), 3.78 (mi.) and 3.72 (ma.) (s,
3H), 3.51 (m, 2H), 3.28 (m, 2H), 1.87 (s, 3H), 1.41 (s, 9H). 13C NMR
(62.9 MHz, CDCl3): δ 170.5, 168.3 (mi.) and 168.0 (ma.), 165.1, 156.6,
151.8, 141.6, 111.0, 80.2, 53.3 (mi.) and 52.8 (ma.), 50.6 (mi.) and
49.2 (ma.), 49.0, 48.3, 39.1, 28.8, 12.7. HRMS (CI) (M + H)+: calcd
for C17H27N4O7 399.1879, found 399.1849. Anal. Calcd for
C17H26N4O7: C, 51.25; H, 6.58; N, 14.06. Found: C, 50.75; H, 6.25;
N, 13.96.
Methyl N-[2-(Boc-amino)ethyl]-N-[(4-thiothymin-1-yl)acetyl]-
glycinate (14). Derivative 14 was prepared according to general
procedure D, from 7b (1.29 g, 6.45 mM) using 22 mL of dry DMF.
The alkaline solution was prepared using 10 mL of DMF. The residue
was purified by silica gel chromatography using a gradient of methanol
in CH2Cl2 (0f1%) to afford 14 as a yellow foam (2.3 g, 86%). 1H
NMR (250 MHz, CDCl3): δ 10.98 (br s, 1H), 7.07 (mi.) and 7.03
(ma.) (s, 1H), 5.65 (ma.) and 5.18 (mi.) (br t, 1H), 4.60 (ma.) and 4.44
(mi.) (s, 2H), 4.19 (mi.) and 4.03 (ma.) (s, 2H), 3.74 (mi.) and 3.67
(ma.) (s, 3H), 3.47 (m, 2H), 3.23 (m, 2H), 1.99 (s, 3H), 1.37 (s, 9H).
13C NMR (62.9 MHz, CDCl3): δ 191.6, 170.5, 167.8 (mi.) and 167.5
(ma.), 156.6, 149.4, 138.4, 119.7, 80.4, 53.4 (mi.) and 53.0 (ma.), 50.8
(mi.) and 49.4 (ma.), 49.0, 39.1, 28.8, 17.4. HRMS (EI) (M)+: calcd
for C17H26N4O6S 414.1573, found 414.1573. Anal. Calcd for
C17H26N4O6S: C, 49.27; H, 6.32; N, 13.52. Found: C, 49.55; H, 6.44;
N, 12.65.
Methyl N-[2-(Boc-amino)ethyl]-N-[(N3-methyl-4-thiothymin-1-yl)-
acetyl]glycinate (15). Derivative 15 was prepared according to general
procedure D, from 9b (1.30 g, 6.07 mM) using 22 mL of dry DMF.
The alkaline solution was prepared using 10 mL of DMF. The residue,
purified by silica gel chromatography using a gradient of methanol in
ether (0f1%) afforded 15 as a yellow foam (1.67 g, 64%). 1H NMR
(300 MHz, CDCl3): δ 7.05 (mi.) and 7.00 (ma.) (s, 1H), 5.55 (ma.)
and 5.05 (mi.) (br t, 1H), 4.59 (ma.) and 4.42 (mi.) (s, 2H), 4.16 (mi.)
and 4.00 (ma.) (s, 2H), 3.73 (mi.) and 3.66 (ma.) (s, 3H), 3.68 (s, 3H),
3.47 (m, 2H), 3.20 (m, 2H), 2.03 (s, 3H), 1.36 (ma.) and 1.34 (mi.) (s,
9H). 13C NMR (75.5 MHz, CDCl3): δ 192.1, 170.4 (ma.) and 170.2
(mi.), 167.8 (mi.) and 167.4 (ma.), 156.5, 150.3, 135.1, 119.7, 80.3,
53.4 (mi.) and 52.9 (ma.), 50.6 (mi.) and 50.2 (ma.), 49.3, 49.1, 39.1
(ma.) and 38.9 (mi.), 36.1, 28.8, 19.4. HRMS (CI) (M + H)+: calcd
for C18H29N4O6S 429.1819, found 429.1809. Anal. Calcd for
C18H28N4O6S: C, 50.46; H, 6.59; N, 13.08; S, 7.47. Found: C, 50.18;
H, 6.53; N, 12.66; S, 7.26.
3. Dimer Synthesis. Methyl N-[-2-[N′-[2-(Boc-amino)ethyl]-N′-
(thymin-1-ylacetyl)glycyl]amino]ethyl]-N-[(4-thiothymin-1-yl)acetyl]-
glycinate (21). The preparation of 17 was accomplished according to
general procedure A from 14 (2.07 g, 5mM) using 12 mL of 20% TFA
in CH2Cl2. The preparation of 19 was accomplished in 15 min
according to general procedure C from 13 (1.99 g, 5 mM) using 20
mL of NaOH. The coupling reaction was accomplished according to
general procedure D using 5 mM of 19 and 17 and purification was
achieved through silica gel chromatography using a gradient of MeOH
in CH2Cl2 (0f10%) affording 21 (2.2 g, 65%) as yellow foam. 1H
NMR (300 MHz, CD3OD): δ 7.54 (ma.), 7.43 (mi.), 7.34 (mi.) and
7.23 (ma.) (s, 2H), 4.90-4.49 (s, 8H), 4.36 (mi.), 4.34 (mi.), 4.30 (mi.),
4.17 (mi.), 4.15 (ma.), 4.12 (ma.) and 3.99 (mi.) (s, 4H), 3.80 (mi.),
3.78 (mi.), 3.74 ma.), 3.71 (mi.) (s, 3H), 3.69-3.10 (m, 8H), 2.01 (mi.),
1.98 (mi.) and 1.92 (ma.) (s, 3H), 1.85 (mi.), 1.83 (mi.) and 1.78 (ma.)
(s, 3H), 1.42 (s, 9H). 13C NMR (75.5 MHz, CD3OD) (several CH2
were obscured under the solvent peak): δ 192.6, 172.1, 171.2, 171.1,
170.9, 169.7, 168.9, 166.4, 158.0, 152.7, 150.4, 143.4, 140.1 (ma.),
139.7 (mi.), 119.4 (ma.), 110.8, 80.2, 53.0, 52.8, 52.6, 51.4 (mi.) and
51.0 (ma.), 39.2 (ma.), 38.7 (mi.), 37.8 (mi.) and 37.5 (ma.), 28.4, 16.8,
12.0, 11.9. MS (FAB): m/z 703 (M + Na)+.
Methyl N-2-[[N′-[2-(Boc-amino)ethyl]-N′-[(4-thiothymin-1-yl)-
acetyl]glycyl]amino)ethyl]-N-(thymin-1-ylacetyl)glycinate (22). The
preparation of 16 was accomplished according to general procedure A
from 13 (1.98 g, 5 mM) using 12 mL of TFA/CH2Cl2. The preparation
of 20 was accomplished in 15 min according to general procedure C
from 14 (2.07 g, 5 mM) using 20 mL of NaOH. The coupling reaction
was accomplished according to general procedure D using 5 mM of
20 and 16 and the purification was achieved through silica gel
chromatography using a gradient of MeOH in CH2Cl2 (0f10%)
affording 22 (2.18 g, 64%) as yellow foam. 1H NMR (300 MHz, CD3-
OD): δ 7.47 (ma.) and 7.39 (mi.), 7.30 (mi.) and 7.28 (ma.) (s, 2H),
4.90-4.49 (s, 8H), 4.34 (mi.), 4.28 (mi.), 4.17 (mi.), 4.14 (ma.), 4.12
(ma.) and 3.98 (mi.) (s, 4H), 3.79 (mi.), 3.78 (mi.), 3.73 (ma.), 3.71
(mi.) (s, 3H), 3.66-3.12 (m, 8H), 2.01 (mi.), 2.00 (mi.) and 1.99 (ma.)
(s, 3H), 1.84 (mi.), 1.80 (mi.) and 1.76 (ma.) (s, 3H), 1.42 (s, 9H). 13
C
NMR (75.5 MHz, CD3OD) (several CH2 were obscured under the
solvent peak): δ 192.5, 172.0, 171.1, 169.5, 169.2, 166.5, 158.0, 152.7,
150.4, 143.7, 143.5, 139.7, 139.6, 119.7, 119.6, 110.4, 80.2, 53.0, 52.7,
52.6, 51.4, 51.0, 39.2, 38.7, 37.9, 37.5, 37.2, 28.5, 16.8, 16.7, 12.0.
MS (FAB): m/z 703 (M + Na)+.
Methyl N-[2-[[N′-[2-(Boc-amino)ethyl]-N′-(thymin-1-ylacetyl)-
glycyl]amino]ethyl]-N-[(N3-methyl-4-thiothymin-1-yl)acetyl]glyci-
nate (23). Preparation of 18 was accomplished according to general
procedure A from 15 (1.57 g, 3.66 mM) using 12 mL of TFA/CH2Cl2.
For the preparation of 19, see synthesis of 21. The coupling reaction
was accomplished according to general procedure D using 3.47 mM
of 19 and 18. Product purification was achieved through silica gel
chromatography using a gradient of MeOH in CH2Cl2 (0f10%)
affording 23 (1.79 g, 74%) as yellow foam. 1H NMR (300 MHz, CD3-
OD): δ 7.53 (ma.) and 7.44 (mi.) and 7.36 (mi.), 7.31 (mi.) and 7.25
(mi.) and 7.18 (ma.) (s, 2H), 4.86-4.43 (s, 7H), 4.37 (mi.), 4.30 (mi.),
4.18 (mi.), 4.15 (ma.), 4.13 (ma.) and 3.99 (mi.) (s, 4H), 3.81 (mi.),
3.79 (mi.), 3.74 (ma.), 3.73 (mi.), 3.72 (mi.), 3.71 (ma.), 3.68 (mi.) (s,
6H), 3.67-3.12 (m, 8H), 2.08 (mi.), 2.06 (mi.), 2.04 (mi.) and 1.98
(ma.) (s, 3H), 1.83 (mi.), 1.82 (mi.), 1.80 (mi.) and 1.74 (ma.) (s, 3H),
1.42 (s, 9H). 13C NMR (75.5 MHz, CD3OD) (several CH2 were buried
below the solvent peak): δ 192.5, 172.1, 171.0, 169.6, 169.0, 166.3,
156.0, 152.6, 151.2, 143.3, 137.1, 136.7, 119.9, 119.6, 110.8, 110.6,
80.2, 53.0, 52.8, 52.6, 51.4, 51.2, 51.0, 39.2, 38.7, 37.8, 37.5, 35.7,
28.4, 18.6, 12.0, 11.8. MS (FAB): m/z 595 (M + H)+.
N-[2-[[N′-(2-Aminoethyl)-N′-(thymin-1-ylacetyl)glycyl]amino]et-
hyl]-N-[(4-thiothymin-1-yl)acetyl]glycine (1). (1) Methyl ester sa-
ponification of 21 (1.146 g, 1.68 mM) was accomplished in 50 min

Unprecedented (5-4) Photoadduct ReVersion
J. Am. Chem. Soc., Vol. 120, No. 6, 1998 1165
2
after dissolution in 10 mL of MeOH according to general procedure C
using 10 mL of NaOH solution. (2) Boc deprotection was accomplished
according to general procedure B using 10 mL of TFA/CH2Cl2. After
HPLC purification, 690 mg of 1 (53%) was obtained as a yellow
amorphous powder. UV (λmax H2O): 266, 338 nm. 13C NMR (75.5
MHz, D2O): δ 194.3, 194.1, 193.9, 175.7, 175.3, 174.5, 173.5, 173.4,
173.2, 172.0, 171.5, 169.5, 169.2, 155.1, 155.0, 152.6, 146.1, 145.8,
145.6, 143.4, 142.9, 142.5, 123.0, 122.6, 122.5, 122.3, 114.1, 114.0,
113.5, 53.2, 53.0, 52.9, 52.7, 52.5, 52.3, 51.3, 51.1, 50.9, 50.3, 50.2,
49.2, 48.9, 40.7, 40.3, 19.1, 19.0, 14.4. HRMS (FAB) (M + H)+: calcd
for C22H31N8O8S 567.1986, found 567.1985. Anal. Calcd for
C22H30N8O8S‚1.5CF3COOH‚2H2O: C, 38.81; H, 4.62; N, 14.49; S, 4.13.
Found: C, 38.15; H, 4.65; N, 14.16; S, 4.16.
N-[2-[[N′-(2-Aminoethyl)-N′-[(4-thiothymin-1-yl)acetyl]glycyl]-
amino]ethyl]-N-(thymin-1-ylacetyl)glycine (2). (1) Methyl ester
saponification of 22 (1.079 g, 1.59 mM) was accomplished in 50 min
after dissolution in 10 mL of MeOH according to general procedure C
using 10 mL of NaOH solution. (2) Boc deprotection was accomplished
according to general procedure B using 10 mL of TFA/CH2Cl2. After
HPLC purification, 684 mg of 2 (55%) was obtained as a yellow
amorphous powder. UV (λmax H2O): 267, 337 nm. 13C NMR (75.5
MHz, D2O): δ 194.9, 194.6, 176.3, 176.0, 175.1, 174.7, 173.8, 173.6,
173.5, 173.3, 173.1, 172.7, 172.0, 170.2, 170.0, 169.7, 155.5, 153.2,
147.0, 146.7, 146.5, 143.4, 143.2, 123.5, 123.1, 114.2, 114.1, 113.8,
53.9, 53.4, 52.7, 52.4, 52.0, 51.5, 50.8, 49.7, 41.3, 40.9, 40.7, 19.7,
15.0. HRMS (FAB) (M + H)+: calcd for C22H31N8O8S 567.1986,
found 567.1982. Anal. Calcd for C22H30N8O8S‚1.5CF3COOH‚2H2O:
C, 38.81; H, 4.62; N, 14.49; S, 4.13. Found: C, 38.26; H, 4.71; N,
14.04; S, 3.95.
N-[2-[[[2-[(Thymin-1-ylacetyl)amino]ethyl]glycyl]amino]ethyl]-
N-[(4-thiothymin-1-yl)acetyl]glycine (3). (1) Boc deprotection was
accomplished according to general procedure A from 21 (1.012 g, 1.49
mM) in 10 mL of TFA/CH2Cl2. (2) Methyl ester saponification and
acyl migration were accomplished in 60 min according to general
procedure C using 10 mL of NaOH solution. After HPLC purification,
401 mg of 3 (36.5%) was obtained as a yellow amorphous powder
that crystallized from MeOH/H2O (1:1). Mp: 195-198 °C. UV (λmax
H2O) 266, 338 nm. 1H NMR (300 MHz, D2O): δ 7.41 (s, 1H), 7.36
(s, 1H), 4.76 (ma.) and 4.61 (mi) (s, 2H), 4.48 (mi) and (4.46) (ma.)
(s, 2H), 4.28 (mi.) and 4.14 (ma.) (s, 2H), 3.98 (ma.) and 3.79 (mi.) (s,
2H), 3.68-3.20 (m, 8H), 2.01 (s, 3H), 1.81 (s, 3H). 13C NMR (75.5
MHz, D2O): δ 193.0, 174.1, 171.9, 170.4, 168.3, 153.8, 151.3, 144.7,
141.8, 141.6, 121.3, 112.6, 52.2, 51.4, 50.6, 49.8, 49.5, 48.6, 48.3, 38.4,
37.6, 17.6, 12.9. HRMS (FAB) (M + H)+: calcd for C22H31N8O8S
567.1986, found 567.2018. Anal. Calcd for C22H30N8O8S‚
1.5CF3COOH: C, 40.71; H, 4.30; N, 15.19; S, 4.34. Found: C, 40.85;
H, 4.54; N, 15.44; S, 4.61.
) 0.049 (with Rw ) [∑w(Fo - |Fc|)2/∑wFo ]1/2 and w ) 1/[σ2(Fo) +
2
0.0002Fo ], goodness of fit 0.95. The residual electron density in the
final difference map was located between -0.35 and 0.22 e Å3.
N-[2-[[[2-[[4-Thiothymin-1-yl)acetyl]amino]ethyl]glycyl]amino]-
ethyl]-N-(thymin-1-ylacetyl)glycine (4). (1) Boc deprotection was
accomplished according to general procedure A from 22 (1.024 g, 1.50
mM) in 10 mL of TFA/CH2Cl2. (2) Methyl ester saponification and
acyl migration were accomplished in 60 min according to general
procedure C using 10 mL of NaOH. After HPLC purification, 386
mg of 4 (35%) was obtained as a yellow amorphous powder. UV (λmax
H2O): 267, 338 nm. 1H NMR (300 MHz, D2O): δ 7.46 (s, 1H), 7.31
(s, 1H), 4.69 (ma.), 4.52 (mi) (s, 2H), 4.52 (mi) and (4.50) (ma.) (s,
2H), 4.19 (mi.), 4.10 (ma.) (s, 2H), 3.98 (ma.) and 3.79 (mi.) (s, 2H),
3.70-3.20 (m, 8H), 1.99 (s, 3H), 1.82 (s, 3H). 13C NMR (75.5 MHz,
D2O): δ 193.0, 174.1, 171.4, 171.0, 168.4, 168.3, 167.8, 153.6, 151.5,
144.9, 144.7, 141.7, 121.6, 112.3, 52.9, 52.7, 50.8, 50.6, 49.7, 49.5,
48.8, 48.5, 38.5, 37.7, 17.7, 13.0. HRMS (FAB) (M + H)+: calcd for
C22H31N8O8S 567.1986, found 567.2010. Anal. Calcd for
C22H30N8O8S‚1.5‚CF3COOH: C, 40.71; H, 4.30; N, 15.19; S, 4.34.
Found: C, 40.21; H, 4.76; N, 15.31; S, 4.53.
N-[2-[[[2-[(Thymin-1-ylacetyl)amino]ethyl]glycyl]amino]ethyl]-
N-[(N3-methyl-4-thiothymin-1-yl)acetyl]glycine (5). (1) Boc depro-
tection was accomplished according to general procedure A from 23
(1.690 g, 2.43 mM) in 10 mL of TFA/CH2Cl2. (2) Methyl ester
saponification and acyl migration were accomplished in 60 min
according to general procedure C using 10 mL of NaOH. After HPLC
purification, 447 mg of 5 (27%) was obtained as a yellow amorphous
powder. UV (λmax H2O): 267, 331 nm. 1H NMR (300 MHz, D2O)
[60:40 rotamer mixture]: δ 7.47 (s, 1H), 7.43 (mi.), 7.41 (ma.) (s, 1H),
4.80 (ma.), 4.71 (mi.) (s, 2H), 4.51 (mi), 4.49 (ma.) (s, 2H), 4.32 (mi.),
4.18 (ma.) (s, 2H), 4.08 (ma.), 3.87 (mi.) (s, 2H), 3.74 (mi.), 3.73 (ma.)
(s, 3H), 3.70-3.20 (m, 8H), 2.12 (s, 3H), 1.87 (s, 3H). 13C NMR (75.5
MHz, D2O): δ 194.9, 176.0, 173.4, 172.4, 172.0, 169.9, 169.4, 155.4,
153.8, 146.4, 140.3, 123.1, 114.2, 54.4, 53.8, 52.9, 51.5, 50.6, 40.3,
39.4, 39.1, 21.5, 14.8. HRMS (FAB) (M + H)+: calcd for C23H33N8O8S
581.2142, found 581.2133. Anal. Calcd for C23H32N8O8S‚CF3COOH‚
4H2O: C, 39.16; H, 5.39; N, 14.62; S, 4.17. Found: C, 38.71; H,
5.17; N, 15.05; S, 4.27.
B. General Photolysis Procedure. Irradiation experiments (20 mg/
100 mL) were performed at pH 3-4, with an Original Hanau
Quarzlampen Fluotest-Forte Ref. 5261 for 30 min (1 and 2), 6 h (3
and 4), and 5.5 h (5) (progress of the reaction was monitored by UV
and 1H NMR spectroscopy). Irradiations were performed under
continuous nitrogen bubbling. See above for purification conditions.
1
Photoproduct 24: Yield, 17.9%; UV (λmax H2O) 267, 317 nm; H
NMR (300 MHz, D2O) [50(a)/30(b)/20(c) rotamer mixture] δ 8.30(c),
8.26(a), 8.14(b) (1H, s), 7.15(a), 6.76(b), 6.34(c) (1H,s), 5.35-3.10
(18H), 2.09(a), 2.06(b), 2.05(c) (3H, s). 13C NMR (75.5 MHz, D2O)
δ 176.1, 174.1, 173.2, 172.1, 172.0, 171.2, 168.1, 158.2, 155.4, 155.2,
154.9, 149.2, 145.8, 144.4, 119.3, 118.3, 113.7, 110.9, 55.3, 55.0, 54.4,
53.9, 53.1, 52.7, 52.3, 51.7, 50.9, 50.5, 49.9, 49.1, 48.5, 48.1, 42.6,
41.7, 40.4, 40.1, 16.0. HRMS (FAB) (M + H)+ calcd for C22H29N8O8
533.2108, found 533.2090.
Crystal Structure of 3. Crystal data: C22H30N8O8‚2H2O, Mw
)
602.62, colorless crystal of 0.06 × 0.30 × 0.30 mm, monoclinic, space
group Cc, Z ) 4, a ) 7.634 (2), b ) 22.258 (4), c ) 17.021 (3) Å, â
) 97.52 (3), V ) 2867 (1) Å3, dcalc ) 1.40 g cm-3, F(000) ) 1272, λ
(Cu KR) ) 1.5418 Å, µ ) 1.54 mm-1. Intensity data were measured
on a Enraf-Nonius CAD-4 diffractometer using graphite-monochro-
mated Cu KR radiation and the (θ - 2θ) scan technique up to θ )
66°. Of the 4835 collected reflections (-8 e h e 8, -26 e k e 26,
0 e l e 20), 2534 were unique (Rint ) 0.057) of which 2479 were
considered as observed having I g 3σ(I). Cell parameters were refined
from 25 well-centered reflections with 13.1 e θ e 20.6°. The structure
was solved by direct methods using SHELXS8635 and refined by full-
matrix least-squares with SHELX76,36 minimizing the function ∑w(Fo
- |Fc|)2. Two water molecules were found on difference Fourier maps.
The coordinates of the hydrogen atoms, located in difference Fourier
maps, were refined. They were assigned an isotropic thermal factor
equivalent to that of the bonded carbon atom, plus 10%. One hydrogen
atom of the water molecules W40 was not located on difference map,
it was set in theoretical position according to the probable hydrogen
bond W40sH‚‚‚O5. Convergence was reached at R ) 0.037 and Rw
1
Photoproduct 25: Yield 8.7%. UV (λmax H2O) 279 nm; H NMR
(300 MHz, D2O) (80:20 isomer mixture) δ 7.57 (mi.) 7.46 (ma.) (1H,
s), 1.42 (mi.) (d, J ) 7.3 Hz), 1.37 (ma.) (d, J ) 6.7 Hz) (3H). 13C
NMR (75.5 MHz, D2O) δ 210.4, 175.9, 175.4, 173.6, 172.5, 168.8,
155.7, 154.2, 149.1, 148.6, 114.3, 112.9, 64.5, 63.5, 54.7, 54.2, 51.5,
51.3, 50.8, 49.0, 47.8, 40.9, 40.6, 39.6, 27.4, 25.4, 17.6. HRMS (FAB)
(M + H)+ calcd for C22H31N8O8S 567.1986, found 567.1971.
Photoproduct 26: Yield 4.3%; UV (λmax H2O) sh 255 nm; 1H NMR
(300 MHz, D2O): δ 6.06 (1H, s), 1.64 (s). 13C NMR (75.5 MHz, D2O)
δ 176.0, 175.5, 174.4, 174.2, 173.7, 155.8, 155.5, 132.4, 108.7, 83.0,
78.2, 53.7, 52.2, 51.4, 50.1, 49.8, 48.6, 47.2, 40.6, 40.2, 30.3, 19.3.
HRMS (FAB) (M + H)+ calcd for C22H31N8O8S 567.1986, found
567.1952.
1
Photoproduct 27: Yield 44.2%; UV (λmax H2O) 268, 317 nm; H
NMR (300 MHz, D2O) (50:50 rotamer mixture) δ 7.82, 7.78 (1H, s),
6.58, 6.18 (1H, s), 5.41 (d, J ) 16.1 Hz), 5.01 (d, J ) 16.7 Hz) (1H),
4.79 (d, J ) 16.1 Hz), 4.66 (2H, m), 4.29 (d, J ) 16.7 Hz), 4.15 (d, J
) 16.3 Hz) (1H), 4.07-3.56 (8H, m), 3.47-3.20 (5H, m), 2.00 (3H,
(35) Sheldrick, G. M. SHELXS86. Program for the solution of crystal
structures. University of Go¨ttingen, Germany, 1986.
(36) Sheldrick, G. M. SHELX76. Program for crystal structure deter-
mination. University of Cambridge, England, 1976.

1166 J. Am. Chem. Soc., Vol. 120, No. 6, 1998
CliVio et al.
s). 13C NMR (75.5 MHz, D2O) δ 181.1, 181.0, 178.8, 178.4, 174.7,
174.6, 172.5, 171.7, 171.6, 168.5, 160.8, 160.4, 154.9, 154.8, 153.0,
152.8, 147.0, 146.7, 119.7, 118.7, 113.5, 113.0, 55.7, 55.1, 54.6, 54.5,
53.0, 52.0, 51.7, 51.5, 50.9, 49.2, 48.6, 40.8, 40.7, 39.3, 35.2, 34.3,
16.2, 16.1. HRMS (FAB) (M + H)+ calcd for C22H29N8O8 533.2108,
found 533.2134.
Preparation of Compounds 30 and 35. The irradiation mixture
(20 mg) was lyophilized and the residue dissolved in D2O (400 µL)
then 40 µL of a 5% solution of CH3SO2SCH3 in CD3OD were added.
Evolution of the reaction was controlled by 1H NMR. After completion,
the solution was freeze-dried then stored at -18 °C. This sequence
was repeated 10 times, then lyophilisates were pooled and purified by
HPLC.
Photoproduct 31: Yield 15.7% from 4 and 7.7% from 3; UV (λmax
1
1
H2O) 267 nm; H NMR (300 MHz, D2O) (60:40 rotamer mixture) δ
Compound 30: Yield 32.2%; UV (λmax H2O) 318 nm; H NMR
7.45 (1H, s), 7.40 (1H, s), 4.76 (mi.), 4.60 (ma.) (2H, s), 4.52 (2H, s),
4.06 (ma.), 3.95 (mi.) (2H, s), 3.87 (mi.) 3.72 (ma.) (2H, s), 3.58 (5H,
br s), 3.45 (1H, m), 3.18 (2H, m), 1.88 (3H, s). HRMS (FAB) (M +
H)+ calcd for C22H31N8O9 551.2214, found 551.2231.
(300 MHz, D2O/TFA) δ 7.50 (1H, s), 5.27 (1H, s), 4.62 (1H, d, J )
16.1 Hz), 4.42 (1H, d, J ) 16.1 Hz), 4.12 (1H, d, J ) 17.3 Hz), 3.92-
3.42 (7H, m), 3.35-2.80 (6H, m), 1.73 (3H, s), 1.20 (3H, s). 13C NMR
(75.5 MHz, D2O/TFA) δ 175.1, 173.9, 173.6, 172.7, 170.0, 168.9, 158.1,
155.2, 153.7, 116.6, 85.6, 57.0, 53.6, 50.6, 50.0, 48.7, 48.3, 38.5, 37.7,
18.5, 16.0. HRMS (FAB) (M + H)+ calcd for C22H31N8O9 551.2214,
found 551.2234.
1
Photoproduct 32: Yield 4.0%. UV (λmax H2O) 269, 315 nm; H
NMR (250 MHz, D2O) δ 7.89 (1H, s), 7.17 (1H, s), 4.78 (2H, s), 4.55
(2H, s), 4.08 (2H, s), 3.74 (2H, s), 3.69 (2H, s), 3.67 (2H, m), 3.54
(2H, m), 3.39 (2H, m), 3.16 (2H, m), 2.10 (3H, s). 13C NMR (75.5
MHz, D2O) δ 180.8, 179.1, 173.8, 173.2, 171.4, 169.7, 160.8, 156.2,
153.2, 146.8, 119.6, 114.8, 56.0, 54.4, 53.4, 52.6, 50.7, 49.8, 40.5, 17.0.
HRMS (FAB) (M + H)+ calcd for C22H29N8O8 533.2108, found
533.2089.
1
Compound 35: Yield 13.7%. UV (λmax H2O) 318 nm; H NMR
(300 MHz, D2O/TFA) (80:20 isomer mixture) δ 7.94 (1H, s), 5.63 (ma.),
5.55 (mi.) (1H, s), 2.22 (mi.), 2.12 (ma.) (3H, s), 1.61 (mi.), 1.57 (ma.),
(3H, s). 13C NMR (75.5 MHz, D2O/TFA) δ 176.2, 174.5, 174.4, 172.2,
171.7, 167.7, 157.9, 154.4, 153.6, 153.3, 116.6, 86.5, 57.9, 55.4, 51.2,
50.3, 49.8, 49.3, 48.8, 39.3, 38.2, 18.3, 15.8. HRMS (FAB) (M +
H)+ calcd for C22H31N8O9 551.2214, found 551.2233.
Photoproduct 36: Yield 21.1%; UV (λmax H2O) 277 nm; 1H NMR
(300 MHz, D2O/TFA) (80:20 isomer mixture) δ 7.41 (mi.), 7.25 (ma.)
(1H, s), 2.40 (2H, m), 1.35 (ma.) (3H, d, J ) 6.4 Hz), 1.31 (mi.) (3H,
d, J ) 6.5 Hz). 13C NMR (62.9 MHz, D2O/TFA) δ 210.7, 210.2, 173.7,
173.5, 172.0, 171.5, 170.8, 168.3, 167.8, 166.7, 166.4, 153.4, 153.2,
152.8, 146.5, 111.0, 110.4, 60.9, 59.0, 53.0, 51.2, 50.8, 50.5, 49.8, 49.0,
48.3, 47.6, 46.4, 45.5, 38.3, 37.8, 37.3, 37.1, 36.9, 25.6, 17.0, 16.7.
HRMS (FAB) (M + H)+ calcd for C23H33N8O8S 581.2142, found
581.2145.
Supporting Information Available: X-ray crystallographic
analysis for 3 and 1H NMR spectra (D2O) of 1 and 2 (11 pages).
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instructions.
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