Studies on the chemical synthesis of oligonucleotides containing the (6- 4) photoproduct of thymine-cytosine and its repair by (6-4) photolyase

Article abstract of DOI:10.1021/ja982004y

To prepare oligonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct between thymine and cytosine, which is formed more efficiently than any counterpart at other dipyrimidine sites, a dimer building block of this photoproduct was synthesized.

Full text of DOI:10.1021/ja982004y

10634
J. Am. Chem. Soc. 1998, 120, 10634-10642
Studies on the Chemical Synthesis of Oligonucleotides Containing the
(6-4) Photoproduct of Thymine-Cytosine and Its Repair by (6-4)
Photolyase
Toshimi Mizukoshi,† Kenichi Hitomi,†,‡ Takeshi Todo,‡ and Shigenori Iwai*,†
Contribution from the Department of Bioorganic Chemistry, Biomolecular Engineering Research Institute,
6-2-3 Furuedai, Suita, Osaka 565-0874, Japan, and Radiation Biology Center, Kyoto UniVersity,
Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
ReceiVed June 8, 1998
Abstract: To prepare oligonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct between
thymine and cytosine, which is formed more efficiently than any counterpart at other dipyrimidine sites, a
dimer building block of this photoproduct was synthesized. Irradiation of thymidylyl(3′-5′)deoxycytidine,
with protecting groups at the phosphate and the 3′-hydroxyl function, gave a hydrate of cytosine as the main
product, and the formation of the (6-4) photoproduct reached a plateau faster than that from the thymidylyl-
(3′-5′)thymidine derivative. After chromatographic purification of the desired (6-4) photoproduct, a
phosphoramidite building block was synthesized in three steps. When this compound was used for the usual
oligonucleotide assembly on a solid support, it was found that acylation of the amino group of the 5′-pyrimidine
component occurred at the capping step. Oligonucleotides were synthesized successfully by omitting the capping
steps after coupling of the (6-4) photoproduct unit. These synthetic oligonucleotides were applied to the
characterization of the (6-4) photolyase. By HPLC analyses of the photoreactivation product and its nucleoside
composition, it was demonstrated that this (6-4) photoproduct was repaired to its original pyrimidine components
by this enzyme.
Introduction
photoreactivate this damage, (6-4) photolyase, was discovered.6
The (6-4) photoproduct is converted to its Dewar valence
isomer by absorbing UVB light with wavelengths between 280
and 320 nm.7
Base moieties of nucleic acids are subject to modification
by ionizing radiation, ultraviolet (UV) light, and various
chemicals. Among these damages, those caused by UV
radiation at dipyrimidine sites in DNA, namely cis-syn
cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone
photoproducts (abbreviated as (6-4) photoproducts), have been
studied most intensively.1 The (6-4) photoproduct is produced
by ring opening of an oxetane or azetidine intermediate formed
by a 2π + 2π photocycloaddition of the excited carbonyl or
iminyl group of the 3′-pyrimidine onto the C5-C6 double bond
of the 5′ component. Since the two rings constituting the (6-
4) photoproduct are perpendicular to each other,2 this damage
causes a large bend in the DNA helix.3 The (6-4) photoproduct
blocks replication, and translesion synthesis induces mutations
at extremely high frequencies, especially on the 3′ side of the
photoproduct.4 This DNA damage is repaired by the nucleotide
excision repair pathway in human cells,5 and an enzyme to
In our previous work, we synthesized a phosphoramidite
building block of the (6-4) photoproduct of thymidylyl(3′-
5′)thymidine (T(6-4)T; Figure 1) and incorporated it into
oligonucleotides,8 which have been applied to studies of the
molecular biology of mutation9 and repair.10 Synthetic oligo-
nucleotides containing such specific damage at a single site in
a defined sequence are useful tools for biochemical studies on
various subjects, including structure,3 mutagenesis,4,9 and
repair.10,11 In most cases, the (6-4) photoproduct used in these
experiments is T(6-4)T, but it has been reported that the (6-
4) photoproduct is formed most efficiently at the TC sequence.
For example, in vivo mapping of (6-4) photoproducts at single-
nucleotide resolution revealed that the major site was TC
(followed by CC),12 and in quantitative experiments using
(6) Todo, T.; Takemori, H.; Ryo, H.; Ihara, M.; Matsunaga, T.; Nikaido,
O.; Sato, K.; Nomura, T. Nature 1993, 361, 371-374.
* Corresponding author. Tel.: +81-6-872-8208. FAX: +81-6-872-8219.
E-mail: iwai@bioorg.beri.co.jp.
(7) Taylor, J.-S.; Cohrs, M. P. J. Am. Chem. Soc. 1987, 109, 2834-
2835.
† Biomolecular Engineering Research Institute.
(8) Iwai, S.; Shimizu, M.; Kamiya, H.; Ohtsuka, E. J. Am. Chem. Soc.
1996, 118, 7642-7643.
(9) (a) Fujiwara, Y.; Iwai, S. Biochemistry 1997, 36, 1544-1550. (b)
Kamiya, H.; Iwai, S.; Kasai, H. Nucleic Acids Res. 1998, 26, 2611-2617.
(10) Hitomi, K.; Kim, S.-T.; Iwai, S.; Harima, N.; Otoshi, E.; Ikenaga,
M.; Todo, T. J. Biol. Chem. 1997, 272, 32591-32598.
(11) (a) Reardon, J. T.; Nichols, A. F.; Keeney, S.; Smith, C. A.; Taylor,
J.-S.; Linn, S.; Sancar, A. J. Biol. Chem. 1993, 268, 21301-21308. (b)
Kim, S.-T.; Malhotra, K.; Smith, C. A.; Taylor, J.-S.; Sancar, A. J. Biol.
Chem. 1994, 269, 8535-8540. (c) Zhao, X.; Liu, J.; Hsu, D. S.; Zhao, S.;
Taylor, J.-S.; Sancar, A. J. Biol. Chem. 1997, 272, 32580-32590.
(12) Pfeifer, G. P.; Drouin, R.; Riggs, A. D.; Holmquist, G. P. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 1374-1378.
‡ Kyoto University.
(1) (a) Taylor, J.-S. Pure Appl. Chem. 1995, 67, 183-190. (b) Pfeifer,
G. P. Photochem. Photobiol. 1997, 65, 270-283.
(2) (a) Rycyna, R. E.; Alderfer, J. L. Nucleic Acids Res. 1985, 13, 5949-
5963. (b) Taylor, J.-S.; Garrett, D. S.; Wang, M. J. Biopolymers 1988, 27,
1571-1593.
(3) Kim, J.-K.; Choi, B.-S. Eur. J. Biochem. 1995, 228, 849-854.
(4) (a) LeClerc, J. E.; Borden, A.; Lawrence, C. W. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 9685-9689. (b) Smith, C. A.; Wang, M.; Jiang, N.;
Che, L.; Zhao, X.; Taylor, J.-S. Biochemistry 1996, 35, 4146-4154.
(5) For reviews see: (a) Sancar, A. Annu. ReV. Biochem. 1996, 65, 43-
81. (b) Wood, R. D. Annu. ReV. Biochem. 1996, 65, 135-167.
10.1021/ja982004y CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/01/1998

Synthesis of Oligonucleotides by Photolyase
J. Am. Chem. Soc., Vol. 120, No. 41, 1998 10635
of 2′-deoxycytidine, as well as its 3′-hydroxyl function, was
protected to prepare thymidylyl(3′-5′)deoxycytidine. The
protecting group for this amino function should be removed
without detaching the other base-labile protecting groups
because the base moiety must be deprotected before the
formation of the (6-4) photoproduct by UV irradiation. The
4,4′-dimethoxytrityl (DMT) group was chosen for this purpose,
and 4-N-DMT-3′-O-levulinyl-2′-deoxycytidine (2) was prepared
in three steps from 2′-deoxycytidine (1). After coupling with
5′-O-DMT-thymidine 3′-(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite, the two DMT groups were removed with trichlo-
roacetic acid. It took 25 h at room temperature to deprotect
the amino group completely. The resulting dinucleoside mono-
phosphate 3 had a 2-cyanoethyl protecting group, which is used
generally in oligonucleotide synthesis, and a levulinyl group,
which was used successfully in the T(6-4)T case,8 at the
internucleoside phosphate and the 3′-hydroxyl function, respec-
tively. The diastereomers of 3, due to the chiral phosphorus
atom, could not be separated even by high-performance liquid
chromatography (HPLC).
Figure 1. (6-4) Photoproducts formed at TT and TC sequences.
dinucleoside monophosphates, the (6-4) photoproduct of
thymidylyl(3′-5′)deoxycytidine (T(6-4)C; Figure 1) was formed
about 3 times more efficiently than T(6-4)T.13
Consequently, the synthesis of oligonucleotides containing
T(6-4)C at a defined site has great significance. Such
oligonucleotides have been prepared by UV irradiation of
undamaged dodecamers containing a single TC site.9a,11c Since
the chain length and the sequence are limited in this method,
direct solid-phase oligonucleotide synthesis using a phosphor-
amidite building block appears to be much more efficient, as
demonstrated in the T(6-4)T case.8 However, due to the
different chemical structures of thymine and cytosine, there are
several problems to be solved. (1) The amino function of the
cytosine must be protected to prepare the dinucleoside mono-
phosphate, but this protecting group must be removed prior to
UV irradiation. (2) Although the T(6-4)T derivative was the
major product in our previous work,8 there may be side reactions
during UV irradiation in the T(6-4)C case. (3) The chemical
stability of T(6-4)C may be different from that of T(6-4)T.
(4) The amino group of T(6-4)C may undergo side reactions
during oligonucleotide assembly. In this article, we describe
an approach to the synthesis of oligonucleotides containing T(6-
4)C via a dimer building block, as well as our solutions to the
above problems.
In the last part of this article, the application of the T(6-
4)C-containing oligonucleotides to the characterization of the
(6-4) photolyase is described. The (6-4) photolyase was first
discovered in a cell extract of Drosophila melanogaster,6 and
subsequently was found in Xenopus laeVis and Arabidopsis
thaliana.14 This enzyme contains flavin adenine dinucleotide
(FAD) as a chromophore,14a and analyses of its reaction using
several enzymes strongly suggested that this enzyme repairs the
(6-4) photoproducts to their original pyrimidines by utilizing
blue to near-UV light.6,11b Recently we demonstrated that this
enzyme restored the pyrimidines of T(6-4)T to their normal
form.10 Here we present direct evidence that T(6-4)C is also
repaired to its intact components, thymine and cytosine.
In our previous study, the solubility of the protected thymid-
ylyl(3′-5′)thymidine (the counterpart of 3) in water was low,
and thus, acetonitrile was added to make a solution for UV
irradiation.8 In the T(6-4)C case, compound 3 could be
dissolved in water at least up to 4 mM. As shown in Figure
2A, irradiation of 3 on a commercially available UV cross-linker
equipped with six 15-W germicidal lamps gave two peaks with
an absorption maximum at 314 nm, which were eluted much
earlier than the starting material, in an HPLC analysis using a
reversed phase column. In addition, several peaks with an
absorption maximum at about 240 nm (peaks 3-5 in Figure
2A) were detected, which were not found in the T(6-4)T case
(Figure 2B; the two large peaks in the elution profile monitored
at 254 nm are the diastereomers of TpT). The UV absorption
spectra and the molecular weights determined by mass spec-
trometry of these products showed that they were photohydrates
of cytosine (diastereomers of the dinucleoside monophosphate
containing one of the two stereoisomers of 6-hydroxy-5,6-
dihydrocytosine).16 Probably due to the formation of these
photohydrates as byproducts, the formation of the T(6-4)C
photoproduct reached a plateau earlier than that of T(6-4)T,
as shown in Figure 3, although the quantum yield of T(6-4)C
at the dinucleoside monophosphate level was reported to be
higher.13 For preparative UV irradiation, a 2 mM aqueous
solution of 3 was used, and the two diastereomers of 4 (peaks
1 and 2 in Figure 2A) were separated by chromatography on
alkylated silica gel. The major isomer (peak 2) was purified
further by anion-exchange chromatography to remove an
impurity, which was close to this product in reversed phase
chromatography and was probably a byproduct without the
2-cyanoethyl group protecting the internucleoside linkage. Since
the minor isomer (peak 1) could not be purified satisfactorily,
this major isomer, which was purified to the level shown in
Figure 2C, was used to synthesize the phosphoramidite building
block.
Results and Discussion
Synthesis of the Building Block. A phosphoramidite
building block of the T(6-4)C photoproduct (7), which would
be prepared by a route similar to that for T(6-4)T,8 was
designed, and its synthesis was the first purpose of this study
(Scheme 1). Since the amino functions of nucleobases react
with a tetrazole-activated phosphoramidite,15 the amino function
1
The structure of the photoproduct 4 was confirmed by H
and 13C NMR spectroscopy including HMQC and HMBC
techniques. The stereochemistry of the pyrimidine-pyrimidone
structure was determined using ROESY spectra measured at a
mixing time of 300 ms. The two cross-peaks between the proton
signals of the base and sugar moieties in the 3′ component, H6-
H3′ and H6-H2′, indicated that the pyrimidone base had an
(13) (a) Lemaire, D. G. E.; Ruzsicska, B. P. Photochem. Photobiol. 1993,
57, 755-769. (b) Douki, T.; Zalizniak, T.; Cadet, J. Photochem. Photobiol.
1997, 66, 171-179.
(14) (a) Todo, T.; Kim, S.-T.; Hitomi, K.; Otoshi, E.; Inui, T.; Morioka,
H.; Kobayashi, H.; Ohtsuka, E.; Toh, H.; Ikenaga, M. Nucleic Acids Res.
1997, 25, 764-768. (b) Nakajima, S.; Sugiyama, M.; Iwai, S.; Hitomi, K.;
Otoshi, E.; Kim, S.-T.; Jiang, C.-Z.; Todo, T.; Britt, A. B.; Yamamoto, K.
Nucleic Acids Res. 1998, 26, 638-644.
(15) Wada, T.; Moriguchi, T.; Sekine, M. J. Am. Chem. Soc. 1994, 116,
9901-9911.
(16) Boorstein, R. J.; Hilbert, T. P.; Cunningham, R. P.; Teebor, G. W.
Biochemistry 1990, 29, 10455-10460.

10636 J. Am. Chem. Soc., Vol. 120, No. 41, 1998
Mizukoshi et al.
Scheme 1
anti to high-anti conformation. Taking this into account, the
cross-peaks between H6 and H3′ of the 5′ component and
between H6 of the 5′ component and H5 of the 3′ component
demonstrated that the configuration at the C6 position of the
pyrimidine was R. The configuration at the C5 position of the
pyrimidine was determined as R, by using the cross-peak
between H6 and CH3 of the 5′ component.
phosphoramidite 7 was obtained in good yield without the
byproduct formation. Tetrahydrofuran also gave a satisfactory
result in the phosphitylation reaction to prepare the T(6-4)T
phosphoramidite.
Synthesis of Oligonucleotides Containing T(6-4)C. The
phosphoramidite of T(6-4)C (7) was used as a building block
to synthesize oligonucleotides containing this photoproduct at
a specific site in defined sequences. This compound was
dissolved in anhydrous acetonitrile at a concentration slightly
higher than that of the other phosphoramidites, and the reaction
time for its coupling on the synthesizer was extended to 20 min
to ensure chain elongation. For adenine, guanine, and cytosine,
nucleoside phosphoramidite units in which the exocyclic amino
function was protected with the (4-tert-butylphenoxy)acetyl
(tBPA) group18 were used because the (6-4) photoproduct is
labile in hot alkali.19 Our analysis of the stability of (6-4)
photoproduct-containing oligonucleotides revealed that they
decomposed when heated at 55 °C in concentrated aqueous
ammonia but were quite stable at room temperature, i.e., under
conditions for the removal of the tBPA groups with simultaneous
deprotection of the phosphates and cleavage from the solid
support. As a capping reagent for termination of failed
sequences, (4-tert-butylphenoxy)acetic anhydride was used to
avoid the replacement of the tBPA group of guanines by the
acetyl group which is more stable in the ammonia treatment.18
As a first step, a 30-mer, d(GTATGATTAATGTAT(6-4)-
CATGTAATTAGTATG), was synthesized to examine whether
The isolation yield of 4 was low, and about 10 mmol of the
starting material 3 was required to obtain an amount sufficient
for the following steps. The 5′-hydroxyl function of 4 was
protected with the DMT group again, and the levulinyl group
at the 3′-position was removed with hydrazine monohydrate to
give 6. Finally, the 3′-hydroxyl function was phosphitylated
with (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite in
the presence of diisopropylethylamine.17 In our previous study
on T(6-4)T, pyridine was used as a solvent for this phos-
phitylation reaction, due to a solubility problem;8 however, the
formation of a byproduct, which had an Rf value lower than
that of the desired phosphoramidite, was observed frequently.
In the present study, pyridine was again the only solvent in
which compound 6 could be dissolved, and the formation of a
similar byproduct reduced the yield of 7 to a great extent.
Separation and analysis of this byproduct revealed that it
contained a phosphoramidate residue produced by oxidation of
the obtained 3′-phosphoramidite. Therefore, tetrahydrofuran
was tested although compound 6 was precipitated when it was
mixed with this solvent. As the reaction with the phosphit-
ylating reagent proceeded, the precipitate disappeared, and the
(18) Sinha, N. D.; Davis, P.; Usman, N.; Pe´rez, J.; Hodge, R.; Kremsky,
J.; Casale, R. Biochimie 1993, 75, 13-23.
(19) Franklin, W. A.; Lo, K. M.; Haseltine, W. A. J. Biol. Chem. 1982,
257, 13535-13543.
(17) Sinha, N. D.; Biernat, J.; McManus, J.; Ko¨ster, H. Nucleic Acids
Res. 1984, 12, 4539-4557.

Synthesis of Oligonucleotides by Photolyase
J. Am. Chem. Soc., Vol. 120, No. 41, 1998 10637
Figure 3. Formation of (6-4) photoproducts at varying UV doses at
254 nm. HPLC peak areas of the major diastereomers of protected T(6-
4)C (solid line) and T(6-4)T (broken line) were plotted after
normalization with each ꢀ value at the detector wavelength. Concentra-
tions of the initial dinucleoside monophosphates were 1 mM (crosses
and circles), 2 mM (triangles), and 4 mM (squares).
a significant impurity, that gave a large peak eluted at a high
acetonitrile concentration (peak 2 in Figure 4A). The relative
size of this peak was not reduced by heating the mixture at 55
°C in aqueous ammonia or by treating it with methanolic
ammonia. To identify this impurity, HPLC was used to purify
it, and nuclease P1 and alkaline phosphatase were used to
degrade it to its nucleoside components. HPLC analysis of the
degradation products revealed that the impurity in question
contained a highly hydrophobic substance (Figure 4B), which
had a UV absorption maximum at 316 nm and smaller
absorption peaks between 260 and 290 nm. These results
strongly suggested that this component was a T(6-4)C deriva-
tive modified with the tBPA group. Therefore, the partially
protected T(6-4)C derivative 4 was treated with (4-tert-
butylphenoxy)acetic anhydride in the presence of 1-methylimid-
azole, and the acylation sites were determined by NMR analysis
of the product. In the NOESY spectra, a cross-peak was found
between H5 of the 3′-pyrimidone and the methylene proton in
the tBPA group, which showed that the amino group of the
5′-pyrimidine, but not the N3 position, was acylated with the
capping reagents, as shown in Scheme 2. This fully protected
compound 8 was deprotected with ammonia, and it was
demonstrated that this modified T(6-4)C 9 had a UV absorption
spectrum identical to that of the unknown component in the
impurity and coeluted with it from an HPLC column (Figure
4C).
Since this side reaction was found at the capping step in
oligonucleotide synthesis, the synthesizer program was changed
to omit this step in each cycle after coupling of the T(6-4)C
unit. A short oligonucleotide, d(CAT(6-4)CAGCACGAC),
which was prepared previously by UV irradiation of the
undamaged 12-mer,9a was synthesized by this method. After
deprotection with aqueous ammonia, this crude mixture was
analyzed by reversed phase HPLC, which gave the elution
profile shown in Figure 5A. The highest peak had UV absorp-
tion maxima at 256 and 314 nm and coeluted with the authentic
12-mer containing T(6-4)C.9a Following the successful syn-
thesis of this 12-mer, one 14-mer, d(AAAAAAAAT(6-4)-
Figure 2. (A) HPLC analysis of a UV-irradiated solution of protected
TpdC, monitored at 225 nm (solid line) and 314 nm (broken line). The
total UV dose was about 40 J/cm2, and the gradient was 7 to 19%
acetonitrile in 0.1 M TEAA. Peaks 1 and 2, diastereoisomers of the
protected T(6-4)C; peaks 3, 4, and 5, cytosine hydrates; peak 6, the
starting material. (B) Formation of T(6-4)T. Elution from the HPLC
column was monitored at 254 nm (solid line) and 325 nm (broken line),
and the gradient was 11 to 19% acetonitrile in 0.1 M TEAA. (C) HPLC
analysis of the purified T(6-4)C (peak 2 in panel A), monitored at
254 nm (solid line) and 314 nm (broken line).
the T(6-4)C building block could be used successfully in the
same way as the T(6-4)T phosphoramidite. This synthesis was
carried out on a synthesizer using the reagents and the reaction
time described above, and after deprotection of the hydroxyl
function at the 5′ end with trichloroacetic acid on the synthesizer,
cleavage from the support and removal of the 2-cyanoethyl and
tBPA groups were performed by treatment with 28% ammonia
water at room temperature for 2 h. An aliquot of the deprotected
oligonucleotide was analyzed by reversed phase HPLC, as
shown in Figure 4A, which revealed that this sample contained

10638 J. Am. Chem. Soc., Vol. 120, No. 41, 1998
Mizukoshi et al.
Scheme 2
scale. An HPLC analysis of the deprotected mixture of
d(CCTGGTTAGTACAT(6-4)CGATTTAGGTGACACT) is
shown in Figure 5B, as an example. In addition to the highest
peak with the largest 314-nm absorption, which eluted at 17.0
min, several other peaks were detected. From the elution profile
monitored at 314 nm, the impurity eluted before the highest
peak is supposed to be a 28-mer lacking T(6-4)C, which was
produced due to the omission of the capping steps. The peaks
with longer retention times, which were also found in the
synthesis of T(6-4)T-containing oligonucleotides, may be
attributed to the coupling of phosphoramidites with the imino
function of the 5′-pyrimidine in the (6-4) photoproduct, as
discussed previously.8 Purification was performed under the
same HPLC conditions, and the yield of this 30-mer was 4.8
A260 units, which means an overall yield of ca. 9% from the
3′-terminal nucleoside on the solid support. The oligonucleo-
tides purified by reversed phase HPLC were analyzed further
by anion-exchange HPLC, which confirmed extremely high
purity (Figure 5C).
Figure 4. Detection and analysis of the byproduct produced in the
oligonucleotide synthesis. (A) A 30-mer, d(GTATGATTAATGTAT-
(6-4)CATGTAATTAGTATG), was synthesized on a synthesizer by
the usual method, and the deprotected mixture was analyzed by HPLC
using a 5-25% acetonitrile gradient in 0.1 M TEAA. Peaks 1 and 2
are the desired 30-mer and the byproduct discussed in the text,
respectively. (B) After purification by HPLC, the byproduct (peak 2
in panel A) was treated with nuclease P1 and alkaline phosphatase,
and the degraded products were analyzed by HPLC. The acetonitrile
gradient was 0-10% during the first 20 min and 10-50% during the
following 15 min, and the elution was monitored at 254 nm (broken
line) and 314 nm (solid line, magnified by a factor of 5). X represents
the unknown component discussed in the text. (C) Prior to injection,
the T(6-4)C bearing the tert-butylphenoxyacetyl group at the amino
function (compound 9) was added to the degradation products.
To confirm that the desired oligonucleotides were obtained,
the purified 30-mers were degraded with nuclease P1 and
alkaline phosphatase, and the components were separated by
HPLC. A typical result is shown in Figure 6. The peak detected
at 314 nm coeluted with the authentic T(6-4)C, prepared by
UV irradiation of deprotected TpdC, and had the same UV
absorption spectrum as T(6-4)C. The nucleoside composition
obtained from the peak areas was dA:dG:dC ) 9.3:5.9:12.8 (the
calculated ratio is 10:6:12) in this case.
CAAAA), and five 30-mers, d(GCACGACCAACGCAT(6-
4)CACGCAACCAGCACG), d(GTATGATTAATGTAT(6-
4)CATGTAATTAGTATG), d(AAAGTCAGCCTTGTGCT(6-
4)CGCCTACGGCCAT),
d(CCTGGTTAGTACAT(6-4)-
CGATTTAGGTGACACT), and d(CTCGTCAGCATCT(6-4)-
CCATCATACAGTCAGTG), were synthesized on a 0.2 µmol

Synthesis of Oligonucleotides by Photolyase
J. Am. Chem. Soc., Vol. 120, No. 41, 1998 10639
Figure 6. HPLC analysis of the components of the 30-mer, d(GCAC-
GACCAACGCAT(6-4)CACGCAACCAGCACG), produced by deg-
radation with nuclease P1 and alkaline phosphatase. The gradient was
0-10% acetonitrile in 0.1 M TEAA during 20 min, and the elution
was monitored at 260 nm (broken line) and 314 nm (solid line,
magnified by a factor of 5).
demonstrated that the T(6-4)T photoproduct is repaired to
normal thymines by Xenopus (6-4) photolyase, by direct HPLC
analysis of a photoreactivation product.10 An important problem
remaining to be solved is whether this enzyme repairs other
types of (6-4) photoproducts, especially the most abundant
T(6-4)C. On this subject, only a primer extension assay has
been reported previously.11c
For the direct analysis of the photoreactivation product with
this enzyme, an oligonucleotide in which the T(6-4)C photo-
product was incorporated into an oligo(deoxyadenylate),
d(AAAAAAAAT(6-4)CAAAA), was synthesized. The chain
length and the site of the photoproduct were chosen on the basis
of the results of a footprinting experiment reported previously,10
to give a minimal DNA for recognition by the (6-4) photolyase.
This 14-mer was treated with Xenopus (6-4) photolyase, which
was produced in Escherichia coli and purified as described
previously,14a and the reaction mixture was subjected to HPLC
analysis on a reversed phase column. As shown in Figure 7A,
two large peaks were detected. Peak 1 was determined to be
FAD contained by the enzyme, from its retention time and
absorption spectrum, and peak 2 appeared to be a photoreac-
tivation product because it had no absorption in the long
wavelength region, which is characteristic of the (6-4) photo-
product. To confirm this, the starting 14-mer containing T(6-
4)C was co-injected into the column, and it was shown that
this photoproduct-containing oligonucleotide was separated from
peak 2, as shown in Figure 7B. By the addition of the
undamaged 14-mer, d(AAAAAAAATCAAAA), to the photo-
reactivation mixture prior to injection, it was demonstrated that
this undamaged oligonucleotide coeluted with peak 2 (Figure
7C).
Figure 5. (A) HPLC analysis of a deprotected mixture of the 12-mer,
d(CAT(6-4)CAGCACGAC). The gradient was 5-15% acetonitrile in
0.1 M TEAA during 20 min. (B) HPLC analysis of a deprotected
mixture of the 30-mer, d(CCTGGTTAGTACAT(6-4)CGATTTAG-
GTGACACT). The gradient was the same as above, and the elution
was monitored at 254 nm (solid line) and 314 nm (broken line,
magnified by a factor of 20). (C) Anion-exchange HPLC analysis of
the purified 30-mer. A TSK-gel DEAE-2SW column was used with a
linear gradient of ammonium formate (0.4-1.2 M during 20 min) in
20% aqueous acetonitrile.
The photoreactivation product (peak 2 in Figure 7A) was
purified under the same HPLC conditions. This oligonucleotide
had a UV absorption spectrum identical to that of the undamaged
14-mer. To confirm this identity, the nucleoside components
of both oligonucleotides were analyzed by HPLC. As shown
in Figure 8, two peaks, corresponding to deoxycytidine and
thymidine, were detected in the sample obtained by degradation
of the photoreactivation product with nuclease P1 and alkaline
phosphatase. These two components had UV absorption spectra,
as well as retention times, identical to those of deoxycytidine
and thymidine, and the ratio calculated from the peak areas was
dC:T:dA ) 1.00:1.08:11.6 (The actual ratio for d(AAAAAAAAT-
CAAAA) is 1:1:12). These results clearly demonstrate that the
Photoreactivation of T(6-4)C with (6-4) Photolyase. As
the first application of the chemically synthesized oligonucleo-
tides containing the T(6-4)C photoproduct, we analyzed the
photoreactivation of this photoproduct with the (6-4) photo-
lyase.6 This enzyme contains FAD as a chromophore14a and
specifically repairs the (6-4) photoproduct in DNA using blue
to near-UV light. Enzyme activities from D. melanogaster,6,11b,c
X. laeVis,10,14a and A. thaliana14b have been characterized, and
several model studies toward the elucidation of its reaction
mechanism have been published.20 In our previous work, we
(20) (a) Prakash, G.; Falvey, D. E. J. Am. Chem. Soc. 1995, 117, 11375-
11376. (b) Heelis, P. F.; Liu, S. J. Am. Chem. Soc. 1997, 119, 2936-2937.

10640 J. Am. Chem. Soc., Vol. 120, No. 41, 1998
Mizukoshi et al.
Figure 8. HPLC separation of the nucleoside components of the
photoreactivation product from d(AAAAAAAAT(6-4)CAAAA) (a)
and those of the undamaged 14-mer, d(AAAAAAAATCAAAA) (b).
T(6-4)C to a great extent in the UV irradiation of partially
protected thymidylyl(3′-5′)deoxycytidine, whereas the quantum
yield of T(6-4)C is reported to be higher than that of T(6-
4)T.13 The other problem was the acylation of the amino
function in the T(6-4)C photoproduct with the capping reagents,
and thus, the capping steps were omitted to improve the yields
of the desired oligonucleotides. Despite these difficulties, we
believe that T(6-4)C-containing oligonucleotides are worth
synthesizing because this photoproduct is formed more ef-
ficiently than T(6-4)T in human cells and is more important
in terms of DNA repair.
One of the oligonucleotides synthesized in this study was
used to characterize a repair enzyme, (6-4) photolyase. Direct
HPLC analysis of the photoreactivation product demonstrated
that this enzyme restored the pyrimidines in the T(6-4)C
photoproduct to their original structures. This result also proves
that the oligonucleotides synthesized by this method contain
the naturally occurring T(6-4)C photoproduct.
Experimental Section
General Methods. All solvents and reagents were obtained from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan), except for N,
N-diisopropylethylamine and (2-cyanoethyl)-N,N-diisopropylchloro-
phosphoramidite, which were obtained from Aldrich Chemical Co., Inc.
(Milwaukee, WI) and Sigma Chemical Company (St. Louis, MO),
respectively. Nucleic acid synthesis grade solvents were used for
reactions. Reagents for the DNA/RNA synthesizer were purchased
from Perkin-Elmer Japan (Chiba, Japan) and PerSeptive Biosystems,
Inc. (Framingham, MA). TLC analyses were carried out on Merck
silica gel 60 F254 plates, which were visualized by UV illumination at
254 nm and spraying of an anisaldehyde/sulfuric acid solution, followed
by heating. Merck silica gel 60 F254 0.5 mm plates were used for
preparative TLC. For column chromatography, either Wakogel C-200,
C-300 (Wako Pure Chemical Industries), or Merck silica gel 60 was
used. Preparative C18 125 Å (Waters Corporation, Milford, MA) was
used on a Bio-rad Econo System for reversed phase chromatography.
UV and fluorescence spectra were recorded on a Beckman DU-64
spectrophotometer and a Hitachi F-4500 fluorescence spectrophotom-
eter, respectively. NMR spectra were measured on either a Bruker
DPX 300 or DMX 750 spectrometer. Proton signals were assigned
by using homonuclear 2D NMR techniques, COSY and NOESY,
measured on the DPX 300 spectrometer. For dinucleoside monophos-
phates, DQF-COSY, ROESY, HMQC, and HMBC spectra were used
for the signal assignments. Carbon assignments were carried out by
heteronuclear 2D NMR, HMQC, and HMBC. 31P NMR spectra were
measured on a Bruker DPX 300 spectrometer using trimethyl phosphate
Figure 7. HPLC analysis of the photoreactivation product. (A) The
14-mer, d(AAAAAAAAT(6-4)CAAAA), was mixed with Xenopus
(6-4) photolyase, and the mixture was illuminated with fluorescent
light for 4 h. An aliquot was analyzed by reversed phase HPLC, using
an Inertsil ODS-2 column with a linear gradient of acetonitrile (9-
12% during 20 min) in 0.1 M TEAA. (B) The T(6-4)C-containing
14-mer was added to the reaction mixture prior to injection. Peak 1,
FAD contained by the enzyme; peak 2, the photoreactivation product;
peak 3, the T(6-4)C-containing 14-mer. (C) The undamaged 14-mer,
d(AAAAAAAATCAAAA), was added to the reaction mixture.
T(6-4)C photoproduct is repaired to its original pyrimidine
components by the photoreactivation with Xenopus (6-4)
photolyase in the same way as T(6-4)T.
Conclusions
In this article, we described the synthesis of oligonucleotides
containing the (6-4) photoproduct formed between thymine and
cytosine. Although the procedure was basically the same as
that for oligonucleotides containing the thymine-thymine (6-
4) photoproduct,8 two different points were found in this study.
The formation of cytosine hydrates reduced the actual yield of

Synthesis of Oligonucleotides by Photolyase
J. Am. Chem. Soc., Vol. 120, No. 41, 1998 10641
as an internal standard. Mass spectra were obtained on a JEOL HX-
110 spectrometer.
60 min. This mixture was diluted with chloroform (250 mL) and was
washed with 1 M aqueous Na2S2O3 (180 mL). After being dried with
Na2SO4 and after the organic layer was concentrated, the product was
purified by silica gel column chromatography eluted with a step gradient
of 0-4% methanol in chloroform. The fully protected dinucleoside
monophosphate was obtained as a foam by evaporation. This com-
pound was treated with 3.5% trichloroacetic acid in dichloromethane
(270 mL) at room temperature for 25 h, and the mixture was neutralized
and extracted with 0.33 M aqueous NaHCO3 (150 mL) and with water
(80 mL). The combined aqueous layer was concentrated to 40 mL
and desalted on a column of alkylated silica gel (2.5 cm × 7 cm) with
a linear gradient of 9-25% acetonitrile in water. The product was
purified on a column of alkylated silica gel (1.5 cm × 48 cm) with a
linear gradient of 5-22.5% acetonitrile. The fractions were analyzed
by reversed phase HPLC, and those containing the desired product were
collected. Compound 3 was obtained as a white powder by lyophiliza-
tion (2.47 g, 65%). 1H NMR (750 MHz, pyridine-d5) δ 13.23 (1H, br
s, -NH-/T), 8.54-8.20 (2H, m, -NH2/C), 8.02 (1H, s, H6/T), 7.99 (1H,
d, J ) 7.4 Hz, H6/C), 6.93 (1H, dd, J ) 8.2, 6.0 Hz, H1′/T), 6.88-
6.78 (1H, m, H1′/C), 6.22-6.12 (1H, m, H5/C), 5.75-5.67 (1H, m,
H3′/T), 5.65-5.57 (1H, m, H3′/C), 4.75-4.64 (2H, m, H5′,5′′/C), 4.65-
4.57 (1H, m, H4′/T), 4.52, 3.08 (2H each, t, J ) 5.9 Hz, -CH2CH2-
CN), 4.52-4.44 (1H, m, H4′/C), 4.23-4.13 (2H, m, H5′,5′′/T), 2.94-
2.72 (2H, m, H2′,2′′/T), 2.77, 2.66 (2H each, t, J ) 5.8 Hz,
-OCOCH2CH2CO-), 2.73-2.44 (2H, m, H2′,2′′/C), 2.09, 2.08 (3H in
total (two diastereomers), s, -COCH3), 1.84, 1.83 (3H in total (two
diastereomers), s, -CH3/T). 31P NMR (121.5 MHz, pyridine-d5) δ
-4.43, -4.53. HRMS (FAB) calcd for C27H36N6O13P (M + H+)
683.2078, found 683.2087.
HPLC analyses were carried out on a Gilson gradient-type analytical
system equipped with a Waters 996 photodiode array detector.
A
µBondasphere 5µ C18 300 Å column (3.9 mm × 150 mm; Waters)
was used at a flow rate of 1.0 mL/min with a linear gradient of
acetonitrile in 0.1 M triethylammonium acetate (TEAA, pH 7.0). An
Inertsil ODS-2 column (4.6 × 250 mm; GL Sciences Inc., Tokyo,
Japan) was used in the case mentioned in the figure legend. For anion-
exchange HPLC, a TSK-gel DEAE-2SW column (4.6 mm × 250 mm;
Tosoh Corporation, Tokyo, Japan) was used at a flow rate of 1.0 mL/
min with a linear gradient of ammonium formate in 20% aqueous
acetonitrile.
4-N-(4,4′-Dimethoxytrityl)-3′-O-levulinyl-2′-deoxycytidine (2). To
2′-deoxycytidine hydrochloride (6.00 g, 22.8 mmol), which was dried
three times by coevaporation with pyridine and was suspended in
anhydrous pyridine (115 mL), were added 4,4′-dimethoxytrityl chloride
(19.3 g, 57.0 mmol), triethylamine (9.45 mL, 68.0 mmol), and
(dimethylamino)pyridine (117 mg, 95.8 µmol).21 After stirring at room
temperature for 40 min, the mixture was diluted with ethyl acetate (400
mL) and washed with saturated aqueous NaHCO3 (600 mL) and then
with water (400 mL). The organic layer was dried with Na2SO4 and
concentrated, and the residue was subjected to silica gel column
chromatography eluted with a step gradient of 0-3% methanol in
chloroform. The product was precipitated with hexane (2 L) from a
chloroform solution to give the 4-N,5′-O-ditrityl derivative as a white
powder (16.3 g, 19.6 mmol, 86%). This compound (16.3 g, 19.6 mmol)
was dissolved in 1,4-dioxane (160 mL) containing pyridine (4 mL)
and mixed with 4-(dimethylamino)pyridine (191 mg, 1.57 mmol) and
1,3-dicyclohexylcarbodiimide (10.1 g, 49.0 mmol). To this solution,
levulinic acid (4.01 mL, 39.2 mmol) was added. After stirring at room
temperature for 4 h, the mixture was diluted with chloroform (700 mL)
and washed with saturated aqueous NaHCO3 (300 mL), water (300
mL), and brine (300 mL). The organic layer was dried with Na2SO4
and concentrated, and the residue was subjected to silica gel column
chromatography eluted with a step gradient of 0-2% methanol in
chloroform. The product was precipitated with hexane from a
chloroform solution to give the 3′-levulinyl derivative as a white powder
(16.3 g, 17.5 mmol, 77% from 2′-deoxycytidine). This compound (16.1
g, 17.3 mmol) was treated with 1 M ZnBr2 in dichloromethane
containing 2-propanol (158 mL, 158 mmol) at room temperature for 6
h. The reaction mixture was diluted with chloroform (800 mL) and
washed with 1.2 M aqueous ammonium acetate (1000 mL), water (800
mL), and brine (800 mL). The organic layer was dried with Na2SO4
and concentrated, and the residue was subjected to silica gel column
chromatography eluted with a step gradient of 0-4% methanol in
chloroform. The product was precipitated with hexane from a
chloroform solution to give 2 as a white powder (10.4 g, 16.6 mmol,
73% from 2′-deoxycytidine). TLC (methanol-chloroform, 1:10) Rf
0.32. 1H NMR (300 MHz, chloroform-d) δ 7.37-6.78, (13H, m,
aromatic), 7.32 (1H, d, J ) 7.5 Hz, H6), 6.03 (1H, t, J ) 7.5, 6.0 Hz,
H1′), 5.34-5.28 (1H, m, H3′), 5.08 (1H, d, J ) 7.5 Hz, H5), 4.10-
4.04 (1H, m, H4′), 3.91-3.73 (2H, m, H5′,5′′), 3.80 (6H, s, -OCH3 ×
2), 3.18 (1H, br s, -OH), 2.75, 2.56 (2H each, t, J ) 6.0 Hz,
-OCOCH2CH2CO-), 2.58-2.37 (2H, m, H2′,2′′), 2.18 (3H, s, -COCH3).
HRMS (FAB) calcd for C35H38N3O8 (M + H+) 628.2659, found
628.2668.
Formation of the photoproduct 4. A 2 mM solution of compound
3 in water (150 mL) was irradiated for 2 h in an ice-cooled aluminum
tray (23 × 32 cm) on a Spectrolinker XL-1500 UV cross-linker
(Spectronics Corp., Westbury, NY) equipped with six 15-W germicidal
lamps (the total UV dose was ca. 40 J/cm2). After this irradiation was
repeated 36 times, using a total of 5.4 L of the solution, the mixture
was concentrated and applied to a column of alkylated silica gel (1.5
cm × 48 cm) equilibrated with 5% aqueous acetonitrile. Elution was
performed with a linear gradient of acetonitrile (5-17%), and the
fractions were analyzed by reversed phase HPLC using a µBondasphere
5µ C18 300 Å column with an acetonitrile gradient (7-19% during
20 min) in 0.1 M TEAA. The major diastereomer of the photoproduct
4 was separated, and the starting material 3 was recovered (4.79 g,
67%). The recovered starting material was used for reirradiation in
the same manner as described above, and the desired photoproduct was
combined with that from the first preparation. The product was passed
through a column of DEAE-Toyopearl 650 M (1.0 cm × 17 cm; Tosoh
Corporation), equilibrated and eluted with water, and was obtained as
a glassy solid by evaporation (280 mg, 3.9%). UV (H2O) λmax 314 nm
(ꢀ ) 7.8 × 103), λmin 265 nm. Fluorescence (H2O) λem 384 nm (at λex
313 nm). 1H NMR (750 MHz, D2O) δ 8.25 (1H, d, J ) 6.9 Hz, H6/
C), 6.75 (1H, d, J ) 6.9 Hz, H5/C), 6.40 (1H, dd, J ) 7.5, 0.7 Hz,
H1′/C), 6.05 (1H, d, J ) 8.7, 1.4 Hz, H1′/T), 5.42-5.38 (1H, m, H3′/
C), 4.93 (1H, s, H6/T), 4.31-4.24 (3H, m, H4′/C and -OCH2CH2CN),
4.22-4.18 and 4.15-4.11 (1H each, m, H5′,5′′/C), 4.02-3.97 (1H,
m, H3′/T), 3.90 (1H, dd, J ) 13.2, 2.1 Hz, H5′ or H5′′/T), 3.84-3.81
(1H, m, H4′/T), 3.73 (1H, dd, J ) 13.2, 3.3 Hz, H5′ or H5′′/T), 3.06
(1H, br dd, H2′ or H2′′/C), 2.88-2.84 (4H, m, -OCOCH2CH2CO- and
-OCH2CH2CN), 2.76 (1H, ddd, J ) 15.4, 9.1, 7.5 Hz, H2′ or H2′′/C),
2.57 (2H, t, J ) 6.1 Hz, -OCOCH2CH2CO-), 2.38 (1H, m, H2′ or H2′′/
T), 2.16 (s, 3H, -COCH3), 1.82 (br dd, 1H, H2′ or H2′′/T), 1.51 (3H,
s, -CH3/T). 31P NMR (121.5 MHz, D2O) δ -2.93. HRMS (FAB) calcd
for C27H36N6O13P (M + H+) 683.2078, found 683.2054.
P-(2-Cyanoethyl)thymidylyl-(3′f5′)-(2′-deoxycytidine) 3′-levul-
inate (3). Compound 2 (3.51 g, 5.60 mmol) and 5′-O-(4,4′-dimethoxy-
trityl)thymidine 3′-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (5.00
g, 6.72 mmol) were dissolved in anhydrous acetonitrile (37.0 mL), and
to this solution was added 0.86 M tetrazole in acetonitrile (31.3 mL,
26.9 mmol). After stirring at room temperature for 10 min, the mixture
was diluted with chloroform (300 mL) and washed with saturated
aqueous NaHCO3 (250 mL) and with brine (250 mL). The organic
layer was dried with Na2SO4 and evaporated in vacuo. To this residue
was added 0.12 M iodine in tetrahydrofuran-pyridine-water (7:2:1,
v/v/v; 150 mL), and the solution was stirred at room temperature for
5′-O-(4,4′-Dimethoxytrityl)-P-(2-cyanoethyl)-T(6-4)C 3′-levul-
inate (5). To a solution of compound 4 (168 mg, 246 µmol) in dry
pyridine (2.7 mL), was added 4,4′-dimethoxytrityl chloride (162 mg,
479 µmol). After the mixture stirred at room temperature for 3.5 h,
methanol (1.0 mL) was added, and the mixture was concentrated. The
residue was dissolved in chloroform (20 mL) and was washed with
water. The organic layer was dried with Na2SO4 and concentrated,
and the product was purified by preparative TLC, using a developing
solvent of chloroform-methanol (8:1, v/v). The tritylated derivative
(21) Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1982,
104, 1316-1319.

10642 J. Am. Chem. Soc., Vol. 120, No. 41, 1998
Mizukoshi et al.
5 was eluted from the silica gel with 4% methanol in chloroform and
was obtained as a glassy solid by evaporation (177 mg, 180 µmol,
73%). TLC (methanol-chloroform, 1:10) Rf 0.23. 1H NMR (300
MHz, chloroform-d) δ 8.49 (1H, s, -NH-), 7.88 (1H, d, J ) 6.9 Hz,
H6), 7.54-6.88 (13H, m, aromatic), 6.62 (1H, dd, J ) 7.5, 3.3 Hz,
H1′/C), 6.51 (1H, d, J ) 6.9 Hz, H5/C), 6.30 (1H, br dd, H1′/T), 5.52-
5.42 (1H, m, H3′/C), 4.73 (1H, s, H6/T), 4.70-4.55 (1H, m, H3′/T),
4.30-3.97 (5H, m, H5′,5′′/C, -OCH2CH2CN, and H4′/C), 3.80 (6H, s,
-OCH3 × 2), 3.74-3.65 (1H, m, H4′/T), 3.58 (1H, dd, J ) 11.1, 1.7
Hz, H5′ or H5′′/T), 3.23 (1H, dd, J ) 11.1, 3.9 Hz, H5′ or H5′′/T),
2.95-2.82, 2.68-2.56 (1H each, m, H2′,2′′/C), 2.76 (2H, t, J ) 5.9
Hz, -OCOCH2CH2CO-), 2.73-2.65 (2H, m, -OCH2CH2CN), 2.56 (2H,
t, J ) 5.9 Hz, -OCOCH2CH2CO-), 2.18 (3H, s, -CH2COCH3), 2.18-
2.01, 1.70-1.56 (1H each, m, H2′,2′′/T), 1.55 (3H, s, -CH3/T). 31P
NMR (121.5 MHz, chloroform-d) δ -4.67. HRMS (FAB) calcd for
C48H54N6O15P (M + H+) 985.3385, found 985.3412.
5′-O-(4,4′-Dimethoxytrityl)-P-(2-cyanoethyl)-T(6-4)C (6). The
5′- and 3′-protected T(6-4)C (5; 147 mg, 149 µmol) was dissolved in
pyridine (2.0 mL), and a solution of hydrazine monohydrate (69 µL,
1.42 µmol) in pyridine-acetic acid (3:2, v/v; 2 mL) was added. After
stirring at room temperature for 5 min, the mixture was cooled in an
ice bath and mixed with acetone (1.5 mL). This mixture was diluted
with chloroform (20 mL) and washed with 2% aqueous NaHCO3 (12
mL) and with water (12 mL). The organic layer was dried with Na2SO4
and concentrated. After coevaporation with toluene, the residue was
chromatographed on silica gel with a step gradient of 0-6% methanol
in chloroform, and the 3′-deprotected product 6 was obtained as a glassy
solid after evaporation (110 mg, 124 µmol, 83%). TLC (methanol-
chloroform, 1:10) Rf 0.14. 1H NMR (300 MHz, pyridine-d5) δ 12.54
(1H, s, -NH-), 8.29 (1H, d, J ) 7.0 Hz, H6/C), 7.85-7.05 (13H, m,
aromatic), 6.84 (1H, br dd, H1′/C), 6.76 (1H, dd, J ) 8.5, 2.0 Hz,
H1′/T), 6.72 (1H, d, J ) 7.0 Hz, H5/C), 5.15 (1H, s, H6/T), 5.17-
5.07 (1H, m, H3′/C), 4.96-4.84 (1H, m, H3′/T), 4.53-4.43, 4.41-
4.30 (1H each, m, H5′,5′′/C), 4.30-4.20 (2H, m, -OCH2CH2CN), 4.28-
4.15 (1H, m, H4′/C), 4.07-3.97 (1H, m, H4′/T), 3.91-3.82 (1H, m,
-OH), 3.75 (6H, s, -OCH3 × 2), 3.78-3.64 (1H, m, H5′ or H5′′/T),
3.59 (1H, dd, J ) 11.0, 4.6 Hz, H5′ or H5′′/T), 2.99-2.84, 2.80-2.66
(1H each, m, H2′,2′′/C), 2.88 (2H, t, J ) 6.1 Hz, -OCH2CH2CN), 2.36-
2.11 (2H, m, H2′,2′′/T), 1.78 (3H, s, -CH3/T). 31P NMR (121.5 MHz,
pyridine-d5) δ -4.58. HRMS (FAB) calcd for C43H48N6O13P (M +
H+) 887.3017, found 887.3007.
MHz, pyridine-d5) δ 12.64 (1Η, s, -NH-), 8.29 (1H, d, J ) 6.8 Hz,
H6/C), 7.92-7.07 (13H, m, aromatic), 6.92-6.69 (3H, m, H1′/C, H1′/
T, and H5/C), 5.18-5.02 (1H, m, H3′/C), 5.10 (1H, s, H6/T), 4.95-
4.85 (1H, m, H3′/T), 4.59-4.48 (1H, m, H5′ or H5′′/C), 4.48-4.17
(4H, m, H4′/C, H5′ or H5′′/C, and -CH2-), 4.11-3.45 (7H, m, H4′/T,
-CH2-, H5′,5′′/T, and -CH(CH3)2 × 2), 3.76 (6H, s, -OMe × 2), 3.12-
2.64 (6H, m, -CH2- × 2, and H2′,2′′/C), 2.37-2.06 (2H, m, H2′,2′′/T),
1.78 (3H, s, -CH3/T), 1.26-1.08 (12H, m, -CH(CH3)2 × 2). 31P NMR
(121.5 MHz, pyridine-d5) δ 145.82, 154.75, -4.54, -4.94. HRMS (FAB)
calcd for C52H65N8O14P2 (M + H+) 1087.4100, found 1087.4169.
Enzymatic Digestion of Oligonucleotides. An aliquot (0.04 A260
unit) of each oligonucleotide was incubated with nuclease P1 (4 µg)
in 30 mM ammonium acetate (pH 5.3, 20 µL) at 37 °C for 14 h. This
mixture was diluted with water (18 µL) and 0.5 M Tris-HCl (pH 9.0,
10 µL), and alkaline phosphatase (from calf intestine; 2 µL, 2 units)
was added. After an incubation at 37 °C for 2 h, ethanol (300 µL)
was added, and the mixture was kept at -20 °C for 2 h. The proteins
were pelleted by centrifugation, and the supernatant was concentrated
in vacuo. The residue was dissolved in water (100 µL), and an aliquot
(25 µL) was analyzed by HPLC using a µBondasphere 5µ C18 300 Å
column, as shown in Figures 4, 6, and 8.
Analysis of the Oligonucleotide Photoreactivated with (6-4)
Photolyase. Xenopus (6-4) photolyase was produced in E. coli and
was purified as described previously.14a This enzyme was illuminated
with fluorescent light in advance to obtain a reduced form of FAD. A
14-mer, d(AAAAAAAAT(6-4)CAAAA), (0.51 A260 units) was dis-
solved in a 1.6 µM solution of the (6-4) photolyase (1.0 mL) in 10
mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol, and this solution
was illuminated with fluorescent light. After 4 h, the reaction mixture
was heated at 75 °C for 15 min. The enzyme was pelleted by
centrifugation, and aliquots of the supernatant were analyzed by HPLC
using an Inertsil ODS-2 column, as shown in Figure 7. The product,
to which the T(6-4)C-containing 14-mer was converted quantitatively,
was purified under the same HPLC conditions (9-13% acetonitrile in
0.1 M TEAA during 20 min). This oligonucleotide and the undamaged
14-mer, d(AAAAAAAATCAAAA), were degraded to their nucleoside
components, as described above, and the results of the HPLC analysis
are shown in Figure 8.
Acknowledgment. We thank Ms. U. Tagami, Ms. R. Yuuji,
and Dr. K. Hirayama of Ajinomoto Co., Inc. and Drs. K.
Nakatani and I. Saito of Kyoto University for obtaining the high-
resolution mass data. We also thank Drs. H. Hiroaki and D.
Kohda for technical assistance with the 750 MHz NMR
measurements.
5′-O-(4,4′-Dimethoxytrityl)-P-(2-cyanoethyl)-T(6-4)C 3′-(2-Cy-
anoethyl)-N,N-diisopropylphosphoramidite (7). A solution of com-
pound 6 (111 mg, 125 µmol) in anhydrous tetrahydrofuran (1.8 mL)
was treated with (2-cyanoethyl)-N,N-diisopropylchlorophosphoramidite
(56 µL, 250 µmol) in the presence of N,N-diisopropylethylamine (87
µL, 500 µmol) at room temperature for 30 min. The reaction mixture
was diluted with chloroform (20 mL) and washed with 2% aqueous
NaHCO3 and with water. The organic layer was dried with Na2SO4
and evaporated in vacuo. The product was purified on a silica gel
column with a step gradient of 0-3% methanol in chloroform
containing 0.1% pyridine and precipitated with pentane from a
chloroform solution to give 7 as a white powder (81.5 mg, 74.0 µmol,
59%). TLC (methanol-chloroform, 1:10) Rf 0.48. 1H NMR (300
Supporting Information Available: 13C NMR data for 3
and 4, reaction of T(6-4)C with capping reagents, and a
procedure for oligonucleotide synthesis (3 pages print/PDF). See
any current masthead page for ordering information and Web
access instructions.
JA982004Y