Synthesis of a phosphoramidite coupling unit of the pyrimidine (6-4) pyrimidone photoproduct and its incorporation into oligodeoxynucleotides

Full text of DOI:10.1021/ja9603158

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7642
J. Am. Chem. Soc. 1996, 118, 7642-7643
Scheme 1a
Synthesis of a Phosphoramidite Coupling Unit of
the Pyrimidine (6-4) Pyrimidone Photoproduct and
Its Incorporation into Oligodeoxynucleotides
Shigenori Iwai,*,† Masato Shimizu,† Hiroyuki Kamiya,‡,§ and
Eiko Ohtsuka‡
Biomolecular Engineering Research Institute
6-2-3 Furuedai, Suita, Osaka 565, Japan
Faculty of Pharmaceutical Sciences
Hokkaido UniVersity, Kita-ku, Sapporo 060, Japan
ReceiVed January 30, 1996
Ultraviolet (UV) light causes lesions in DNA, which induce
mutations, cellular transformation, and cell death. At adjacent
pyrimidine sites, two major types of photoproducts, namely the
cis-syn cyclobutane pyrimidine dimer and the pyrimidine (6-
4) pyrimidone photoproduct, are formed.1 It appears that the
(6-4) photoproduct, which induces 3′ thymine-to-cytosine or
cytosine-to-thymine transitions,2 is much more mutagenic than
the cis-syn dimer.3 The (6-4) photoproduct is also important
in studies of DNA repair, because it has been suggested that
this lesion is removed in human cells via the nucleotide excision
repair pathway.4 For these studies, oligonucleotides containing
the damaged base at a single site are of great use, as
demonstrated previously.5 Smith and Taylor reported the
preparation of an oligonucleotide containing the (6-4) photo-
product at a single site, in which a hexamer was irradiated with
UV and then purified by HPLC.6 Although this hexamer was
elongated by ligation to a 49-mer, which was used for analyses
of protein binding7 and enzyme reactions,8 this procedure suffers
from limitations in chain length, sequence, and yield. One way
to solve this problem is to use a dinucleotide building block, as
developed for the cis-syn thymine dimer.9 In this communica-
tion, we describe the synthesis of a phosphoramidite coupling
unit of the (6-4) photoproduct of thymidylyl(3′-5′)thymidine
and its incorporation into 8-mer and 30-mer oligodeoxynucle-
otides.
The phosphoramidite coupling unit of the (6-4) photoproduct
(6) was designed in order that it would be used generally on
DNA synthesizers. The 4,4′-dimethoxytrityl and 2-cyanoethyl
groups were used for the protection of the 5′-hydroxyl group
and the internucleoside phosphate, respectively. At the begin-
ning, protection of the hydroxyl group generated by photoprod-
uct formation was planned, but it was found that the reactivity
of this hydroxyl was extremely low, as described below. A
levulinyl group, which was used successfully in the preparation
of the coupling unit of the cis-syn thymine dimer,10 was chosen
for the transient protection of the 3′-hydroxyl group.
a Reagents and yields: (a) UV (254 nm), 16%; (b) 4,4′-dimethoxy-
trityl chloride, pyridine, 84%; (c) (CH3CO)2O, DMAP, 49%; (d)
NH2NH2‚H2O, pyridine-AcOH, 85%; (e) NCCH2CH2OP(Cl)N(iPr)2,
EtN(iPr)2, 85%. Abbreviations: DMT, 4,4′-dimethoxytrityl; Lev,
levulinyl.
The 3′-levulinyl thymidylyl(3′-5′)thymidine 2-cyanoethyl
phosphotriester (1) was prepared as described previously.10 The
two diastereomers, due to the chiral phosphorus, could not be
separated on silica gel. Irradiation of a 1 mM solution of 1 in
20% aqueous acetonitrile, on a UV-cross-linker equipped with
six 15 W germicidal lamps, resulted in the production of two
peaks with retention times shorter than that of the starting
material, as well as several other peaks, as analyzed by reversed-
phase HPLC. These two products each had a UV absorption
spectrum with a maximum at 326 nm, which was exactly the
same as that reported for the unprotected (6-4) photoproduct
of thymidylyl(3′-5′)thymidine.11 The amounts of these peaks
reached a plateau at a UV dose of 30 J/cm2, while the starting
materials were still decreasing. The formation of the photo-
product was performed on a preparative scale, and the products
were purified by reversed-phase chromatography on alkylated
silica gel. The diastereomers mentioned above were separated
at this step and obtained in a ratio of 1:2. The product showed
a maximum emission at 397 nm, at an excitation of 313 nm, in
its fluorescence spectrum, as reported previously for the
unprotected photoproduct.11 The pyrimidine-pyrimidone struc-
ture, including the stereochemistry, was confirmed by NMR
spectroscopy, as described in the supporting information. The
yield of 2 was not high (16%, in the total of both isomers), but
a practical amount was obtained.
* Author to whom correspondence should be addressed. Phone: +81-
6-872-8208. Fax: +81-6-872-8219. E-mail: iwai@bioorg.beri.co.jp.
† Biomolecular Engineering Research Institute.
‡ Hokkaido University.
§ Present address: Institute of Industrial Ecological Sciences, University
of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku,
Kitakyushu 807, Japan.
(1) For review, see: Friedberg, E. C.; Walker, G. C.; Siede, W. DNA
repair and mutagenesis; ASM Press: Washington, DC, 1995.
(2) (a) LeClerc, J. E.; Borden, A.; Lawrence, C. W. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 9685-9689. (b) Horsfall, M. J.; Lawrence, C. W. J.
Mol. Biol. 1994, 235, 465-471.
The following procedure is shown in Scheme 1. The 5′-
hydroxyl group of 2 was protected with the 4,4′-dimethoxytrityl
group, and then we tried protection of the hydroxyl group at
the base moiety. An acetyl group was chosen to avoid steric
hindrance, and the protected dimer (3) was treated with an
excess amount of acetic anhydride in the presence of 4-(di-
methylamino)pyridine. However, the reaction proceeded very
(3) Naegeli, H. FASEB J. 1995, 9, 1043-1050.
(4) Jones, C. J.; Wood, R. D. Biochemistry 1993, 32, 12096-12104.
(5) Szymkowski, D. E.; Lawrence, C. W.; Wood, R. D. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 9823-9827.
(6) Smith, C. A.; Taylor, J.-S. J. Biol. Chem. 1993, 268, 11143-11151.
(7) 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.
(8) Kim, S.-T.; Malhotra, K.; Smith, C. A.; Taylor, J.-S.; Sancar, A. J.
Biol. Chem. 1994, 269, 8535-8540.
(10) Murata, T.; Iwai, S.; Ohtsuka, E. Nucleic Acids Res. 1990, 18, 7279-
7286.
(11) Franklin, W. A.; Lo, K. M.; Haseltine, W. A. J. Biol. Chem. 1982,
257, 13535-13543.
(9) Taylor, J.-S.; Brockie, I. R.; O’Day, C. L. J. Am. Chem. Soc. 1987,
109, 6735-6742.
S0002-7863(96)00315-0 CCC: $12 00 © 1996 American Chemical Society

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Communications to the Editor
J. Am. Chem. Soc., Vol. 118, No. 32, 1996 7643
Figure 1. HPLC analysis of crude d(GTAT(6-4)TATG). Lines A,
B, and C indicate the retention times of d(GTAT(6-4)TATG) prepared
by UV irradiation of d(GTATTATG), d(GTAT(Dewar)TATG), and
d(GTATTATG), respectively. A µBondasphere C18 300 Å column
(3.9 mm × 150 mm) was used at a flow rate of 1.0 mL/min with a
linear gradient of 7%-11% acetonitrile for 20 min in 0.1 M triethyl-
ammonium acetate (pH 7.0).
Figure 2. HPLC analysis of the crude (6-4) 30-mer, monitored at
254 and 325 nm. The elution conditions are described in the legend
to Figure 1, except that the acetonitrile gradient was from 7% to 13%
and the column temperature was 50 °C.
4) octamer was 6.8 A260 units (0.10 µmol, 50% from the 3′-
terminal dG).
In order to demonstrate that long oligonucleotides can be
obtained by this method without sequence limitations, a 30-
mer, d(CTCGTCAGCATCT(6-4)TCATCATACAGTCAGTG),
was synthesized. In the HPLC analysis of the deprotected
mixture, a main peak, which had absorption maxima at 259 and
327 nm, was detected, together with several impurities (Figure
2). The impurities with retention times longer than that of the
main peak had a smaller A325/A254 ratio, and prolonged reaction
with tetrazole-activated phosphoramidites resulted in production
of many peaks and increase in their relative amount. These
results suggested that these impurities were branching byprod-
ucts caused by coupling with the (6-4) photoproduct. The yield
after HPLC purification was 6.0 A260 units, and this product
was coeluted with d(CTCGTCAGCATCTTCATCATACAGT-
CAGTG) by anion-exchange HPLC.
As described above, we have developed a direct method for
the preparation of oligonucleotides containing the (6-4) pho-
toproduct. Oligonucleotides prepared with the coupling unit
of the cis-syn thymine dimer have been used in studies of the
molecular biology of DNA repair,14 and we have applied this
method to the elucidation of the substrate recognition and the
catalytic mechanism of bacteriophage T4 endonuclease V.15
Similarly, the coupling unit of the (6-4) photoproduct synthe-
sized in this study will contribute significantly to the molecular
and cellular biology of DNA repair.
slowly, and only the N-acetylated product was obtained. Since
the N3 position of thymidine is not protected in oligonucleotide
synthesis, the base moiety was left unprotected. Finally, the
levulinyl group was removed from 3, and the resultant 3′-
hydroxyl group was phosphitylated.
Using the fully protected dimer, the removal of the protecting
groups was tested. Treatment of 4 with 80% acetic acid and
then with 28% aqueous ammonia at room temperature for 2 h
gave a compound which was coeluted from the HPLC column
with the authentic (6-4) photoproduct prepared by UV irradia-
tion of unprotected thymidylyl(3′-5′)thymidine. The stability
of the (6-4) photoproduct was also tested using this dimer. It
was found that the (6-4) photoproduct was stable in 80% acetic
acid and in 28% aqueous ammonia at room temperature but
was decomposed when heated above 40 °C in aqueous ammonia.
The octamer, d(GTAT(6-4)TATG), which was previously
prepared by UV irradiation of d(GTATTATG),12 was synthe-
sized on a 0.2 µmol scale first. Due to the instability of the
(6-4) photoproduct under alkaline conditions, nucleoside 3′-
phosphoramidites with the (4-tert-butylphenoxy)acetyl group for
the protection of the exocyclic amino groups of dA, dG, and
dC, which can be deprotected with ammonia at room temper-
ature,13 were used in combination with 6. The reaction time
for the coupling of 6 was prolonged to 20 min, and the coupling
yield of 6 was 97%. After the chain assembly on a synthesizer,
the solid support containing the oligonucleotide was treated with
aqueous ammonia at room temperature for 2 h, and the product
was analyzed by HPLC (Figure 1). The parent sequence,
d(GTATTATG), the (6-4) octamer prepared by UV irradiation
of d(GTATTATG), and the Dewar octamer, d(GTAT(Dewar)-
TATG), prepared by irradiation of the (6-4) octamer, were
eluted under the same conditions. The retention time and the
UV absorption spectrum, with maxima at 256 and 327 nm, of
the main peak was exactly the same as those of the authentic
(6-4) octamer prepared by the irradiation, and no contamination
by d(GTATTATG) or d(GTAT(Dewar)TATG) was found in
the mixture. After purification by HPLC, the yield of the (6-
Supporting Information Available: Synthetic procedures and 1H-
NMR, 31P-NMR, and HRMS data for all new compounds; determination
of configuration at the (6-4) photoproduct (9 pages). See any current
masthead page for ordering and Internet access instructions.
JA9603158
(14) (a) Svoboda, D. L.; Taylor, J.-S.; Hearst, J. E.; Sancar, A. J. Biol.
Chem. 1993, 268, 1931-1936. (b) Svoboda, D. L.; Smith, C. A.; Taylor,
J.-S. A.; Sancar, A. J. Biol. Chem. 1993, 268, 10694-10700. (c) Donahue,
B. A.; Yin, S.; Taylor, J.-S.; Reines, D.; Hanawalt, P. C. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 8502-8506.
(15) (a) Iwai, S.; Maeda, M.; Shimada, Y.; Hori, N.; Murata, T.; Morioka,
H.; Ohtsuka, E. Biochemistry 1994, 33, 5581-5588. (b) Iwai, S.; Maeda,
M.; Shirai, M.; Shimada, Y.; Osafune, T.; Murata, T.; Ohtsuka, E.
Biochemistry 1995, 34, 4601-4609. (c) Vassylyev, D. G.; Kashiwagi, T.;
Mikami, Y.; Ariyoshi, M.; Iwai, S.; Ohtsuka, E.; Morikawa, K. Cell. 1995,
83, 773-782.
(12) Zao, X.; Taylor, J.-S. J. Am. Chem. Soc. 1994, 116, 8870-8876.
(13) Sinha, N. D.; Davis, P.; Usman, N.; Pe´rez, J.; Hodge, R.; Kremsky,
J.; Casale, R. Biochimie 1993, 75, 13-23.