Efficient chemical synthesis of a pyrimidine (6-4) pyrimidone photoproduct analog and its properties

Article abstract of DOI:10.1016/S0040-4039(00)00127-1

We synthesized an analog of a pyrimidine (6-4) pyrimidone photoproduct with a formacetal linkage instead of the phosphodiester in the (6-4) photoproduct, and investigated the interaction between a monoclonal antibody specific to the natural (6-4) photopro

Full text of DOI:10.1016/S0040-4039(00)00127-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2175–2179
Efficient chemical synthesis of a pyrimidine (6–4) pyrimidone
photoproduct analog and its properties
Kousuke Satou,a Yasuo Komatsu,a,∗ Takuya Torizawa,b Koichi Kato,b Ichio Shimada,b
Osamu Nikaido c and Eiko Ohtsuka d
aGraduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
bGraduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
cKanazawa University, Kanazawa 920-0934, Japan
dHealth Sciences University of Hokkaido, Toubetsu 061-0293, Japan
Received 10 December 1999; revised 13 January 2000; accepted 14 January 2000
Abstract
We synthesized an analog of a pyrimidine (6–4) pyrimidone photoproduct with a formacetal linkage instead
of the phosphodiester in the (6–4) photoproduct, and investigated the interaction between a monoclonal antibody
specific to the natural (6–4) photoproduct and a DNA containing the photoproduct analog. © 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: nucleic acids; nucleic acid analogs; photochemistry; antigens; antibodies.
Ultraviolet-light (UV) causes damage in DNA and generates photoproducts, predominantly in tandem
pyrimidine sites such as cyclobutane pyrimidine dimers, pyrimidine (6–4) pyrimidone, and Dewar
photoproducts. The (6–4) photoproducts are formed by UV-light 3–5-fold less frequently than the
cis–syn cyclobutane type of pyrimidine dimer; however, they cause mutations more frequently than the
cyclobutane types.1,2 Enzymes that repair the (6–4) photoproduct have been reported.3 To investigate
the catalytic mechanism of the enzymes, DNA that contains the (6–4) photoproduct at a specific site is
required as a probe. Although the chemical synthesis of the photoproduct dimer unit has been reported,4
the yield of the photoreaction is not satisfactory. A large amount of the photoproduct is required for
physicochemical analyses. Recently, a cyclobutane-type thymine dimer in which the phosphodiester
was modified was reported;5 however, the efficient synthesis of the (6–4) photoproduct has yet to be
developed. We now have synthesized an analog of the (6–4) photoproduct and obtained it in a satisfactory
yield.
∗
Corresponding author. E-mail: komatsu@pharm.hokudai.ac.jp (Y. Komatsu)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00127-1
tetl 16406

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1. Results and discussion
We synthesized a thymine dimer (compound 4) with a formacetal linkage using a standard procedure6,7
except a tert-butyldimethylsilyl group was used for the 30-hydroxyl group protection (Scheme 1). First,
we compared the reactivity of compound 4 with that of 6 by HPLC8 (Fig. 1). Compound 4 was converted
to the (6–4) photoproduct 5 faster than the normal thymine dimer 6 as shown in Fig. 1b. The generation
of T[6c4]T 5 became maximal by irradiation with 40 kJ/m2, whereas T[6p4]T 7 with a phosphodiester
required 100 kJ/m2 irradiation for the maximum yield. Compound 4 with a formacetal linkage was found
to be converted to T[6c4]T efficiently. The (6–4) photoproducts decomposed to unknown products by
further UV irradiation.
Scheme 1. (i) CH3SCH2Cl, NaH, THF; (ii) TBDMSCl, imidazole, THF; (iii) 80% CH3COOH/H2O; (iv) NBS, 2,6-lutidine,
molecular sieves 4 Å, CH2Cl2; (v) 80% CH3COOH/H2O
We scaled up the reaction,9 and the yield of the photoreaction of T[6c4]T 510 was 30% and about
four-fold higher than that of T[6p4]T 7. T[6p4]T is a diastereomeric mixture at the phosphotriester
configuration; however, 5 was obtained as a unique product due to its lack of chirality.
We converted 4 to the thymine dimer amidite unit using the same preparation route as the T[6c4]T
amidite 10 shown in Scheme 2, and incorporated TcT in the tetra-oligonucleotide, d(ATcTA). The photo-
reactivity of TcT in the oligonucleotide was also compared with that in a normal tetra-oligonucleotide,
d(ATpTA). The analog d(AT[6c4]TA) was obtained more efficiently than the natural oligonucleotide (data
not shown). This result is consistent with that obtained in the dimer reaction (T[6c4]T), and indicates
that the thymine dimer with a formacetal linkage is also useful in post-synthetic photoreactions. It is
thought that the high reactivity of TcT is derived from the two proximal thymine bases connected by the
formacetal linkage.
After T[6c4]T 5 was converted to the amidite unit 10 (Scheme 2), 10 was inserted in the center of a tetra
(AT[6c4]TA-biotin) or an octa (CAAT[6c4]TAAG-biotin) oligonucleotide,11 using a 30-biotin support for
immobilization on a streptavidin surface.
Analysis of monoclonal antibody binding: The monoclonal antibody, 64M5, specifically binds to
oligonucleotides with the (6–4) photoproduct,12–14 and thus we investigated whether 64M5 would
recognize the (6–4) photoproduct analog with a formacetal linkage using surface plasmon resonance.
64M5 bound with oligonucleotides containing a T[6c4]T photoproduct, although the binding affinities
of 64M5 to those antigens were about 10-fold lower than those to the cognate T[6p4]T antigens (Table
1). These low affinities may be due to the formacetal linkage of the T[6c4]T photoproduct. Since there
might be some electrostatic contacts between the phosphodiester of the natural T[6p4]T and the basic
amino acid residues in the antibody, the binding affinity of 64M5 for the T[6c4]T antigens might be
decreased. However, the binding magnitude of 64M5 to the T[6c4]T antigens was significant, because the
dissociation constant is in the order of 10−8 M. This result indicates that the phosphodiester of T[6p4]T is

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Fig. 1. (a) Photoreactions to give both T[6p4]T (compound 7) and T[6c4]T (compound 5) photoproducts. (b) Plots of the
concentration of the (6–4) photoproduct versus UV dose. T[6p4]T (ꢀ) and T[6c4]T (ꢁ). Analysis of photoreactions from
compound 4 to 5 (c) and from compound 6 to 7 (d) by HPLC using a reverse phase column after the irradiation of UV-light (80
kJ/m2)
important for the binding, but is not absolutely required for the recognition by the monoclonal antibody.
The dissociation rate constants (kdiss), rather than the association rate constants (kass), of T[6c4]T were
decreased as compared with those of T[6p4]T. If there are some conformational changes in the 64M5-
T[6p4]T complex, such as an induced fit, then the structural change might be difficult to accommodate
in the 64M5–T[6c4]T complex. Then, the antibody would easily dissociate from the complex.
Various organisms have repair enzymes that recognize UV-damaged DNA, such as the (6–4) photo-
product. For physicochemical analyses of enzyme-photoproduct interactions, large amounts of the
photoproducts are required. The thymine dimer connected by a formacetal linkage increased the yield

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Scheme 2. (i) DMTrCl, pyridine; (ii) TBAF, THF; (iii) i-Pr2NP(OCE)Cl, i-Pr2NEt, CH2Cl2
Table 1
Kinetic and thermodynamic parameters for antigen–antibody complexes
of the (6–4) photoreaction, and simplified the preparation of the photoproduct. Furthermore, T[6c4]T
may be useful as a probe for analyzing the electrostatic contacts within repair enzymes.
Acknowledgements
We thank Drs. Hiroyuki Kobayashi and Jon D. Stewart for helpful discussions. This work was
supported by a Grant-in-Aid by the Monbusho International Scientific Research funding from the
Ministry of Education, Science, Culture and Sports of Japan.
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8. Compounds 4 and 6 were each dissolved in a 20% aqueous acetonitrile solution. The 1 mM solutions containing 4 or 6 were
irradiated with UV-light (254 nm). After aliquots were taken from the solutions at each UV dose, the generation of the (6–4)
photoproducts was analyzed by HPLC using a reverse phase column (µ-Bondasphere, C-18, 3.9 mm i.d.×150 mm, Waters),
and the gradient was 10–100% B buffer content over 20 min (A buffer, 5% aqueous acetonitrile, 0.1 M triethylammonium
acetate (TEAA); B buffer, 60% aqueous acetonitrile, 0.1 M TEAA). The elution was monitored by a photodiode array

2179
detector (Waters TM 996). Since both 5 and 7 absorb UV-light at 325 nm efficiently, the concentrations of those products
were calculated from the peak areas at 325 nm.
9. After compound 4 (274 mg, 0.45 mmol) was dissolved in 20% aqueous acetonitrile (450 ml), the 1 mM solution of 4 (50
ml×9) was irradiated with UV-light (254 nm, 50 kJ/m2). Compound 5 (82 mg, 0.13 mmol) was isolated by silica gel column
chromatography (Wakogel, C-300).
1
10. Chemical shifts and coupling constants of 5 were as follows; H NMR (CDCl3) δ (ppm); 7.19 (s, 1H, cT H6), 6.63 (d,
1H, cT H10, J=6.6 Hz), 6.20 (d, 1H, Tc H10, J=8.5 Hz), 5.03 (s, 1H, Tc H6), 4.82–4.79 (m, 1H, cT H30), 4.68 (AB, 2H
-OCH2O-), 4.24 (m, 1H, cT H40), 4.01 (m, 2H, cT H50), 3.96 (m, 2H, Tc H50), 3.83 (dd, 1H, Tc H30, J=4.6, 12.9 Hz),
3.72 (m, 1H, Tc H40), 2.83 (m, 1H, cT H20), 2.56 (m, 1H, cT H200), 2.27 (s, 3H, cT -CH3), 2.01 (m, 1H, Tc H20), 1.62 (s,
3H, Tc -CH3), 1.57 (m, 1H, Tc H200), 0.87 (s, 9H, TBS t-Bu), 0.05 (s, 6H, TBS -CH3). Tc and cT indicate 50- and 30-side
thymidines, respectively. The base structure was confirmed by ROESY (CDCl3; Tc H30 and cT -CH3, Tc H6 and cT -CH3,
cT H6 and cT -CH3, cT H6 and cT H20, cT H6 and -CH2-). FAB-HRMS calcd for C27H43N4O10Si [M+H]+ 611.2745, found
611.2741.
11. Oligonucleotides containing T[6c4]T were synthesized using a 30 biotinon CPG support (CLONTECH) identical to the
natural (6–4) photoproduct,4 and were purified on a reverse phase column (µ-Bondasphere, 3.9 mm i.d.×150 mm, Waters).
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B buffer content, respectively, over 20 min (A buffer, 5% aqueous acetonitrile, 0.1 M TEAA; B buffer, 25% aqueous
acetonitrile, 0.1 M TEAA). The overall yields of T[6c4]T-4 and T[6c4]T-8 were 31% and 14%, respectively.
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14. Torizawa, T.; Kato, K.; Kato, J.; Kobayashi, H.; Komatsu, Y.; Morioka, H.; Nikaido, O.; Ohtsuka, E.; Shimada, I. J. Mol.
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