Isolation and characterization of a novel product, 2′-deoxyoxanosine, from 2′-deoxyguanosine, oligodeoxynucleotide, and calf thymus DNA treated by nitrous acid and nitric oxide

Full text of DOI:10.1021/ja952550g

J. Am. Chem. Soc. 1996, 118, 2515-2516
2515
Isolation and Characterization of a Novel Product,
′-Deoxyoxanosine, from 2′-Deoxyguanosine,
2
Oligodeoxynucleotide, and Calf Thymus DNA
Treated by Nitrous Acid and Nitric Oxide
†
‡
§
Toshinori Suzuki, Ryohei Yamaoka, Masatoshi Nishi,
†
,†
Hiroshi Ide, and Keisuke Makino*
Department of Polymer Science and Engineering and
Department of Applied Biology
Kyoto Institute of Technology
Matsugasaki, Sakyo-ku, Kyoto 606, Japan
Faculty of Pharmaceutical Sciences, Setsunan UniVersity
Nagaotouge-cho, Hirakata, Osaka 573-01, Japan
ReceiVed July 31, 1995
Figure 1. RPHPLC chromatogram obtained for HNO
detected at 260 nm.
2
-treated dGuo
The inactivation and mutagenic effects of HNO2 on nucleic
acids were first demonstrated around 1960.1 The principal
reaction products were identified as xanthine (Xan), hypoxan-
thine, and uracil.2 Shortly thereafter, it was shown that guanine
moieties (Gua) of tobacco mosaic virus RNA were not
exclusively converted to Xan, but approximately half of the Gua
mentation pattern, NMR spectrum, and substantial similarity
of the UV spectrum to that of 2-nitro-Ino.
4
The product (referred to as compound 1) eluting in the fourth
peak was isolated by preparative RPHPLC and subjected to
structural assignment. Elementary analysis revealed that com-
pound 1 has a chemical composition of C10H12N4O5, identical
to that of dXao. High-resolution mass measurement (HR-EI)
for the base fragment of compound 1 indicated that m/z )
3
was consumed in an unknown reaction. In subsequent efforts
to characterize unidentified products, 2-nitroinosine (2-nitro-
4
4
5
Ino) (maximum yield, 8.5%) and cross-linked compounds
(
6
maximum yield, 2.4%) were isolated and identified. However,
1
52.032 49, which agrees with the theoretical molecular mass
the yields of these two compounds did not fully account for
the remaining half of consumed Gua. Evidently some major
product(s) remain to be identified in the HNO2-Gua systems.
In the present study, we have reexamined such systems and
report isolation and characterization of a new major product
formed from HNO2-treated 2′-deoxyguanosine (dGuo), oligode-
oxynucleotide (dTGTT), and calf thymus DNA. Furthermore,
we show that this compound is also produced in the reaction of
dGuo with NO.
8
for the composition C5H4N4O2 within 1 mmu. These data
indicate that compound 1 and dXao are structural isomers in
the base unit. In IR spectrum, however, a major band at ca.
-
1
1
700 cm , which is attributable to the stretching vibration of
an amide carbonyl group and commonly observed for dXao,
8
13
Xao, and dGuo, disappeared for compound 1. The C NMR
8
spectrum of compound 1 contained 10 resonances. Five among
the 10 resonances existed in the aromatic region (Figure 2).
1
The H NMR (in DMSO-d6) showed an exchangeable singlet
When 10 mM dGuo was incubated in the presence of NaNO2
100 mM) in acetate buffer (3.0 M, pH 3.7) for 2 h at 37 °C,
(
7.90 ppm) attributable to two protons in addition to a set of
(
8
peaks of 2′-deoxyribose and an aromatic proton (Figure 2). A
correlation between this singlet (7.90 ppm) and the N signal
(
HMQC measurement. Thus the singlet at 7.90 ppm is assigned
as a primary amino group. The H and N chemical shifts of
five major peaks appeared in the reversed-phase (RP) HPLC
chromatogram (Figure 1). The third peak is unreacted dGuo.
15
1
5
9
1
15
93.3 ppm relative to NH4 NO3) was observed in the H- N
2
′-Deoxyxanthosine (dXao), which is a major product of this
reaction, eluted in the second peak: NMR and UV data for the
isolated compound were consistent with those reported previ-
ously.7 Xanthine produced by depurination of dXao was
assigned on the basis of the agreement with the RPHPLC
retention time and UV spectrum of the authentic sample. A
yellowish side product eluting in the fifth peak was identified
as 2-nitro-2′-deoxyinosine (2-nitro-dIno) by the EI mass frag-
1
15
the amino group in compound 1 are fairly downfield from those
1
of the primary amino group in dGuo (for dGuo: H, 6.43 ppm;
1
5
N, 82.7 ppm). These downfield shifts suggest that compound
has a ring including an oxygen atom which is located near
1
the amino group. These data imply that compound 1 is 5-amino-
3
-â-(2-deoxy-D-ribofuranosyl)-3H-imidazo[4,5-d][1,3]oxazin-7-
†
one (2′-deoxyoxanosine). Available spectroscopic data includ-
Department of Polymer Science and Engineering, Kyoto Institute of
Technology.
‡
1
Department of Applied Biology, Kyoto Institute of Technology.
(8) Compound 1. H NMR (500 MHz, D2O at 30 °C): δ (ppm/TSP-d4)
§
Faculty of Pharmaceutical Sciences, Setsunan University.
7.98 (s, 1H, H-2), 6.26 (dd, J1′2′ ) 6.9, J1′2′′ ) 6.6, 1H, H-1′), 4.61 (ddd,
J3′4′ ) 4.1, 1H, H-3′), 4.12 (ddd, J4′5′ or J4′5′′ ) 3.7 or 4.9, 1H, H-4′), 3.79
(ABX, J5′5′′ ) 12.4, 2H, H-5′,5′′), 2.77 (ddd, J2′2′′ ) 14.1, J2′3′ ) 7.1, 1H,
*
Author to whom correspondence should be addressed. Phone: +81-
7
5-724-7812. FAX: +81-75-722-2938. E-mail: keisuke@ipc.kit.ac.jp.
1
(
1) (a) Gierer, A.; Mundry, K. W. Nature 1958, 182, 1457-1458. (b)
H-2′), 2.52 (ddd, J2′′3′ ) 3.9, 1H, H-2′′). H NMR (500 MHz, DMSO-d6 at
Schuster, V. H.; Schramm, G. Z. Naturforsch. 1958, 13b, 697-704. (c)
Geiduschek, E. P. Proc. Natl. Acad. Sci. U.S.A. 1961, 47, 950-955. (d)
Horn, E. E.; Herriott, R. M. Proc. Natl. Acad. Sci. U.S.A. 1962, 48, 1409-
30 °C): δ (ppm/TMS) 8.00 (s, 1H, H-2), 7.90 (s, 2H, NH2), 6.05 (dd, 1H,
H-1′), 5.34 (br, 1H, 3′-OH), 4.96 (br, 1H, 5′-OH), 4.34 (ddd, 1H, H-3′),
3.82 (ddd, 1H, H-4′), 3.53 (ABX, 2H, H-5′,5′′), 2.49 (ddd, 1H, H-2′ or
-2′′), 2.23 (ddd, H1, H-2′ or -2′′). 13C NMR (125 MHz, DMSO-d6 at 30
°C): δ (ppm/TMS) 159.7, 153.9, 152.6, 136.4 (C-2), 110.9, 87.7 (C-4′),
82.8 (C-1′), 70.4 (C-3′), 61.4 (C-5′), 39.8 (C-2′). The following measure-
ments were performed for the sample further desalted by RPHPLC. IR
(KBr): 3314, 3125, 2961, 2876, 2791, 2363, 1779, 1640, 1555, 1453, 1381,
1339, 1279, 1238, 1163, 1084, 1051, 1001, 951, 914, 850, 802, 764, 710,
1
416.
(
2) Schuster, V. H. Z. Naturforsch. 1960, 15b, 298-304.
(
3) Schuster, V. H.; Wilhelm, R. C. Biochim. Biophys. Acta 1963, 68,
5
54-560.
(
4) (a) Shapiro, R. J. Am. Chem. Soc. 1964, 86, 2948-2949. (b) Shapiro,
R.; Pohl, S. H. Biochemistry 1968, 7, 448-455.
5) (a) Shapiro, R.; Dubelman, S.; Feinberg, A. M.; Crain, P. F.;
-
1
(
637, 557, 444 cm . UV: λmax 248, 287 nm (pH 1), 245, 286 nm (pH 7),
266 nm (pH 13). CI (i-C4H10) m/z: 153, 117, 99, 81. HR-EI MS m/z:
152.032 49 (Mbasefragment + 1) (calcd for C5H4N4O2, 152.03340). Anal. Calcd
for C10H12N4O5: C, 44.78; H, 4.51; N, 20.89. Found: C, 44.75; H, 4.49;
N, 20.90.
McCloskey, J. A. J. Am. Chem. Soc. 1977, 99, 302-303. (b) Dubelman,
S.; Shapiro, R. Nucleic Acids Res. 1977, 4, 1815-1827.
(
6) (a) Kirchner, J. J.; Hopkins, P. B. J. Am. Chem. Soc. 1991, 113,
4
681-4682. (b) Kirchner, J. J.; Sigurdsson, S. T.; Hopkins, P. B. J. Am.
Chem. Soc. 1992, 114, 4021-4027.
7) Moschel, R. C.; Keefer, L. K. Tetrahedron Lett. 1989, 30, 1467-
468.
(9) The chemical shift was reported downfield from the nitrogen
resonance in nitrate ion of 15N-enriched NH4 NO3 in D2O as an external
standard (30.0 ppm).
15
(
1
0
002-7863/96/1518-2515$12.00/0 © 1996 American Chemical Society

2
516 J. Am. Chem. Soc., Vol. 118, No. 10, 1996
Communications to the Editor
Figure 2. 1H NMR spectrum (A) and aromatic region of 13C NMR
spectrum (B) obtained for compound 1 in DMSO-d
6
13
at 30 °C. The
1
signal assignments were performed by COSY and H- C HMQC. The
numbers designated over the resonances designate the position numbers
of the inset structure. Extra peaks denoted as TEAA were due to the
residual triethylammonium buffer used during the purification process.
Inset: Structure of 2′-deoxyoxanosine.
Figure 3. Reaction products of HNO
denote the percentage yield for each compound at the reaction time of
h.
2
-treated dGuo. The numbers
6
1
0
ing UV, IR, and NMR reported for 2′-deoxyoxanosine and
1
1a
oxanosine, a ribonucleoside form of 2′-deoxyoxanosine, are
essentially consistent with those obtained for compound 1 in
this study (see also supporting information). Furthermore, an
NO. dGuo (10 mM) was dissolved in phosphate buffer (100
mM, pH 7.0) and bubbled with NO until the pH reached 2.9.
RPHPLC analysis revealed that 2′-deoxyoxanosine was formed
in 11.7% yield. In another experiment, aqueous dGuo (10 mM)
in 100 mM phosphate buffer (10 mL, pH 7.0) was exposed to
NO gas (110 mL) in a tightly sealed vessel (inner volume of
the vessel being 120 mL) at room temperature for 4 days (the
final pH being 6.5). The formation of 2′-deoxyoxanosine
(0.22% yield) was also observed in this system.
In conclusion, we have found a new major product in the
reaction of dGuo with HNO2 and NO by RPHPLC analysis.
By spectroscopic measurements, this compound was identified
as 2′-deoxyoxanosine, which was previously reported as a potent
antibiotic. 2′-Deoxyoxanosine is also produced in a single-
stranded oligodeoxynucleotide and double-stranded calf thymus
DNA. Elucidation of genotoxic effects of 2′-deoxyoxanosine
formed in DNA is an important subject of future studies.
8
equilibrium (pKa ) 9.2) which is attributable to saponification
and relactonization of a lactone in 2′-deoxyoxanosine was
observed by the pH titration of UV spectra (data not shown).
From these results we have concluded that compound 1 is 2′-
deoxyoxanosine.
Oxanosine was isolated as a novel antibiotic in 1981 from
the culture broth of Streptomyces capreolus MG265-CF3 and
characterized by the X-ray crystallographic study.11 2′-Deoxy-
oxanosine was synthesized from oxanosine and exhibited a
stronger antineoplastic activity than oxanosine.10 Oxanosine had
weak antibacterial activity against Escherichia coli K-12 and
Proteus mirabilis IFM OM-9.1 It also inhibited the growth
of HeLa cells in culture and induced reversion toward the normal
phenotype of K-ras-transformed rat kidney cells.1
1a
1a,12
2
′-Deoxyoxanosine was produced from dGuo (10 mM) in
2
3
1.5% yield (100 mM NaNO2, 3.0 M acetate buffer (pH 3.7) at
7 °C for 6 h; the percentage yield is based on the value against
Acknowledgment. We are deeply grateful to the referees of this
paper for the valuable suggestions about the identification of the reaction
product. We also thank Mr. K. Kanaori in Kyoto Institute of
Technology for acquiring NMR spectra.
the initial dGuo concentration) (Figure 3). To see if 2′-
deoxyoxanosine is also generated in DNA, dTGTT (0.1 mM)
and calf thymus DNA (1.0 mg/mL) were treated with HNO2.
After incubation for 6 h and subsequent enzymatic digestion
with nuclease P1 and alkaline phosphatase, products were
analyzed by RPHPLC. The yield of 2′-deoxyoxanosine was
Supporting Information Available: HPLC conditions, quantitative
procedures, characterization data of dXao and 2-nitro-dIno, comparison
1
13
of spectroscopic data for 2′-deoxyoxanosine, IR spectrum, H- C and
1
15
2
4.7% for dTGTT and 29.4% for calf thymus DNA, indicating
H- N HMQC spectra of 2′-deoxyoxanosine, and RPHPLC chro-
matograms of HNO -treated dTGTT and calf thymus DNA (9 pages).
2
that this compound is also formed in high yield in the reaction
of DNA with HNO2.
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instructions.
It has been reported that dXao is also produced from dGuo
by the attack of NO.13 Therefore, possible formation of 2′-
deoxyoxanosine from dGuo was explored in the presence of
(
10) Kato, K.; Yagisawa, N.; Shimada, N.; Hamada, M.; Takita, T.;
Maeda, K.; Umezawa, H. J. Antibiot. 1984, 37, 941-942.
11) (a) Shimada, N.; Yagisawa, N.; Naganawa, H.; Takita, T.; Hamada,
JA952550G
(
M.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1981, 34, 1216-1218. (b)
Nakamura, H.; Yagisawa, N.; Shimada, N.; Takita, T.; Umezawa, H.; Iitaka,
Y. J. Antibiot. 1981, 34, 1219-1221.
(13) (a) Wink, D. A.; Kasprzak, K. S.; Maragos, C. M.; Elespuru, R.
K.; Misra, M.; Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A.
W.; Allen, J. S.; Keefer, L. K. Science 1991, 254, 1001-1003. (b) Nguyen,
T.; Brunson, D.; Crespi, C. L.; Penman, B. W.; Wishnok, J. S.; Tannenbaum,
S. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3030-3034.
(
12) Itoh, O.; Kuroiwa, S.; Atsumi, S.; Umezawa, K.; Takeuchi, T.; Hori,
M. Cancer Res. 1989, 49, 996-1000.