544–551
Nucleic Acids Research, 2000, Vol. 28, No. 2
© 2000 Oxford University Press
Formation of 2′-deoxyoxanosine from 2′-deoxyguanosine
and nitrous acid: mechanism and intermediates
Toshinori Suzuki, Hiroshi Ide1, Masaki Yamada, Nobuyuki Endo, Kenji Kanaori2,
Kunihiko Tajima2, Takashi Morii and Keisuke Makino*
Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan, 1Department of Mathematical
and Life Sciences, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8526,
Japan and 2Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku,
Kyoto 606-8585, Japan
Received August 31, 1999; Revised and Accepted November 21, 1999
ABSTRACT
The reaction mechanism for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by nitrous acid
was explored using methyl derivatives of guanosine
and an isolated intermediate of the reaction. When
1-methylguanosine was incubated with NaNO2 under
acidic conditions, N5-methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N2,N2-dimethylguanosine generated no
product. In a similar experiment without NO2–, participation of a Dimroth rearrangement was ruled out. In
the guanosine–HNO2 reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20°C) was
isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was
converted into oxanosine and xanthosine at a molar
ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and
20°C. The ratio was not affected by the incubation pH
between 2 and 10, but increased linearly with temperature from 0.22 (0°C) to 0.32 (50°C). The addition of
acetone also increased the ratio up to 0.85 (98%
acetone). Based on these results, a con-ceivable
pathway for the formation of 2′-deoxyoxanosine from
2′-deoxyguanosine by HNO2 is proposed.
INTRODUCTION
Oxanosine (Oxo), 5-amino-3--(D-ribofuranosyl)-3H-imidazo[4,5-d][1,3]oxazin-7-one, was isolated as a novel antibiotic in
1981 from the culture broth of Streptomyces capreolus
MG265-CF3 and characterized by X-ray crystallography (1,2).
Oxo manifests a wide spectrum of biological effects including
in vitro cytotoxicity against HeLa cells, antibacterial activity
against Escherichia coli K-12 and Proteus mirabilis IFM OM-9,
and induction of reversions to the normal phenotype of K-rastransformed rat kidney cells (1,3). 2-Deoxyoxanosine (dOxo),
a 2-deoxyribonucleoside counterpart of Oxo, has been synthesized from Oxo and found to exhibit stronger antiviral and antineoplastic activities than Oxo (4). Oxo and its analogs have
been chemically synthesized by several defined routes using
4-aminoimidazole derivatives (5–8) or inosine (9) as starting
materials.
We have reported that the reaction of 2-deoxyguanosine
(dGuo) with nitrous acid (HNO2) results in the formation of
dOxo together with 2-deoxyxanthosine (dXao), a deaminated
product of dGuo (10). The maximum yield of dOxo was
21.5%, which corresponded to ~1/3 of the deamination products.
dOxo is also formed in a single-stranded oligodeoxynucleotide
and double-stranded calf thymus DNA by HNO2 treatment
with a comparable yield. Furthermore, dOxo is generated from
dGuo with nitric oxide (NO) at acidic and neutral pH. We have
also demonstrated that the N-glycosidic bond of dOxo is as
stable as that of dGuo and is hydrolyzed 44-fold more slowly
than that of dXao (11). 2-Deoxyoxanosine 5-triphosphate
(dOTP) is more efficiently incorporated opposite cytosine in a
DNA template than 2-deoxyxanthosine 5-triphosphate
(dXTP) (12). Furthermore, dOTP is incorporated opposite
thymine in the template, while dXTP is not incorporated under
the same conditions. These results imply that the dOxo moiety
generated from a dGuo moiety in the nucleotide pool or DNA
may have important and unique roles in mutagenic and lethal
events in cells.
dOxo has a six-membered ring in the base moiety in which
the imino group at N1 of the pyrimidine ring of dGuo is substituted
by an oxygen atom. The formation of dOxo requires opening
and closing of the pyrimidine ring of dGuo. Recent ab initio
molecular orbital calculations predict that a ring-opened cation
is formed synchronously with release of a nitrogen molecule
from the diazonium ion of guanine (13–15). However, direct
evidence as to the reaction mechanism for the formation of
dOxo from dGuo by HNO2 has not been shown so far.
Recently, we reported the detection and isolation of an intermediate in the reaction of 2-deoxycytidine (dCyd) with HNO2
or NO (16). The intermediate was identified from spectrometric
data as a diazoate derivative of dCyd. Under physiological
conditions (pH 7.4, 37C), the diazoate intermediate was fairly
stable (t½ = 330 h) and was exclusively converted into 2-deoxyuridine. However, there is no information on the intermediates
yielding dOxo and dXao in the reaction of dGuo with HNO2. If
the intermediates could be isolated, further insights into the
reaction mechanism could be obtained.
*To whom correspondence should be addressed. Tel: +81 774 38 3517; Fax: +81 774 38 3524; Email: [email protected]
Nucleic Acids Research, 2000, Vol. 28, No. 2
We report herein an analysis of the products formed by the
reaction of methyl-substituted guanosines with HNO2. Furthermore, the diazoate intermediate of Guo was isolated and the
influences of pH, temperature and solvent on the formation of
Oxo and Xao from the intermediate were studied. The reaction
mechanism of dOxo formation by HNO2 is discussed based on
these results.
MATERIALS AND METHODS
Materials
Guanosine (Guo) was purchased from Kohjin (Tokyo, Japan).
N2,N2-Dimethylguanosine (N2,N2-diMe-Guo), N2-methylguanosine
(N2-Me-Guo), 1-methlyguanosine (1-Me-Guo) and xanthosine
(Xao) were obtained from Sigma (St Louis, MO). All other
chemicals of reagent grade were purchased from Wako Pure
Chemicals (Osaka, Japan) or Nacalai Tesque (Osaka, Japan),
and used without further purification. Water was purified with
a Millipore Milli-QII deionizer.
HPLC conditions
545
293 nm (pH 7); APCI-LC/MS (negative) m/z 325 (M – H)–,
296 (M – NO)–.
1-Methylxanthosine (1-Me-Xao). 1H NMR (500 MHz, DMSO-d6
at 30C) (p.p.m./TMS) 7.82 (s, 1H, H-8), 5.73 (dd, 1H, H-1),
5.42 (br, 1H, 5- or 2-OH), 5.19 (br, 1H, 3-OH), 4.30 (m, 1H,
H-2), 4.07 (ddd, 1H, H-3), 4.00 (ddd, 1H, H-4), 3.64 (ABX,
2H, H-5,5), 3.17 (d, 3H, CH3). [We measured the 1H NMR
spectrum in the range –1 to 17 p.p.m. but could not observe the
signal of the NH proton. In H2O at neutral pH, 1-Me-Xao exists
as the 6-keto-2-enol anion form (17). We suspect that 1-Me-Xao
existed as the 6-keto-2-enol anion form in DMSO-d6 due to a
trace of H2O present in DMSO or the sample, thus obscuring
the NH signal. We observed only two OH proton signals
derived from the ribose moiety. One (5.19 p.p.m.) was identified
as the 3-OH proton by COSY measurement. However, for
another (5.42 p.p.m., a fairly broadened signal), it could not be
determined whether it arose from the 2-OH or 5-OH since no
correlation was observed for this resonance in the COSY spectrum.
It is likely that the third OH signal was completely obscured by
further broadening.] UV max 251 nm, 277 nm (pH 7); APCI-LC/
MS (negative) m/z 297 (M – H)–, 165 (Mbase fragment)–.
The HPLC system consisted of Shimadzu LC-6A pumps and a
CTO-6A system controller. On-line UV spectra were obtained
with a Shimadzu SPD-M6A UV-Vis photodiode array
detector. For reversed phase (RP-)HPLC, a Hypersil ODS-5
octadesylsilane column (4.6 150 mm, particle size 5 m; GL
Sciences) was used. The eluant was 100 mM triethylammonium
acetate (TEAA) buffer (pH 7.0) containing acetonitrile
(CH3CN). For measurement of the hydrolysis rate constant of
the intermediate only, 50 mM sodium phosphate buffer
(pH 7.0) containing CH3CN was used as the eluant. The
CH3CN concentration was increased from 0 to 20% over 20 min
as a linear gradient. RP-HPLC analysis was performed at a
flow rate of 1.0 ml/min and ambient temperature.
N5-Methyloxanosine (N5-Me-Oxo). 1H NMR (500 MHz, DMSO-d6
at 30C) (p.p.m./TMS) 8.28 (br, 1H, NH), 8.01 (s, 1H, H-2),
5.68 (dd, J = 6.0 Hz, 1H, H-1), 5.41 (d, J = 6.1 Hz, 1H, 2-OH),
5.17 (d, J = 4.9 Hz, 1H, 3-OH), 4.93 (dd, J = 4.1 Hz, J = 4.1
Hz, 1H, 5-OH), 4.47 (m, 1H, H-2), 4.11 (ddd, 1H, H-3), 3.90
(ddd, 1H, H-4), 3.60 (ABX, 2H, H-5,5), 2.81 (d, J = 3.9 Hz,
3H, CH3). [A correlation between the NH proton signal
(8.28 p.p.m.) and the CH3 proton signal (2.81 p.p.m.) was
observed in the COSY measurement.] UV max 248 nm,
292 nm (pH 7); APCI-LC/MS (negative) m/z 297 (M – H)–, 165
(Mbase fragment)–; HR-EI (positive) m/z 166.049 72 (Mbase fragment + H)+
(calculated for C6H6N4O2, 166.049 08).
Spectrometric measurements
Quantitative procedures
NMR spectra were measured on a Bruker ARX-500 NMR
spectrometer (500 MHz) at 30C. The chemical shifts were
referenced to sodium 3-(trimethylsilyl)[2,2,3,3-d4]propionate
(TSP) in D2O or tetramethylsilane (TMS) in DMSO-d6 as an
internal standard. The high resolution (HR) EI mass spectrum
was recorded on a JEOL DX-300 at 30 eV. Negative ion APCI
(atmospheric pressure chemical ionization)-LC mass spectra
were obtained with a Hitachi M-2000 MS system. The sample
was directly injected into the MS system by an HPLC pump
without separation columns. The LC/MS conditions were as
follows: eluant, 100% CH3CN (isocratic); flow rate, 1 ml/min;
vaporization temperature, 300C; desolvation temperature,
300–370C; drift voltage, –195 V. UV spectra for hydrolysis
of the intermediate were measured on a Shimadzu UV-260
UV-Vis spectrophotometer equipped with an SPR-5 temperature
controller.
Guo or 1-Me-Guo (10 mM) was incubated with NaNO2
(100 mM) in 1 ml sodium acetate buffer (3.0 M, pH 3.7) at
37C for 6 h. For the Guo–HNO2 system, the concentration of
each reaction product was determined from the peak area of the
RP-HPLC chromatogram monitored at 260 nm and the molar
extinction coefficient. The molar extinction coefficients [260: Guo,
11 500; Oxo, 5100; Xao, 7800] were taken from the literature
(4,18,19). The initial RP-HPLC peak area was used as a
standard. Since the molar extinction coefficients of the methyl
derivatives (1-Me-Guo, N5-Me-Oxo and 1-Me-Xao) were not
available, the amounts of these products were determined
using 1H NMR integration signals as follows. After reaction
with HNO2, 1-Me-Guo solution was neutralized with concentrated
NaOH. Then the sample was lyophilized and dissolved in
400 l of D2O (98%). The 1H NMR spectrum of the sample
was measured with a 45 pulse with a delay time of 10 s. The
yields of products were determined by the relative intensities of
the integral values for the methyl proton resonances of 1-Me-Guo
(3.48 p.p.m./TSP), N5-Me-Oxo (2.96 p.p.m./TSP) and 1-Me-Xao
(3.35 p.p.m./TSP).
Spectrometric data
N2-Methyl-N2-nitrosoguanosine (N2-Me-N2-nitroso-Guo). 1H
NMR (500 MHz, DMSO-d6 at 30C) (p.p.m./TMS) 12.86
(br, 1H, NH), 8.35 (s, 1H, H-8), 5.55 (dd, 1H, H-1), 5.48
(br, 1H, OH), 5.21 (br, 1H, OH), 4.97 (br, 1H, OH), 4.55
(m, 1H, H-2), 4.16 (ddd, 1H, H-3), 3.95 (ddd, 1H, H-4), 3.61
(ABX, 2H, H-5,5), 3.41 (s, 3H, CH3); UV max 240 nm,
Kinetics
pH dependence. The RP-HPLC column eluant containing the
diazoate (20 l) was directly dropped into 100 mM buffer
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Nucleic Acids Research, 2000, Vol. 28, No. 2
(480 l) and incubated at 37C. The buffers used were sodium
acetate buffer (pH 2–3.7, concentrated HCl was added for pH 2
and 3), sodium phosphate buffer (pH 5–8) and sodium
carbonate buffer (pH 9 and 10). The incubation periods were
10 min for pH 2 and 3, 30 min for pH 4–6, 1 h for pH 7 and 8,
1.5 h for pH 9 and 2 h for pH 10. The samples in acidic or alkaline
buffer were neutralized by addition of 1 M NaOH or HCl and
analyzed by RP-HPLC.
Temperature dependence. The RP-HPLC column eluant
containing the diazoate (20 l) was directly dropped into
temperature controlled 100 mM sodium phosphate buffer
(480 l) at pH 7.0. The incubation periods were 4 h for 0C,
2 h for 10C, 1 h for 20 and 30C and 30 min for 37 and 50C.
The samples were analyzed by RP-HPLC.
Solvent effect. The RP-HPLC column eluant containing the
diazoate (20 l) was directly dropped into the mixture of
acetone and H2O (480 or 980 l) at 20C. The incubation
periods were 30 min for 0% (acetone % v/v), 1 h for 25%, 1.5 h
for 50%, 2 h for 75% and 48 h for 96 and 98%. Acetone was
removed from each sample by aspirator and the samples were
analyzed by RP-HPLC.
RESULTS AND DISCUSSION
Determination of the reaction site using methyl derivatives
of Guo
The reaction site leading to the conversion of dGuo into dOxo
by HNO2 was explored using Guo (the ribonucleoside counterpart of dGuo) and its methyl derivatives to avoid cleavage of
the labile N-glycosidic bond of deoxyribonucleosides. Guo
(10 mM) was incubated with 100 mM NaNO2 in 3.0 M sodium
acetate buffer (pH 3.7) at 37C for 6 h and analyzed by RP-HPLC
(data not shown). The first peak (retention time tR = 9.1 min)
was identified as Xao by agreement with the retention time and
the on-line UV spectrum of the authentic sample. The second peak
(tR = 10.6 min) was unreacted Guo. The third peak (tR = 12.0 min)
was identified as Oxo by coincidence with the UV spectrum in
a previous paper (1). The yields of reaction products were
17.9% Oxo and 79.3% Xao with 4.0% unreacted Guo. Figure 1A
shows the structures of these compounds with atomic
numbering. It should be noted that the atomic numbering of the
base moiety of Oxo and its analog is different from that of
purines. Although the numbering of purines is customary, a
systematic numbering following the IUPAC rules is applied to
Oxa and its analog since they are not purines (2,20). For dGuo,
it has been reported that the yields were 21.5% dOxo and
58.7% dXao and xanthine (a base moiety of dXao) with 7.6%
unreacted dGuo under the same reaction conditions (10).
Accordingly, substitution of a hydroxyl group (Guo) for a
hydrogen atom (dGuo) at the 2 sugar position had little influence
on the formation of Oxo derivatives.
Concerning the mechanism of dOxo formation, two conceivable
pathways exist in the initial step of the reaction (Scheme 1). In
Path I, the reaction is initiated by attack of NOx (probably
N2O3) produced by protonation of nitrite on the N1-imino
group of dGuo. In this case, dGuo bearing N1 substituents like
nitroso, nitro or diazo groups are potential precursors en route
to dOxo. dOxo is formed by subsequent attack of a water
Figure 1. Reaction products of HNO2-treated (A) guanosine (Guo), with the
atomic numbering, (B) N2,N2-dimethylguanosine (N2,N2-diMe-Guo), (C) N2methylguanosine (N2-Me-Guo) and (D) 1-methylguanosine (1-Me-Guo). Note
that the atomic numbering of the base moieties of oxanosine (Oxo) and its analog
is different from that of purines (see text). The numbers in parentheses denote
percentage yields of products at a reaction time of 6 h.
Scheme 1. Two possible reaction pathways for the formation of 2-deoxyoxanosine (dOxo) from 2-deoxyguanosine (dGuo) by HNO2 treatment. dR in
the structures denotes a 2-deoxyribose moiety.
molecule on N1 of the intermediates. To obtain 15N-labeled
purine nucleosides, an N1-nitropurine compound has been
Nucleic Acids Research, 2000, Vol. 28, No. 2
employed as a precursor for N1 substitution by 15N compounds
(21). Path II includes attack of N2O3 on the exocyclic amino
group of dGuo. In this case, a nitroso group is introduced on a
N2 atom. Subsequently several intermediates are formed, such
as diazohydroxide, diazoate and diazonium derivatives, based
on the diazo chemistry of aromatic primary amines (Scheme 2;
22,23). To determine which reaction pathway is responsible for
dOxo formation, N2,N2-diMe-Guo, N2-Me-Guo and 1-Me-Guo
were treated with HNO2 and the products were analyzed. If
Path I is involved, substitution of N1 by an oxygen atom will
not occur for 1-Me-Guo since the methyl group protects N1
against the attack of N2O3. If Path II is involved, the reaction
will not proceed for N2,N2-diMe-Guo since the methyl groups
protect the exocyclic amino group on C2 against attack by
N2O3. The reaction of N2-Me-Guo carrying a secondary amine
will stop at the step of the nitroso derivative.
Scheme 2. Proposed reaction pathways for the nitrosative deamination of aromatic
amines by HNO2.
N2,N2-diMe-Guo (1.0 mM), N2-Me-Guo (10 mM) and 1-Me-Guo
(10 mM) were incubated with NaNO2 (100 mM) in sodium
acetate buffer (3.0 M, pH 3.7) for 6 h at 37C, and the reactions
monitored by RP-HPLC. No product was observed in the reaction
of N2,N2-diMe-Guo (Fig. 1B). One product was detected in the
reaction of N2-Me-Guo and isolated by RP-HPLC. From spectrometric data, the product was identified as a nitroso derivative,
N2-methyl-N2-nitrosoguanosine (N2-Me-N2-nitroso-Guo) (Fig. 1C).
N2-Me-Guo was completely converted to N2-Me-N2-nitroso-Guo
after incubation for 6 h. When 1-Me-Guo was incubated with
HNO2, two major product peaks with tR = 10.2 and 13.7 min
appeared in the RP-HPLC chromatogram (data not shown).
The UV spectra of the peaks were similar to those of Xao and
Oxo, respectively. From the spectrometric data, these products
were identified as 1-Me-Xao, a deamination product of 1-Me-Guo,
and N5-methyloxanosine (N5-Me-Oxo), an Oxo analog bearing
an exocyclic methyl amino group (Fig. 1D). The yields of the
products (HNO2 treatment for 6 h) were 38.0% N5-Me-Oxo
and 22.3% 1-Me-Xao with 39.7% unreacted 1-Me-Guo.
Unlike the results for Guo and dGuo, treatment of 1-Me-Guo
with HNO2 resulted in a higher yield of the Oxo derivative than
the Xao derivative.
547
Consideration of rearrangements
In the formation of N5-Me-Oxo from 1-Me-Guo (Fig. 1D), the
N1-methyl group shifted from the endocyclic to exocyclic
position. This type of methyl group shift, which is known as a
Dimroth rearrangement, can take place in heterocyclic
compounds when an exocyclic amino or imino group has a
neighboring N-alkyl group in a ring (24,25). Dimroth
rearrangement proceeds under alkaline or acidic conditions for
pyrimidines, purines and pteridine derivatives (26–31). If
Dimroth rearrangement is involved in the present reaction
system, generation of N5-Me-Oxo from 1-Me-Guo would
require initial formation of N2-Me-Guo from 1-Me-Guo
followed by substitution of the N1 atom by an oxygen atom. To
examine this possibility, 1-Me-Guo (10 mM) was incubated in
3.0 M sodium acetate buffer (pH 3.7) in the absence of NO2–
for 48 h at 37C. No peak other than the starting 1-Me-Guo
was observed in the RP-HPLC chromatogram. Consequently,
it is evident that intramolecular rearrangement from 1-Me-Guo
to N2-Me-Guo is not involved in the present system.
Since the base moieties of the Oxo and Xao derivatives are
structural isomers of each other, we also explored the possibility of
interconversion between these derivatives. An aqueous solution
containing Xao or 1-Me-Xao was incubated in the presence or
absence of 100 mM NaNO2 in 3.0 M sodium acetate buffer
(pH 3.7) at 37C for 48 h. However, Oxo or N5-Me-Oxo was
not observed in the RP-HPLC chromatograms (data not
shown). In similar experiments, no conversion from Oxo and
N5-Me-Oxo to Xao and 1-Me-Xao occurred, respectively.
Intramolecular rearrangement from 1-Me-Guo to N2-Me-Guo
and conversion between Oxo and Xao derivatives were not
involved in the present system. These results clearly show that
the formation of dOxo is initiated by the attack of N2O3 on the
exocyclic amino group of dGuo. It is also evident that the
amino group of dOxo was derived from the N1-imino group of
dGuo. Thus, cleavage of the C6–N1 amido bond in the pyrimidine
ring of dGuo occurs during the reaction.
Detection and separation of a reaction intermediate
To obtain further information on the reaction mechanism of dOxo
formation, we attempted to identify the reaction intermediates by
RP-HPLC analysis. Guo (10 mM) and NaNO2 (100 mM) was
incubated in sodium acetate buffer (3.0 M, pH 3.7) at 37C for
a short period (5 min). Immediately after neutralization, the
reaction mixture was analyzed by RP-HPLC (Fig. 2A). In
addition to the major products, several small peaks were
detected. Among these peaks, only the peak with tR = 11.7 min,
which showed a UV spectrum with max = 315 nm (Fig. 2A,
inset), decreased at an incubation time of 30 min (data not
shown). Therefore, a compound contained in this peak
(referred to as 1) was assumed to be a reaction intermediate. In
order to obtain 1 in large scale, another reaction system was
employed. Guo (0.5 M) in 10 l of 5 M H2SO4 was added to
1.2 l of 4 M NaNO2 and incubated at 0C for 5 s. The solution
was immediately neutralized with 10 M NaOH (10 l) and
100 mM phosphate buffer (pH 7.0, 100 l) and analyzed by
RP-HPLC. As shown in Figure 2B and its inset, a large peak
appeared which had the same retention time and UV spectrum
as 1, indicating that the peak contained 1. The RP-HPLC fraction
of 1 obtained by the above method was used in the following
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Nucleic Acids Research, 2000, Vol. 28, No. 2
Figure 3. Structure of 1, a diazoate derivative of guanosine [9-(-D-2-ribofuranosyl)-6-oxopurine-2-diazoate].
Figure 2. (A) RP-HPLC profile of the reaction mixture of guanosine (Guo)
when 10 mM Guo and 100 mM NaNO2 were incubated in 3.0 M sodium acetate
buffer (pH 3.7) for 5 min at 37C. (B) RP-HPLC profile of the reaction mixture of
Guo when 0.5 M Guo in 10 l of 5 M H2SO4 was added to 1.2 l of 4 M
NaNO2 and incubated at 0C for 5 s. The inset shows the UV spectrum of 1
indicated by the arrow. The CH3CN concentration was increased in the linear
gradient mode (0 min; 0%, 20 min; 20%).
experiments. The fraction also contained elution buffer
(100 mM TEAA buffer and ~10% CH3CN).
Identification of the intermediate
To identify the intermediate, several measurements were
performed. 1 was trapped completely by an anion exchange
resin (Sephadex A-25, OH– form) but passed directly through
the same bed volume of a cation exchange resin (Bio-Rad
AG50W-X8, H+ form). Among all of the expected intermediates
in the reaction of Guo with HNO2, only the diazoate derivative
is an anion (Scheme 2). Negative ion APCI-LC/MS using
100% CH3CN as eluant showed a signal with m/z = 283 for 1.
However, the signal at m/z = 311 expected for the intact
diazoate anion could not be detected in the LC/MS measurements, probably due to its instability (vide infra). In general,
aromatic diazoate compounds (R–NNO–) are known to give
rise to a strong signal of the [M– – 28] fragment which is
produced by loss of a molecular nitrogen through four-center
skeletal rearrangement (32,33). Thus, the signal with m/z = 283
was attributed to the –N2 product. The on-line UV spectrum of
1 (max = 315 nm) was similar to those of the diazoate derivatives
of 2-deoxycytidine (dCyd) (max = 305 nm) (16) and 9-alkyladenine (max = ~310 nm) (23). Combining these data, we have
tentatively assigned 1 as a diazoate derivative of Guo, 9-(-D2-ribofuranosyl)-6-oxopurine-2-diazoate (Fig. 3).
Hydrolysis of the diazoate intermediate
The RP-HPLC fraction of the diazoate (100 l) was incubated
in 50 mM phosphate buffer (pH 7.0, 500 l) at 37C for 20 min
and analyzed by RP-HPLC. The peak of the diazoate disappeared
and two peaks of Oxo and Xao appeared (data not shown). The
Figure 4. Plot of the absorbance at 340 nm against incubation time. The diazoate
was incubated at 20C in 50 mM sodium phosphate buffer containing ~3%
CH3CN. The inset is the change in the UV spectrum of the reaction mixture
measured at 3 min intervals.
ratio of the yield of Oxo to Xao was 0.29. This result clearly
shows that the diazoate is the common precursor of Oxo and
Xao in the Guo–HNO2 system. Incubation of the diazoate in
0.5 M H2SO4 at 37C for 20 min resulted in Guo as the main
product with small amounts of Oxo and Xao. In a previous
study on the 9-alkyladenine diazoate derivative, similar
conversion from the diazoate group to the starting amino group
has been reported under strongly acidic conditions (23). No
change in the UV spectrum was observed when the diazoate
derivative of Guo was incubated in 1 M NaOH at 37C for 2 h.
Therefore, the diazoate is converted to Oxo and Xao at neutral
pH, but to the starting Guo under acidic conditions. The
diazoate is stable under basic conditions.
The rate constant for hydrolysis of this intermediate was
measured at neutral pH. To avoid the influence of co-existing
TEAA contained in the RP-HPLC eluant on the reaction rate,
the diazoate was RP-HPLC purified using 50 mM phosphate
buffer (pH 7.0) as eluant. The RP-HPLC fraction of the
diazoate (200 l) was diluted with 50 mM phosphate buffer
(400 l, pH 7.0) and then UV spectra were measured at intervals of 3 min at 20C. The diazoate showed an absorption band
at >310 nm, whereas Oxo and Xao showed no absorption.
Thus, as shown in the inset to Figure 4, the absorption at
>310 nm due to the diazoate rapidly decreased with incubation
time. The plot of absorbance at 340 nm against incubation time
followed first order kinetics (Fig. 4). The rate constant of the
reaction was evaluated as 2.1 10–3 s–1 (half life t1/2 = 5.6 min)
from the slope.
Nucleic Acids Research, 2000, Vol. 28, No. 2
549
Ring-closed and ring-opened cations
Generally, diazonium salts generate cations by dissociative
loss of a molecular nitrogen (34–36). Although aromatic diazonium
compounds form ring-closed cations exclusively, some aliphatic
ring compounds generate not only ring-closed but also ringopened cations (37–39). Two types of reaction mechanism
have been proposed for the ring opening process on the basis of
kinetic and stereochemical criteria. One is the one-step reaction
mechanism including synchronous ring opening with elimination
of a nitrogen molecule. Bicycloalkanediazonium ions undergo
a synchronous ring opening reaction (37). The other is the
multi-step reaction mechanism, including formation of the
ring-closed cation and sequential ring opening. Decomposition
of the phenyl-substituted cyclopropanediazonium ion occurs
via a multi-step reaction (39). In recently published papers by
Glaser and colleagues, the reaction mechanism for the formation
of dOxo was explored using ab initio calculation (13–15).
According to their calculations, cleavage of the C6–N1 bond
occurs when the length of the C2–N2+ bond of the guanine
diazonium ion is >2.2 Å. All attempts to search the reaction
channel connecting the diazonium ion to the ring-closed cation
failed. The ring-opened cation was more stable (63.0 kcal/mol)
than the ring-closed cation. These results suggest that release
of a nitrogen molecule from the diazonium ion generates the
ring-opened cation directly without formation of the ring-closed
cation, supporting the one-step mechanism.
Scheme 3. Two conceivable reaction pathways for the formation of 2-deoxyoxanosine (dOxo) and 2-deoxyxanthosine (dXao) from the diazonium intermediate.
If the one-step reaction mechanism is involved in the present
system, only the ring-opened cation would be generated
(Scheme 3A). Attack by H2O on the carbonyl and carbodiimide
groups of the ring-opened cation generate Oxo and Xao,
respectively, after ring closure. In this case the ratio Oxo:Xao
is determined only by the relative reactivity of the carbonyl and
carbodiimide groups with H2O. If the multi-step mechanism is
involved, the ring-closed cation exclusively generating Xao by
reaction with H2O will be present in addition to the ringopened cation (Scheme 3B). To elucidate which reaction
Figure 5. (A) The temperature dependence of the Oxo:Xao ratio at pH 7.0. (B) The
dependence of the Oxo:Xao ratio on acetone content at 20C.
mechanism is operating in the present system, hydrolysis of the
diazoate was carried out under various conditions and the
influences of pH, temperature and solvent on the product ratio
(Oxo:Xao) were determined. The products were quantified by
RP-HPLC analysis. No products other than Oxo and Xao were
detected in all reactions. The RP-HPLC fraction containing the
diazoate was incubated at 20C in the pH range 2–10. The
Oxo:Xao ratio was independent of pH within the range tested,
suggesting that no acid–base equilibrium was present for the
intermediate cation(s). To clarify the temperature effect, the
diazoate was incubated in 100 mM sodium phosphate buffer at
pH 7.0 in the temperature range 0–50C. The Oxo:Xao ratio
increased linearly with temperature from 0.22 (0C) to 0.32
(50C) (Fig. 5A). The previous ab initio calculations showed
that the ring-opened cation was preferred over the ring-closed
cation by 63.0 kcal/mol (13). If an equilibrium exists between
these two cations (Scheme 3B) and the ring-opened cation is
more stable than the ring-closed one, a rise in reaction temperature drives the equilibrium in the endothermic direction,
hence increasing the fraction of the ring-closed cation. In this
case, a rise in incubation temperature will lead to a reduction in
the Oxo:Xao ratio. Thus, the present results showing an opposite
tendency do not support the multi-step reaction. The observed
increase in the Oxo:Xao ratio with a temperature rise might be
the result of the activation energy of hydrolysis of the carbonyl
group being greater than that of the carbodiimide group on the
ring-opened cation. To examine the solvent effect, the
Oxo:Xao ratio was measured by incubating the diazoate in
solutions containing 0–98% (v/v) acetone at 20C. The ratio
increased with increasing acetone content from 0.26 (0%
acetone) to 0.85 (98% acetone) (Fig. 5B). If an equilibrium
exists between the two cations and the ring-closed cation is
preferentially stabilized by reducing the dielectric constant of
the solvent, addition of acetone should increase the fraction of
the ring-closed cation. In this case, the addition of acetone
would reduce the Oxo:Xao ratio. However, the observed effect
550
Nucleic Acids Research, 2000, Vol. 28, No. 2
of acetone was the opposite, not supporting the multi-step
reaction. The increase in the Oxo:Xao ratio might result from
the frequency of access of H2O to the cationic carbonyl group
relative to the carbodiimide group of the ring-opened cation
increasing with a rise in acetone content.
intermediate is converted immediately to the diazoate intermediate, which appears to be the most stable intermediate
throughout the reaction pathway. Then the diazoate intermediate is converted to the diazonium intermediate and the
ring-opened cation is formed synchronously by the release of a
nitrogen molecule from the diazonium. Attack by H2O on the
carbodiimide of the ring-opened cation and subsequent ring
closure and tautomerization generates dXao. On the other
hand, attack by H2O on the carbonyl group of the ring-opened
cation and subsequent ring closure generates dOxo.
ACKNOWLEDGEMENTS
We thank Dr M. Nishi (Setsunan University) for acquiring the
HR-EI MS spectrum. This work was supported partly by
Grants-in-Aid for Scientific Research from the Ministry of
Education, Science and Culture, Japan [to K.M. (11101001,
10151219 and 10878092), K.K. (09780545) and H.I.].
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Scheme 4. The proposed overall mechanism for the reaction of 2-deoxyguanosine (dGuo) with HNO2.
All the results concerning the effects of pH, temperature and
solvent failed to support the existence of the ring-closed cation.
It is very likely that the one-step mechanism dominates in the
reaction of Guo with HNO2.
Overall reaction mechanism
A possible overall reaction pathway of dGuo with nitrous acid
is summarized in Scheme 4 on the basis of the present results.
First, a reactive species NOx (probably N2O3) generated from
protonated NO2– attacks the amino group on C2 of dGuo,
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