Smooth and selective formation of the cyclic 1,N2-propano adducts in the reactions of guanine nucleosides and nucleotides with acetaldehyde

Article abstract of DOI:10.1016/S0040-4039(02)01530-7

The treatment of guanine nucleosides and nucleotides with excess acetaldehyde in pH 8.0 phosphate buffer containing a basic amino acid such as arginine and lysine resulted in the smooth and selective formation of the corresponding cyclic 1,N²-propano adducts even under mild conditions.

Full text of DOI:10.1016/S0040-4039(02)01530-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6701–6703
Smooth and selective formation of the cyclic 1,N2-propano
adducts in the reactions of guanine nucleosides and nucleotides
with acetaldehyde
Magoichi Sako,* Isamu Yaekura and Yoshihiro Deyashiki
Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu 502-8585, Japan
Received 2 July 2002; revised 22 July 2002; accepted 25 July 2002
Abstract—The treatment of guanine nucleosides and nucleotides with excess acetaldehyde in pH 8.0 phosphate buffer containing
a basic amino acid such as arginine and lysine resulted in the smooth and selective formation of the corresponding cyclic
1,N2-propano adducts even under mild conditions. © 2002 Elsevier Science Ltd. All rights reserved.
Chemical modifications of nucleosides and nucleotides
by acetaldehyde (AA) have been investigated in connec-
tion with the toxicity, mutagenicity, and carcinogenicity
of AA commonly existing as exogenous and endoge-
nous sources, such as the primary metabolite of etha-
nol, a component in tobacco smoke, and a metabolic
intermediate of sugars, in the human environment.1
From this viewpoint, it has been documented that the
reactions of nucleic acids with AA under physiological
conditions proceed chemo- and regio-selectively at the
exocyclic amino group of the guanine moiety to form
the easily hydrolyzable N2-ethylidene guanines (A) as
the major product, together with trace amounts of
stable adducts such as N2-(6-methyl-1,3-dioxan-4-
yl)guanine adducts (B) and cyclic 1,N2-propano gua-
nine adducts (C).2
recovery of most of the starting deoxyguanosine. Con-
trary to this fact, when the reaction was carried out in
the presence of two equimolar amounts of arginine, the
starting deoxyguanosine was smoothly consumed and
almost quantitatively converted into 1a as a 1:1 mixture
of the diastereomers. Analogous results were obtained
when using other amino acids such as lysine and cys-
teine in place of arginine, though their efficiencies for
the cyclic adduct formation were dependent on the
nature of the functional groups in the employed amino
acids as shown in Table 1.
R
O
O
R
O
N
N
N
N
N
HN
O
O HN
N
N
Me
N
H
N
R
N
N
Me
N
H
N
Sugar
Sugar
Sugar
A
B
C
We now report that the reactions of guanine
nucleosides and nucleotides with AA are significantly
accelerated even under mild conditions by the presence
of a basic amino acid such as arginine and lysine in the
medium to give the corresponding cyclic 1,N2-propano
adducts (1) (cf., C: R=OH) almost quantitatively (Fig.
1).
R= Me, MeCH(OH)CH2-
R= OH, NH-dGuo
O
OH
N
N
N
N
1
N
H
Me
R2O
O
The treatment of 2%-deoxyguanosine with excess AA in
0.1 M phosphate buffer (pH 8.0) at 37°C for 1 day in a
sealed tube resulted in the formation of only a trace
amount of the cyclic 1,N2-propano adduct (1a) with
R1O
R
a: R= R1= R2= H
b: R= R1= H; R2= PO3H2
c: R= R2= H; R1= PO3H2
d: R= OH; R1= H; R2= PO3H2
e: R= OH; R1, R2= -PO(OH)-
Keywords: cyclic 1,N2-propano guanine adduct; guanine nucleosides
and nucleotides; acetaldehyde.
* Corresponding author. Tel.: +81-(58)237-3931; fax: +81-(58)237-
5979; e-mail: sako@gifu-pu.ac.jp
Figure 1.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01530-7

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M. Sako et al. / Tetrahedron Letters 43 (2002) 6701–6703
Table 1. Reactions of 2%-deoxyguanosine with AA in the
presence of amino acids
Amino acids
1,N2-Cyclic adduct 1a (recovered SM)%
L
L
L
L
L
L
L
L
-Alanine
-Serine
35 (58)
39 (57)
26 (70)
3 (97)
-Tyrosine
-Aspartic acid
-Histidine
-Cysteine
-Lysine
2 (98)
46 (54)
59 (41)
85 (15)
Figure 2. pH-Dependency of the reaction of 2%-
deoxyguanosine with AA or 2-butenal in the presence of
arginine.
-Arginine
Reaction conditions: a mixture of 2%-deoxyguanosine (7.2 mg, 0.025
mmol) and an appropriate amino acid (0.05 mmol) in 0.1 M
phosphate buffer (pH 8.0) (1.5 mL) containing AA (50 mL, 0.89
mmol) was heated at 37°C in a sealed tube for 8 h. The formation
of the cyclic adduct 1a and the recovery of the starting
deoxyguanosine were estimated by TLC densitometric analysis [elu-
ent: chloroform/methanol/acetic acid (40:8:1); detector: 254 nm].
O
Me
H2O
NR
NR
X
RHN
RN
- XH
The structure of the adduct 1a was confirmed by com-
RNH2
+ XH
1
Me
Me
parison with the previously reported UV, mass, and H
Me
Me
O
D
E
NMR spectral data2,3 [e.g. its 1H NMR spectrum
showed characteristic signals for the cyclic propano
group at l 1.16 (3H, d, J=6 Hz, NHCHMe; lC 20.6),
1.43 and 2.05 (each 1H, br t and br d, J=each 12 Hz,
CHCH2CHMe; lC 34.7), 3.63 (1H, m, NHCHMe; lC
41.3), and 6.03 and 6.07 (each 1/2H, each br s,
NCHOH; lC 72.0) ppm] and by its NaBH4-reduction
- RNH2
X= OH, NHR
dGuo
RNH2= Arginine
O
NR
NH
N
N
N
N
H
Me
HO
O
to
give
N2-(3-hydroxy-1-methylpropyl)-2%-deoxy-
guanosine.4 The cyclic adduct 1a was very stable in
both acidic and basic medium; for example, even after
heating overnight at 70°C in a pH 2 or pH 10 solution,
the starting adduct was recovered unchanged. However,
the adduct 1a underwent acid hydrolysis at 90°C in the
pH 2 solution leading to the deglycosylated product,3
and its alkaline hydrolysis at 90°C in the pH 10 solu-
tion produced 2%-deoxyguanosine.
H2O
1a
OH
Scheme 1.
The aldol condensation of AA to 3-hydroxybutanal (or
its equivalent, D) followed by dehydration can be accel-
erated by the presence of arginine to form a Shiff base
of 2-butenal, (E), as the key intermediate.5 The Michael
addition of the exocyclic amino group in 2%-
deoxyguanosine to the 2-butenylidene moiety in E fol-
lowed by ring-closure and the subsequent hydrolytic
elimination of arginine affords the cyclic adduct 1a as
the ultimate product.
The formation of the cyclic adduct 1a in the reaction of
2%-deoxyguanosine with AA in the presence of arginine
was pH-dependent, and the employment of a basic
buffer was effective for the adduct formation as shown
in Fig. 2. When the reaction was carried out in a
1
deuterated pH 8.0 buffer solution and followed by H
NMR spectroscopy, the smooth formation of 2-butenal
or its equivalent (detected as 2-butenal after CDCl3-
extraction of the reaction mixtures) during the reaction
was observed, together with the formation of 3-hydroxy-
butanal or its equivalent. The generation of 2-butenal
(or its equivalent) in this reaction was parallel to the
formation of the cyclic adduct 1a under the conditions
employed. The advanced formation of 1a in the pres-
ence of arginine and the pH-dependency for the adduct
formation were also observed in the reaction of 2%-
deoxyguanosine with 2-butenal as shown in Fig. 2.
Analogous results were obtained in the reactions of
guanine nucleotides such as 5%-dGMP, 5%-GMP, and
3%,5%-cyclic GMP with AA in the presence of arginine to
give the corresponding cyclic 1,N2-propano adducts
1b–e in high yields, respectively.6 It should be noted
that no formation of any stable adducts was observed
in the reactions of the adenine nucleosides and pyrim-
idine nucleosides under the same conditions (in the
presence of arginine, pH 8.0 buffer, at 37°C, for 1 day),
suggesting that the cyclic adduct formation with AA in
the presence of the basic amino acids is also chemo-
and regio-selective to the guanine nucleosides and
nucleotides. Recently, the cyclic 1,N2-propano adduct
1c has been detected in the range of 2–3 adducts/109
nucleotides in the tissues of 2-butenal-treated and
On the basis of the above facts and chemical reactivity
of AA, 2-butenal, and the guanine moiety, the forma-
tion of the cyclic 1,N2-propano adduct 1a in the present
reaction can be reasonably explained as illustrated in
Scheme 1.

M. Sako et al. / Tetrahedron Letters 43 (2002) 6701–6703
6703
untreated laboratory animals after enzymatic hydroly-
ses of the liver DNAs.7 The results described above are
interesting in connection with the molecular mecha-
nisms explaining the toxic, mutagenic, and carcinogenic
effects of AA,1 and provide a convenient preparative
method for the cyclic 1,N2-propano adducts of the
guanine nucleosides and nucleotides, 1a–e.
Cyclic 1,N2-propano 5%-dGMP adduct (1b): 75% yield as a
Na salt (as a 3:2 mixture of its diastereomers isolatable on
HPLC); IR (KBr): 3420, 1686, 1637 cm−1; UV (MeOH):
275 (sh), 262 nm; Mass (FAB+): 418 [M+H]+; HR-FAB
MS: 418.1123 (mmu: −0.5; calcd for C14H20N5O8P); 1H
NMR (D2O) l: 1.22 (3H, d, J=6 Hz), 1.55 and 2.13 (each
1H, br t and br d, J=12 Hz), 2.36 and 2.66 (each 1H, each
m), 3.66 (1H, m), 3.90 (2H, m), 4.09 (1H, br s), 4.61 (1H,
br s), 6.17 (1H, br d, J=7 Hz), 6.19 (1H, br s), 7.91 (1H,
s); 13C NMR (D2O) l: 21.0, 34.8, 39.5, 41.3, 65.5, 72.1,
72.4, 84.1, 86.8, 111.6, 137.9, 151.3, 152.1, 161.0.
References
Cyclic 1,N2-propano 3%-dGMP adduct (1c): 80% yield as a
Na salt (as a 1:1 mixture of its diastereomers isolatable on
HPLC); IR (KBr): 3398, 1686, 1638 cm−1; UV (MeOH):
275 (sh), 261 nm; Mass (FAB+): 418 [M+H]+; HR-FAB
MS: 418.1136 (mmu: +0.8; calcd for C14H20N5O8P); 1H
NMR (D2O) l: 1.21 (3H, d, J=6 Hz), 1.54 and 2.12 (each
1H, br t and br d, J=12 Hz), 2.52 and 2.70 (each 1H, each
m), 3.65 (1H, m), 3.67 (2H, m), 4.13 (1H, br s), 4.78 (1H,
br), 6.15 (1H, br t, J=5 Hz), 6.18 (1H, br s), 7.82 (1H, s);
13C NMR (D2O) l: 22.3, 36.4, 40.3, 43.0, 64.1, 73.7, 77.5,
86.3, 88.8, 117.8, 140.2, 152.6, 153.5, 159.9.
1. (a) Hecht, S. S.; McIntee, E. J.; Wang, M. Toxicology
2001, 166, 31–36; (b) Seitz, H. K.; Matsuzaki, S.;
Yokoyama, A.; Homann, N.; Vakevainen, S.; Wang, X.
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Cyclic 1,N2-propano 5%-GMP adduct (1d): 70% yield as a
Na salt (as a 3:7 mixture of its diastereomers isolatable on
HPLC); IR (KBr): 3412, 1686, 1637 cm−1; UV (MeOH):
275 (sh), 262 nm; Mass (FAB+): 434 [M+H]+; HR-FAB
MS: 434.1071 (mmu: −0.6; calcd for C14H20N5O9P); 1H
NMR (D2O) l: 1.21 (3H, d, J=6 Hz), 1.55 and 2.13 (each
1H, br t and br d, each J=12 Hz), 3.70 (2H, m), 3.96 (1H,
m), 4.19 (1H, m), 4.34 (1H, br s), 4.58 (1H, m), 5.77 (1H,
d, J=6 Hz), 6.19 (1H, br s), 7.92 (1H, s).
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Cyclic 1,N2-propano 3%,5%-cGMP adduct (1e): 93% yield as
a Na salt (as a 3:7 mixture of its diastereomers isolatable
on HPLC); IR (KBr): 1685 cm−1; Mass (FAB+): 416
[M+H]+; HR-FAB MS: 416.0978 (mmu: +0.7; calcd for
6. Isolated yields (in 0.1 mmol scale experiments) and spec-
tral data of the cyclic adducts 1a–e are as follows:
Cyclic 1,N2-propano dGuo adduct (1a):3 92% yield (as a 1:1
mixture of its diastereomers isolatable on HPLC); mp
155–160°C (from acetone); IR (KBr): 3394, 1686, 1571
cm−1; UV (MeOH): 275 (sh), 261 nm; Mass (FAB+): 338
[M+H]+; HR-FAB MS: 338.1476 (mmu: +1.1; calcd for
1
C14H18N5O8P); H NMR (D2O) l: 1.21 (3H, d, J=6 Hz),
1.53 and 2.12 (each 1H, br t and br d, J=12 Hz), 3.69
(1H, m), 4.13 (2H, m), 4.34 (1H, m), 4.57 (1H, br), 4.89
(1H, m), 5.80 (1H, s, J=5 Hz), 6.18 (1H, br s), 7.71 (1H,
s); 13C NMR (D2O) l: 24.2, 38.2, 44.7, 71.5, 71.6, 75.5,
75.9, 81.4, 96.3, 120.0, 142.7, 154.4, 155.4, 161.8.
1
C14H20N5O5); H NMR (D2O) l: 1.16 (3H, d, J=6 Hz),
1.43 and 2.05 (each 1H, br t and br d, J=12 Hz), 2.34 and
2.55 (each 1H, each m), 3.63 (3H, m), 3.92 (1H, m), 4.44
(1H, m), 5.97 (1H, m), 6.03 and 6.07 (each 1/2H, each br
s), 7.72 (1H, s); 13C NMR (D2O) l: 20.6, 34.7, 39.4, 41.3,
62.5, 72.0 (2), 84.2, 87.8, 115.8, 137.9, 150.7, 151.7, 157.8.
7. (a) Eder, E.; Budiawan Cancer Epidemiol. Biomarkers
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