Identification of fluorescent 2′-deoxyadenosine adducts formed in reactions of conjugates of malonaldehyde and acetaldehyde, and of malonaldehyde and formaldehyde

Article abstract of DOI:10.1021/tx000155k

2′-Deoxyadenosine was reacted with malonaldehyde in the presence of formaldehyde or acetaldehyde. The reactions were carried out at 37 °C in aqueous solution at acidic conditions. The reaction mixtures were analyzed by HPLC. In both reactions, two major products were formed. The reaction products were isolated and purified by C18 chromatography, and their structures were characterized by UV absorbance, fluorescence emission, ¹H and ¹³C NMR spectroscopy, and mass spectrometry. The reaction products isolated from the mixture containing formaldehyde, malonaldchyde, and deoxyadenosine were identified as 3-(2′-deoxy-β-D-ribofuranosyl)-7H-8-formyl[2,1-i]pyrimidopurine (M₁FA-dA) and 9-(2′-deoxy-β-D-ribofuranosyl)-6-(3,5-diformyl-l,4-dihydro-1-pyrid yl)purine (M2FA-dA). In the reaction mixture consisting of acetaldehyde, malonaldehyde, and deoxyadenosine, the identities of the products were determined to be 3-(2′-deoxy-β-D-ribofuranosyl)-7-methyl-8-formyl[2,1-i]pyrimidopur ine (M₁-AA-dA) and 9-(2′-deoxy-β-D-ribofuranosyl)-6-(3,5-diformyl-4-methyl-1,4-dihydr o-1-pyridyl)purine (M₂AA-dA). The yields of the compounds were 1.8 and 0.7% for M₁FA-dA and M₂FA-dA, respectively, and 6.8 and 10% for M₁AA-dA and M₂AA-dA, respectively. All compounds exhibited marked fluorescent properties. These findings show that in addition to direct reaction of a specific aldehyde with 2′-deoxyadenosine, aldehyde conjugates also may react with the base. Although three of the adducts (M₁FA-dA, M₂FA-dA, and M₁AA-dA) could not be detected in reactions carried out under neutral conditions, the possibility that trace amounts of the adducts may be formed under physiological conditions cannot be ruled out. Therefore, conjugate adducts must be considered in work that aims at clarifying the mechanism of aldehyde genotoxicity.

Full text of DOI:10.1021/tx000155k

1
228
Chem. Res. Toxicol. 2000, 13, 1228-1234
Id en tifica tion of F lu or escen t 2′-Deoxya d en osin e Ad d u cts
F or m ed in Rea ction s of Con ju ga tes of Ma lon a ld eh yd e
a n d Aceta ld eh yd e, a n d of Ma lon a ld eh yd e a n d
F or m a ld eh yd e
†
‡
Frank Le Curieux, Donata Pluskota, Tony Munter, Rainer Sj o¨ holm, and
Leif Kronberg*
Department of Organic Chemistry, A° bo Akademi University, Biskopsgatan 8,
FIN-20500 Turku/ A° bo, Finland
Received J uly 18, 2000
2
′-Deoxyadenosine was reacted with malonaldehyde in the presence of formaldehyde or
acetaldehyde. The reactions were carried out at 37 °C in aqueous solution at acidic conditions.
The reaction mixtures were analyzed by HPLC. In both reactions, two major products were
formed. The reaction products were isolated and purified by C18 chromatography, and their
1
13
structures were characterized by UV absorbance, fluorescence emission, H and C NMR
spectroscopy, and mass spectrometry. The reaction products isolated from the mixture
containing formaldehyde, malonaldehyde, and deoxyadenosine were identified as 3-(2′-deoxy-
â-D-ribofuranosyl)-7H-8-formyl[2,1-i]pyrimidopurine (M
syl)-6-(3,5-diformyl-1,4-dihydro-1-pyridyl)purine (M FA-dA). In the reaction mixture consisting
of acetaldehyde, malonaldehyde, and deoxyadenosine, the identities of the products were
determined to be 3-(2′-deoxy-â-D-ribofuranosyl)-7-methyl-8-formyl[2,1-i]pyrimidopurine (M
AA-dA) and 9-(2′-deoxy-â-D-ribofuranosyl)-6-(3,5-diformyl-4-methyl-1,4-dihydro-1-pyridyl)purine
AA-dA). The yields of the compounds were 1.8 and 0.7% for M FA-dA and M FA-dA,
respectively, and 6.8 and 10% for M AA-dA and M AA-dA, respectively. All compounds exhibited
1
FA-dA) and 9-(2′-deoxy-â-D-ribofurano-
2
1
-
(M
2
1
2
1
2
marked fluorescent properties. These findings show that in addition to direct reaction of a
specific aldehyde with 2′-deoxyadenosine, aldehyde conjugates also may react with the base.
Although three of the adducts (M FA-dA, M FA-dA, and M AA-dA) could not be detected in
1 2 1
reactions carried out under neutral conditions, the possibility that trace amounts of the adducts
may be formed under physiological conditions cannot be ruled out. Therefore, conjugate adducts
must be considered in work that aims at clarifying the mechanism of aldehyde genotoxicity.
In tr od u ction
Aldehydes are ubiquitously distributed in the human
aldehyde was listed in group 2A, i.e., carcinogenic to
animals and probably carcinogenic to humans (24).
Acetaldehyde was classified in group 2B, i.e., carcinogenic
to animals and possibly carcinogenic to humans (23).
Chemical mutagens and carcinogens generally react with
DNA base units and form stable adducts. The formation
of exocyclic adducts in DNA is, along with the formation
of DNA-protein cross-links, one of the most probable
early types of damage in the carcinogenicity process.
Aldehydes such as malonaldehyde, acrolein, or crotonal-
dehyde are known to form stable DNA adducts (25, 30).
Formaldehyde was shown to produce stable adducts in
reaction with guanosine, adenosine, or cytidine (31, 32).
In reactions with nucleosides, acetaldehyde was shown
to produce unstable Schiff bases that can be stabilized
by reduction (33, 34). At present, the early events leading
to the carcinogenicity of malonaldehyde, formaldehyde,
and acetaldehyde in animals are not clearly identified.
Recently, it was reported that acetaldehyde forms a
stable fluorescent protein adduct in the liver of ethanol-
fed rats (35). The adduct was produced through the initial
condensation of one molecule of acetaldehyde with two
molecules of malonaldehyde. We have previously shown
that this malonaldehyde-aldehyde conjugate also reacts
with deoxyadenosine, forming a strongly fluorescent
dihydropyridine adduct (36).
environment. Malonaldehyde is present as a lipid me-
tabolite in human and animal tissues (1, 2) and can occur
at high levels in rancid foods. Formaldehyde is a potent
irritating and sensitizing compound widely used in
industry and can be released from several manufactured
items such as resins, building materials, paints, disin-
fectants, carpets, or personal care products (3, 4). Ac-
etaldehyde is used in the manufacturing of acetic acid,
perfumes, and flavors, but the main human exposure to
acetaldehyde is through alcohol consumption. Malonal-
dehyde is mutagenic in bacteria and mammalian cells
5-7). Formaldehyde and acetaldehyde induce genotoxic
effects in various biological assays (8-21).
Malonaldehyde has been shown to be carcinogenic in
rats (22), and consequently, the compound is classified
as possibly carcinogenic to animals (group 3) by the
International Agency for Research on Cancer (23). Form-
(
*
To whom correspondence should be addressed. E-mail:
leif.kronberg@abo.fi. Phone: +358-2-21 54 138. Fax: +358-2-21 54 866.
†
Present address: Department of Biosciences, Karolinska Institute,
Novum, 141 57 Huddinge, Sweden.
‡
Present address: Faculty of Chemistry, Adam Mickiewicz Uni-
versity, Grunwaldzka 6, 60-780 Poznan, Poland.
1
0.1021/tx000155k CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/23/2000

Malonaldehyde-Acetaldehyde/ Formaldehyde dA Adducts
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1229
In the current work, we have reacted 2′-deoxyadeno-
sine with malonaldehyde in the presence of acetaldehyde
and in the presence of formaldehyde. The aim of the
present study is twofold: to find out whether the mal-
onaldehyde-acetaldehyde condensation pattern is ob-
served when acetaldehyde is replaced with formaldehyde
and to determine whether other products are formed
following other condensation patterns.
F-2000 fluorescence spectrophotometer (Hitachi Ltd., Tokyo,
J apan).
P r ep a r a t ion of 3-(2′-Deoxy-â-D-r ib ofu r a n osyl)-7H -8-
for m yl[2,1-i]p yr im id op u r in e (M
â-D-r ibofu r a n osyl)-6-(3,5-d ifor m yl-1,4-d ih yd r o-1-p yr id yl)-
p u r in e (M F A-d A). Malonaldehyde hydrolyzed from TEP (4
1
F A-d A) a n d 9-(2′-Deoxy-
2
g, 18,2 mmol) and formaldehyde (2.04 g, 68 mmol) were reacted
with 2′-deoxyadenosine (1 g, 4 mmol) in 250 mL of 0.5 M
phosphate buffer solutions at pH 3.5. The reaction was per-
formed at 37 °C. M
1
FA-dA was isolated from a reaction stopped
after 24 h, and M FA-dA was isolated from a reaction stopped
2
Ma ter ia ls a n d Meth od s
after 3 days. The progress of the reaction was followed by HPLC
analyses on the C18 analytical column. After the reactions were
stopped, the reaction mixtures were filtered and finally passed
through the preparative C18 column. The column was eluted
with 200 mL of water and then with 100 mL of 5, 10, 15, 20,
Ch em ica ls. Malonaldehyde was prepared by acid hydrolysis
of 1,1,3,3-tetraethoxypropane (TEP) in 0.1 mL of HCl for 30 min
at 40 °C. 2′-Deoxyadenosine and 1,1,3,3-tetraethoxypropane
(
97% pure) were purchased from Sigma Chemical Co. (St. Louis,
2
5, 30, and 35% acetonitrile solutions in water. Fractions (30
mL) were collected. The M FA-dA eluted from the column with
the 5 to 15% acetonitrile washes, and the M FA-dA eluted with
MO). Formaldehyde (37% solution stabilized with 10-15%
methanol) and acetaldehyde (>99% pure) were obtained from
J . T. Baker B. V. (Deventer, Holland). The water was Aqua
Sterilisata from Pharmacia AB (Stockholm, Sweden). Acetoni-
trile was gradient grade for chromatography from Merck
1
2
the 25% wash. The fractions containing each product were
combined and concentrated by rotary evaporation to about 20
mL. Both compounds were further purified by using the semi-
preparative HPLC column with a flow rate of 4 mL/min; for M -
1
FA-dA the column was eluted with a gradient from 10 to 30%
acetonitrile in 0.01 M phosphate buffer (pH 7.1) over the course
(
Darmstadt, Germany). 1,1,1-Trichloroethane was obtained from
Fluka Chemie AG (Buchs, Switzerland).
Ch r om a togr a p h ic Meth od s. HPLC analyses were per-
formed on a Kontron Instruments liquid chromatographic
system consisting of a model 322 pump, a 440 diode array
detector (UV), a J asco FP-920 fluorescence detector, and the
KromaSystem 2000 data handling program (Kontron Instru-
ments S. P. A., Milan, Italy). The reaction mixtures were
chromatographed on a 5 µm, 4 mm × 125 mm reversed phase
C18 analytical column (Hypersil BDS-C18, Hewlett-Packard,
Espoo/Esbo, Finland). The column was eluted isocratically for
of 9 min and for M
acetonitrile in water over the course of 28 min. The collected
FA-dA fractions were desalted by use of the preparative C18
2
FA-dA with a gradient from 20 to 40%
M
1
column. The solutions containing the pure compounds were then
rotary evaporated to dryness, and the residues (a yellow-green
powder for M
dA) were subjected to spectroscopic and spectrometric studies.
The isolated amount of compound was 6 mg for M FA-dA and
mg for M FA-dA.
The isolated compounds had the following spectral charac-
teristics: UV spectrum for M FA-dA, UVmax 212, 240, 408 nm
ꢀ ) 23 300 M cm ), UVmin 284 nm (HPLC eluent, 10%
acetonitrile in 0.01 M phosphate buffer at pH 7.1); UV spectrum
1 2
FA-dA and a slightly yellow powder for M FA-
1
5
min with 0.01 M phosphate buffer (pH 7.1) and then with a
2
2
gradient from 0 to 30% acetonitrile over the course of 25 min at
a flow rate of 1 mL/min. Preparative isolation of the products
was performed by column chromatography on a 4 cm × 4 cm
column of preparative C18 bonded silica grade (40 µm, Bondesil,
Analytichem International, Harbor City, CA). The products were
further purified on a semipreparative 8 µm, 10 mm × 250 mm
1
-
1
-1
(
-
1
-1
for M
UVmin 273 nm (HPLC eluent, 23% acetonitrile in 0.01 M
phosphate buffer at pH 7.1); fluorescence spectrum (H O) for
FA-dA, λex,max 379 nm, λem,max 462 nm; fluorescence spectrum
O) for M FA-dA, λex,max 320 nm, λem,max 449 nm.
In the positive ion electrospray mass spectra, the following
ions were observed (m/z, relative abundance, formation): M
2
FA-dA, UVmax 320 nm (ꢀ ) 20 200 M cm ), 224 nm,
(
Hyperprep ODS, Hypersil, Krotek, Tampere/Tammerfors, Fin-
2
land) reversed phase C18 column. The column was coupled to
a Shimadzu HPLC system, which consisted of two Shimadzu
LC-9A pumps and a variable-wavelength Shimadzu SPD-6A UV
spectrophotometric detector (Shimadzu Europe).
M
1
(H
2
2
1
-
1
Sp ectr oscop ic a n d Sp ectr om etr ic Meth od s. The H and
+
+
FA-dA 318 (35, MH ), 202 (100, MH - deoxyribosyl + H), high-
resolution mass spectrometry gave the protonated molecular
(MH 318.1179, calcd 318.1202); M
dA 372 (82%, MH ), 256 (76%, MH - deoxyribosyl + H ), high-
resolution mass spectrometry gave the protonated molecular
1
3
C NMR spectra were recorded at 30 °C on a J EOL J NM-A500
Fourier transform NMR spectrometer at 500 and 125 MHz,
respectively (J EOL). The samples were dissolved in Me SO-d
+
formula as C14
H
16
+
N
5
O
4
2
FA-
2
6
,
+
+
1
and TMS was used as an internal standard. The H NMR signal
assignments were based on chemical shifts and H-H and C-H
correlation data. The determinations of the shifts and the
coupling constants of the multiplets of the proton signals in the
deoxyribose unit were based on a first-order approach and are
given with an accuracy of (0.3 Hz. Assignment of carbon signals
was based on chemical shifts, C-H correlations, and carbon-
proton couplings.
+
formula as C17
H
18
N
5
O
5
(MH 372.1290, calcd 372.1307).
1
The H NMR spectroscopic data of M
1
FA-dA were as follows:
δ 9.28 (t, 1 H, CHO, J ) 0.4 Hz), 8.49 (m, 1 H, H-2), 8.30 (m, 1
H, H-5), 7.50 (t, 1 H, H-9, J ) 0.6 Hz), 5.00, 5.01 (m, 2 H, H-7a,
H-7b, J ) 14.4 Hz), 6.32 (dd, 1 H, H-1′, J ) 7.0, 6.5 Hz), 4.40
(
4
ddd, 1 H, H-3′, J ) 5.9, 3.7, 3.1 Hz), 3.87 (dt, 1 H, H-4′, J )
.6, 3.1 Hz), 3.59 (dd, 1 H, H-5′, J ) 11.8, 4.6 Hz), 3.52 (dd, 1 H,
The electrospray ionization mass spectra were recorded on a
Fisons ZabSpec-oaTOF instrument (Manchester, U.K.). Ioniza-
tion was carried out using nitrogen as both the nebulizing and
bath gas. A potential of 8.0 kV was applied to the ESI needle.
The temperature of the pepperpot counter electrode was 90 °C.
The isolated compound was introduced by loop injection at a
H-5′′, J ) 11.8, 4.6 Hz), 2.64 (ddd, 1 H, H-2′, J ) 13.3, 7.2, 6.1
Hz), 2.33 (ddd, 1 H, H-2′′, J ) 13.3, 6.3, 3.5 Hz). The NMR data
for M
al. (37).
The 1H and 13C NMR spectroscopic data of M
1
FA-dA were described in detail in the work of Munter et
2
FA-dA are
presented in Table 1.
2 3
flow rate of 20 µL/min (80/20/1 H O/CH CN/acetic acid mixture).
1
PEG 200 was used as the standard for the exact mass
determination. The mass spectrometer was working at a resolu-
tion of 7000.
1
Abbreviations:
M
1
FA-dA, 3-(2′-deoxy-â-D-ribofuranosyl)-7H-8-
AA-dA, 3-(2′-deoxy-â-D-ribofuranosyl)-
-methyl-8-formyl[2,1-i]pyrimidopurine; M FA-dA, 9-(2′-deoxyribosyl)-
6-(3,5-diformyl-1,4-dihydro-1-pyridyl)purine; M AA-dA, 9-(2′-deoxyribo-
formyl[2,1-i]pyrimidopurines; M
1
7
2
The UV spectra of the isolated compounds were recorded with
the diode array detector as the peak eluted from the HPLC
column. A Shimadzu UV-160 spectrophotometer (Shimadzu
Europa) was used to determine the molar extinction coefficient
2
syl)-6-(3,5-diformyl-4-methyl-1,4-dihydro-1-pyridyl)purine; COLOC, C-H
shift correlation NMR spectroscopy via long-range coupling; COSY,
correlation spectroscopy (H-H); CHSHF, C-H shift correlation NMR
spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Over-
hauser enhancement spectroscopy; PEG, poly(ethylene glycol).
(ꢀ). The fluorescence spectra were recorded with a Hitachi

1
230 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Le Curieux et al.
Ta ble 1. H a n d 13C Ch em ica l Sh ifts (δ),a Sp in -Sp in
Cou p lin g Con sta n ts, a n d J H,H Va lu es of P r oton s in
M2F A-d A
1
0.01 M phosphate buffer at pH 7.1 over the course of 15 min at
a flow rate of 4 mL/min. The fractions containing each pure
compound were combined and then desalted by use of the
preparative C18 column. The desalted solutions were rotary
evaporated to dryness. The residues (a yellow-green powder for
δ
multi-
(ppm) plicity
J H,H
(Hz)
δ
proton
carbon (ppm)
M
1
AA-dA and a slightly yellow powder for M
2
AA-dA) were
1
,4-dihydropyridine unit
H-2/H-6 (2H) 9.10
subjected to spectroscopic and spectrometric studies. The iso-
s
C-2/C-6 141.8
C-3/C-5 121.2
lated amounts of M
respectively.
1 2
AA-dA and M AA-dA were 37 and 52 mg,
CHO (2H)
9.63
3.03
s
br
CHO
C-4
190.9
17.3
H-4 (2H)
The isolated compounds had the following spectral charac-
teristics: UV spectrum for M AA-dA, UVmax 208, 240, 400 nm
ꢀ ) 20 900 M cm ), UVmin 232, 288 nm (HPLC eluent, 10%
acetonitrile in 0.01 M phosphate buffer at pH 7.1); UV spectrum
purine unit
H-2p (1H)
1
8.78
s
s
C-2p
C-4p
C-5p
C-6p
C-8p
151.2
152.4
121.4
146.2
143.5
-1
-1
(
-1
-1
for M AA-dA, UVmax 320 nm (ꢀ ) 46 500 M cm ), 224 nm,
2
UVmin 272, 208 nm, shoulder between 348 and 378 nm (HPLC
eluent, 25% acetonitrile in 0.01 M phosphate buffer at pH 7.1);
H-8p (1H)
′-deoxyribosyl unit
8.89
2
fluorescence spectrum (H
56 nm; and fluorescence spectrum (H
319 nm, λem,max 444 nm.
In the positive ion electrospray mass spectra, the following
ions were observed (m/z, relative abundance, formation): M
2
O) for M
1
AA-dA, λex,max 408 nm, λem,max
H-1′ (1H)
H-2′ (1H)
H-2′′ (1H)
H-3′ (1H)
H-4′ (1H)
H-5′ (1H)
H-5′′ (1H)
OH (1H)
OH (1H)
6.52
2.76
2.40
4.46
3.92
3.64
3.56
5.38
5.01
t
6.6
13.4, 6.3
13.4, 6.3, 3.7
C-1′
C-2′
84.0
39.5
4
2 2
O) for M
AA-dA λex,max
dt
ddd
m
dt
m
m
br
br
C-3′
C-4′
C-5′
70.4
88.1
61.3
4.3, 3.7
1
-
+
+
AA-dA 332 (47, MH ), 216 (100, MH - deoxyribosyl + H), high-
resolution mass spectrometry gave the protonated molecular
+
formula as C15
H
18
+
N
5
O
4
(MH 332.1326, calcd 332.1359); M
2
AA-
+
+
dA 386 (100, MH ), 270 (54, MH - deoxyribosyl + H ), high-
resolution mass spectrometry gave the protonated molecular
a
Relative to TMS.
+
formula as C18
H
20
N
5
O
5
(MH 386.1460, calcd 386.1464).
P r ep a r a tion of 3-(2′-Deoxy-â-D-r ibofu r a n osyl)-7-m eth yl-
-for m yl[2,1-i]p yr im id op u r in e (M AA-d A) a n d 9-(2′-Deoxy-
â-D-r ibofu r a n osyl)-6-(3,5-d ifor m yl-4-m eth yl-1,4-d ih yd r o-1-
p yr id yl)p u r in e (M AA-d A). Malonaldehyde hydrolyzed from
TEP (4 g, 18,2 mmol) and acetaldehyde (0.8 g, 18.2 mmol) were
reacted with 2′-deoxyadenosine (1 g, 4 mmol) in 250 mL of 0.5
M phosphate buffer solutions at pH 4.6. The reaction was carried
out at 37 °C, and the progress of the reaction was followed by
HPLC analyses on the C18 analytical column. After 7 days, the
reaction was stopped and the reaction mixture was filtered and
finally passed through the preparative C18 column. The column
was eluted with 200 mL of water and then with 100 mL of 5,
0, 15, 20, 25, 30, and 35% acetonitrile solutions in water.
Fractions (30 mL) were collected. The M AA-dA eluted from the
column with the 10 and 15% acetonitrile washes, and the M
AA-dA eluted with the 25 and 30% washes. The fractions
containing the product were combined and concentrated by
rotary evaporation to about 20 mL. Further purification was
carried out by using the semipreparative HPLC column as
The 1H NMR spectroscopic data of M
AA-dA were as fol-
2
8
1
lows: δ 9.59 (s, 2 H, CHO), 9.06 (s, 2 H, H-2/H-6), 8.89 (s, 1 H,
H-8p), 8.78 (s, 1 H, H-2p), 3.70 (q, 1 H, H-4, J ) 6.7 Hz), 1.08
2
(
(
d, 3 H, CH
3
, J ) 6.7 Hz), 6.51 (t, 1 H, H-1′, J ) 6.5 Hz), 4.46
dt, 1 H, H-3′, J ) 6.5, 4.0 Hz), 3.92 (dt, 1 H, H-4′, J ) 4.5, 4.0
Hz), 3.64 (dd, 1 H, H-5′, J ) 11.5, 4.5 Hz), 3.56 (dd, 1 H, H-5′′,
J ) 11.5, 4.5 Hz), 2.75 (dt, 1 H, H-2′, J ) 13.5, 6.5 Hz), 2.40
(
(
ddd, 1 H, H-2′′, J ) 13.5, 6.5, 4.0 Hz), 5.37 (br, 1 H, OH), 5.02
br, 1 H, OH). The detailed NMR data for M AA-dA were
2
reported in a previous study (36).
The 1H and 13C NMR spectroscopic data of M
presented in Table 2.
AA-dA are
1
1
1
Sm a ll-Sca le Rea ction s. 2′-Deoxyadenosine (10 mg, 0.04
mmol) and malonaldehyde hydrolyzed from TEP (40 mg, 0.18
mmol) were mixed with formaldehyde (20 mg, 0.67 mmol) or
with acetaldehyde (8 mg, 0.18 mmol) in 3 mL of 0.5 M phosphate
buffer at pH 3.5 (with formaldehyde) or 4.6 (with acetaldehyde).
The appropriate aldehyde (formaldehyde or acetaldehyde) was
added to malonaldehyde, and this mixture was then added to
the solution of 2′-deoxyadenosine. The reactions were performed
at 37 °C, and the progress of the reaction was followed daily by
HPLC analyses of aliquots of the reaction mixtures.
2
-
follows: for M
from 11 to 35% acetonitrile in 0.01 M phosphate buffer (pH 7.1)
over the course of 10 min at a flow rate of 4 mL/min; for M
1
AA-dA, the column was eluted with a gradient
2
-
AA-dA, the elution gradient was from 17 to 30% acetonitrile in
Ta ble 2. H a n d 13C Ch em ica l Sh ifts (δ),a Sp in -Sp in Cou p lin g Con sta n ts, a n d J H,H Va lu es of P r oton s in M1AA-d A
1
proton
H-2 (1H)
δ
multiplicity
s
J H,H (Hz)
carbonb
δ
multiplicity
1J C,H (Hz)
>1J C,H (Hz)
8.50
C-2
C-3a
C-5
C-7
C-8
141.5
145.9
147.3
50.9
119.1
154.6
147.2
124.0
187.4
23.8
dd
dddd
dd
dm
dm
dtd
m
dd
dddd
qd
215.8
4.1
13.3, 7.7, 5.0, 2.1
3.2
H-5 (1H)
H-7 (1H)
8.54
5.54
s
dq
211.7
148.0
6.4, 1.7
0.8
24.3
3.4, 1.4
H-9 (1H)
7.53
d
C-9
176.9
C-10a
C-10b
CHO
CH3
11.5, 4.6
5.5, 5.3, 1.9
2.3
CHO (1H)
CH3 (3H)
9.27
1.30
s
dd
171.8
128.5
6.5, 2.8
2
′-deoxyribosyl unit
H-1′ (1H)
H-2′ (1H)
H-2′′ (1H)
H-3′ (1H)
H-4′ (1H)
H-5′ (1H)
OH
6.32
2.65
2.33
4.40
3.87
3.60
4.98
5.34
dt
ddd
6.6, 1.6
6.8, 6.5, 6.3
C-1′
C-2′
83.7
39.7
d
t
166.8
134.3
ddd
m
m
br
br
3.3, 3.1, 2.7
C-3′
C-4′
C-5′
70.5
88.0
61.4
d
d
td
148.4
148.0
140.2
OH
a
Relative to TMS. b The signals of the sugar carbons were further split into complex multiplets.

Malonaldehyde-Acetaldehyde/ Formaldehyde dA Adducts
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1231
1
Deter m in a tion of P r od u ct Yield s. Quantitative H NMR
analysis, using 1,1,1-trichloroethane as an internal standard,
was performed on an aliquot of M FA-dA and M AA-dA. Then
2 1
a HPLC standard solution was prepared by taking an exact
volume of the NMR sample and diluting it with an appropriate
volume of water. The quantitative determination of the amount
2 1
of M FA-dA and M AA-dA in the reaction mixtures was made
by comparing the chromatographic peak area (recorded at the
wavelength corresponding to the maximum UV absorbance) of
the compound in the HPLC standard solution with the peak
area of the compound in the reaction mixtures. The molar yield
was calculated from the original amount of 2′-deoxyadenosine
in the reaction mixtures. Quantitative NMR was not performed
for M FA-dA and M AA-dA because their extinction coefficients
1 2
were determined in previous studies (refs 37 and 36, respec-
tively).
Resu lts a n d Discu ssion
HPLC analyses of the reaction mixture that contained
formaldehyde, malonaldehyde, and 2′-deoxyadenosine
(
formaldehyde reaction) showed the formation of three
major product peaks. The product peaks are marked M
FA-Ade, M FA-dA, and M FA-dA, respectively, in Figure
A. Also in the HPLC chromatogram of the reaction
1
-
1
2
1
mixture containing acetaldehyde, malonaldehyde, and 2′-
deoxyadenosine (acetaldehyde reaction), three major
product peaks were observed. These product peaks are
1 1 2
marked M AA-Ade, M AA-dA, and M AA-dA, respec-
tively, in Figure 1B. Large-scale reactions were per-
formed to isolate sufficient amounts of the products for
structural determination by spectrometric and spectro-
scopic methods.
On the basis of UV, fluorescence and NMR spectros-
copy, and electrospray mass spectrometry, the products
of the formaldehyde reaction were identified as 8-formyl-
[
2,1-i]-7H-pyrimidopurine (M
ribofuranosyl)-7H-8-formyl[2,1-i]pyrimidopurine (M
dA), and 9-(2′-deoxyribosyl)-6-(3,5-diformyl-1,4-dihydro-
1
FA-Ade), 3-(2′-deoxy-â-D-
1
FA-
1
-pyridyl)purine (M
The compound M
2
FA-dA).
FA-Ade exhibited UV and fluores-
FA-
1
cence spectra identical to those of the compound M
1
dA. In the ESI mass spectrum, the compound generated
a molecular ion at 202 m/z, corresponding to the aglycone
of M
hydrolyzed M
analyzed by HPLC, the only peak that was detected was
1
FA-dA. Further, when a solution of M
1
FA-Ade and
1
FA-dA (30 min at 40 °C in 0.1 M HCl) was
F igu r e 1. C18 analytical column HPLC chromatogram of the
reaction mixture of 2′-deoxyadenosine with malonaldehyde and
formaldehyde (pH 3.5, 3 days) (A) or acetaldehyde (B) (pH 4.5,
the one representing M
The UV, fluorescence, H NMR, and mass spectra of
1
FA-Ade.
7
days) held at 37 °C. For analysis conditions, see Materials
1
and Methods.
1
M FA-dA were identical to those recorded for the com-
pound previously reported to be formed in reaction of the
hydroxyfuranones 3-chloro-4-(dichloromethyl)-5-hydroxy-
at δ 8.89 and 8.78 ppm and two-proton singlets at δ 9.63,
9
.10, and 3.03 ppm (Table 1). This set of signals is almost
2
(5H)-furanone (MX) or 3-chloro-4-(chloromethyl)-5-hy-
droxy-2(5H)-furanone (CMCF), and named pfA-dR (37,
8). Thus, the compound M FA-dA is identical to the
1
identical to the ones observed in the H NMR spectrum
of M AA-dA (36). The only marked differences are that
FA-dA displays no signal at δ 1.08 ppm (signal which
had been assigned to the methyl protons in M AA-dA;
2
3
1
M
2
compound formed by the hydroxyfuranones. However, the
adduct is not produced in the formaldehyde reaction by
the same mechanism as in the reaction of the hydroxy-
furanones (see the later discussion).
2
see Scheme 1) and that the signal at δ 3.03 ppm is now
a two-proton singlet (whereas the corresponding signal
was a one-proton quartet in M
2
AA-dA). In analogy with
The compound M
2
FA-dA exhibited a UV absorption
the NMR data of M AA-dA, the signals at δ 8.89 and 8.78
2
maximum at 320 nm (Figure 2) and displayed a fluores-
cence spectrum with an emission maximum at 449 nm
ppm were assigned to H-8p and H-2p, respectively, and
the signals at δ 9.63 and 9.10 ppm were attributed to
the aldehyde proton and H-2/H-6 (equivalent protons),
respectively.
(
Figure 3). These features are very similar to those of
the compound M AA-dA formed in the acetaldehyde
reaction and characterized previously (36). The H NMR
spectrum of M FA-dA displayed, besides the signals from
the protons of the deoxyribose moiety, one-proton singlets
2
1
The 13C NMR spectrum of M
2
FA-dA showed, besides
2
the signals from the purine and the deoxyribose moieties,
carbon signals at δ 190.9, 141.8, 121.2, and 17.3 ppm

1
232 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Le Curieux et al.
Sch em e 1
trometric, and chromatographic characteristics as M
Ade. Collectively, these findings show that M AA-Ade
represents the aglycone of M AA-dA.
The compound M AA-dA and the compound M
from the formaldehyde reaction exhibited very similar
UV spectra. The UV absorption maximum of M AA-dA
was found at 400 nm (Figure 2), a slightly shorter
wavelength (8 nm) than that for M FA-dA (37, 38). The
fluorescence spectrum of M AA-dA exhibited an emission
maximum at 456 nm (Figure 3), while the emission
1
AA-
1
1
1
1
FA-dA
F igu r e 2. UV absorbance spectra of M
The UV spectra were recorded with the diode array detector as
the compounds eluted from the column.
2
FA-dA and M
1
AA-dA.
1
1
1
1
maximum was 462 nm for M
1
FA-dA (37, 38). The H
NMR spectrum of M AA-dA exhibited, besides the signals
1
from the protons of the deoxyribose moiety, one-proton
signals at δ 9.27, 8.54, 8.50, 7.53, and 5.54 ppm and a
three-proton signal at δ 1.30 ppm (Table 2). The main
difference in the resonance signals of M
AA-dA is that M FA-dA displayed a two-proton signal at
δ 5.005 ppm, whereas M AA-dA exhibited a one-proton
1 1
FA-dA and M -
1
1
signal at δ 5.54 ppm and a three-proton signal at δ 1.30
ppm. The signal at δ 1.30 ppm was assigned to three
methyl protons on the basis of the chemical shift and of
the C-H connectivity to the carbon signal at δ 23.8 ppm.
The signal at δ 5.54 ppm was attributed to the methine
proton, H-7 (Scheme 1). The proton signal appeared as a
quartet and showed a H-H (COSY) correlation with the
methyl protons at δ 1.30 ppm. The assignment of the
signal at δ 8.54 ppm to H-5 was done on the basis of the
chemical shift and the NOE interaction between H-7 and
H-5 (two-dimensional NOESY experiment). On the basis
F igu r e 3. Fluorescence emission spectra of M
20 nm) and M AA-dA (λex ) 408 nm).
2
FA-dA (λex )
3
1
(Table 1). In analogy with the NMR spectra published
1
for M AA-dA (36), the signal at δ 190.9 ppm was assigned
2
of chemical shifts and in analogy with the H NMR data
to the formyl carbons and the signals at δ 141.8 and 121.2
ppm were assigned to C-2/C-6 and C-3/C-5, respectively.
The signal at δ 17.3 ppm was assigned to the methylene
carbon, C-4. The NMR, UV and fluorescence spectro-
scopic, and the mass spectrometric data were consistent
1
of M FA-dA, we assigned the signal at δ 7.53 ppm to H-9.
The signal at δ 9.27 ppm was attributed to the aldehyde
proton on the basis of the downfield chemical shift and
the one-bond C-H shift correlation with the carbon
signal at δ 187.4 ppm. A two-dimensional NOESY
experiment confirmed NOE interactions between the
aldehyde proton and H-9 and between the methyl protons
and H-5. The signal at δ 8.50 ppm was then assigned to
H-2, and it exhibited NOE interactions with protons H-1′,
H-2′, and H-3′ in the deoxyribose moiety. The signal H-9
appeared as a doublet because of the coupling (J ) 0.8
Hz) to the aldehyde proton (this coupling is also observed
in the H-H correlation spectrum), and H-7 was split into
a doublet and a quartet due to the couplings to H-5 and
to the methyl protons, respectively. The methyl protons
gave rise to a doublet of doublets because of their coupling
to H-7 and H-5.
with the structure of M
In the acetaldehyde reaction, the products were identi-
fied as 8-formyl-7-methyl[2,1-i]pyrimidopurine (M AA-
Ade), 3-(2′-deoxy-â-D-ribofuranosyl)-7-methyl-8-formyl-
2,1-i]pyrimidopurine (M AA-dA), and 9-(2′-deoxyribosyl)-
-(3,5-diformyl-4-methyl-1,4-dihydro-1-pyridyl)purine
AA-dA). The compounds M AA-Ade and M AA-dA
2
FA-dA presented in Scheme 1.
1
[
1
6
(M
2
1
1
exhibited identical UV and fluorescence spectra. The
mass unit difference of the molecular ions was 116 units,
which corresponds to the loss of the deoxyribose unit.
Finally, it was found that hydrolysis of M
1
AA-dA yielded
a compound with exactly the same spectroscopic, spec-

Malonaldehyde-Acetaldehyde/ Formaldehyde dA Adducts
The 13C NMR spectrum of M
AA-dA exhibited, besides
the signals from the purine and the deoxyribose moieties,
carbon signals at δ 187.4, 154.6, 147.3, 147.2, 145.9,
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1233
1
carbons in the intermediates, and finally, the substituted
propeno adducts are obtained by loss of water and a
proton. The fluorescence intensities of the propeno ad-
ducts are much weaker than those of the dihydropyridine
adducts.
1
41.5, 124.0, 119.1, 50.9, and 23.8 ppm (Table 2). This
1
3
set of signals is quite similar to the C NMR data from
FA-dA except that it displays one additional carbon.
M
1
The mechanism we suggest for the formation of M
1
-
The additional carbon signal, observed at δ 23.8 ppm,
was attributed to the methyl carbon on the basis of the
upfield chemical shift and the C-H correlation with the
methyl protons (CHSHF experiments). The CHSHF
correlation data allowed the assignment of the signals
at δ 154.6, 147.3, and 141.5 ppm to the three olefinic
methine carbons (C-9, C-5, and C-2, respectively). The
signal at δ 50.9 ppm was attributed to the methine
carbon C-7, and the signal at δ 187.4 ppm was assigned
to the aldehyde carbon. The signal at δ 147.2 ppm was
attributed to C-10a on the basis of chemical shifts and
of C-H connectivities (COLOC) with the signals of H-9
and H-5. The assignment of the signal at δ 145.9 ppm to
C-3a was based on the chemical shift and on the C-H
correlation between C-3a and H-5. Further, C-H cor-
relation data (COLOC experiment) displayed correlations
between the aldehyde carbon and H-9, between C-10a
and H-2, and between C-8 and H-9, the methyl protons,
and the aldehyde protons and between C-7 and H-9, the
methyl protons, and the aldehyde protons. The NMR, UV
and fluorescence spectroscopic, and mass spectrometric
AA-dA, M FA-dA, and M AA-dA implies that the conju-
1
2
gates are formed in the first step and in the second step
the conjugates react with adenosine. However, a mech-
anism that is based on sequential addition of the alde-
hydes to amino groups has been suggested in the
literature (42, 43). At present, there is no conclusive data
in favor of either mechanism.
Malonaldehyde is the main byproduct of lipid peroxi-
dation and is therefore ubiquitous in human tissues.
Acetaldehyde and formaldehyde occur in human tissues
following alcohol intake and occupational or environmen-
tal exposures. Malonaldehyde and acetaldehyde or form-
aldehyde may thus be present at the same time in the
same biological tissues, and adduct formation with DNA
is possible. However, since the deoxyadenosine adducts
were mainly formed under slightly acidic conditions, it
is not known to what extent the adducts are formed in
biological fluids.
In conclusion, the current study has shown that
malonaldehyde in the presence of acetaldehyde or form-
aldehyde reacts with 2′-deoxyadenosine to produce stable
fluorescent adducts. We have identified and determined
data were consistent with the structure of M
presented in Scheme 1.
1
AA-dA
the structure of two novel adducts:
produced in the reaction of 2′-deoxyadenosine with ma-
lonaldehyde and acetaldehyde, and M FA-dA was formed
1
M AA-dA was
The work on the structural characterization of M
2
AA-
dA has been presented in a previous paper from our
laboratory (36).
2
in the reaction of 2′-deoxyadenosine with malonaldehyde
The yields of the adducts from the formaldehyde
and formaldehyde. In addition, we found that the adduct
reaction were found to be 1.8 and 0.7% for M
FA-dA, respectively, when reacted at pH 3.5. The
yields of the adducts derived from acetaldehyde were 6.8
and 10% for M AA-dA and M AA-dA, respectively, in
reactions performed at pH 4.6. The yields decreased
markedly under higher-pH conditions, and with the
exception of M AA-dA (36), the compounds could not be
2
detected by the UV detector in reactions performed under
neutral conditions.
1
FA-dA and
1
M FA-dA, previously identified in the reaction of 2′-
M
2
deoxyadenosine with the chlorohydroxyfuranone MX or
CMCF (37, 38), is also one of the main products in the
reaction of the base with malonaldehyde and formalde-
hyde. The marked fluorescent properties of the adducts
may have interesting biological or pharmaceutical ap-
plications. The reactions described in the present paper
must be taken into account in works that aim at
elucidating the mechanism behind the genotoxicity of
aldehydes.
1
2
G o´ mez-S a´ nchez et al. described the formation of the
2
/1 conjugate (Scheme 1) from malonaldehyde and ac-
etaldehyde and found that the conjugate readily reacts
with amines (39). We previously reported that the 2/1
conjugate forms with deoxyadenosine a strongly fluores-
Ack n ow led gm en t. We are very grateful to Mr.
Markku Reunanen for the mass spectra. This work was
financially supported by the European Commission (Con-
tract ERBFMBICT961394, F.L.C.), by the Foundation of
J enny and Antti Wihurin in Helsinki (D.P.), and by the
Graduate School of Bio-Organic Chemistry at A° bo Aka-
demi University (T.M.).
2
cent methyl-substituted dihydropyridine adduct (M AA-
dA, Scheme 1) (36). Moreover, it was shown that the
analogous dihydropyridine protein adduct is formed in
the presence of malonaldehyde and acetaldehyde (35, 40).
In the current work, we demonstrate that when acetal-
dehyde is replaced with formaldehyde the unsubstituted
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TX000155K