Identification of an ethenoformyl adduct formed in the reaction of the potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone with guanosine

Article abstract of DOI:10.1021/tx9801514

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting bacterial mutagen and a rodent carcinogen occurring in chlorine-disinfected drinking water. In this study, we have reacted MX with guanosine, cytidine, thymidine, and calf thymus DNA in aqueous solutions, HPLC analyses of the reaction mixture of MX with guanosine showed that one main product peak was formed. In the reactions of MX with cytidine or thymidine, no product peaks representing base-modified nucleosides could be observed. The product from the MX guanosine reaction mixture was isolated by preparative chromatography on reversed phase C18 columns, and its structure was determined by UV absorbance, ¹H and ¹³C NMR spectroscopy, and mass spectrometry. The product was identified as 3-(β-D-ribofuranosyl)-7- formylimidazo[1,2-a]purin-9(4H)-one (εfGuo), and the yield for the reaction carried out at pH 7.4 and 37 °C was about 0.3 mol %. The adduct could not be observed at the detection limit of five adducts per 10⁷ bases in the hydrolysate of the calf thymus DNA reacted with MX. However, this failure does not rule out the possibility that lower amounts of the adduct might be involved in the observed mispairing (adenine incorporated opposite an adducted guanine base) caused by MX in the Salmonella typhimurium strain TA100.

Full text of DOI:10.1021/tx9801514

46
Chem. Res. Toxicol. 1999, 12, 46-52
Id en tifica tion of a n Eth en ofor m yl Ad d u ct F or m ed in th e
Rea ction of th e P oten t Ba cter ia l Mu ta gen
3-Ch lor o-4-(d ich lor om eth yl)-5-h yd r oxy-2(5H)-fu r a n on e
w ith Gu a n osin e
Tony Munter, Frank Le Curieux, Rainer Sjo¨holm, and Leif Kronberg*
Department of Organic Chemistry, Åbo Akademi University, Biskopsgatan 8,
FIN-20500 Turku/ Åbo, Finland
Received J une 22, 1998
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting bacterial
mutagen and a rodent carcinogen occurring in chlorine-disinfected drinking water. In this study,
we have reacted MX with guanosine, cytidine, thymidine, and calf thymus DNA in aqueous
solutions. HPLC analyses of the reaction mixture of MX with guanosine showed that one main
product peak was formed. In the reactions of MX with cytidine or thymidine, no product peaks
representing base-modified nucleosides could be observed. The product from the MX guanosine
reaction mixture was isolated by preparative chromatography on reversed phase C18 columns,
1
and its structure was determined by UV absorbance, H and13C NMR spectroscopy, and mass
spectrometry. The product was identified as 3-(â-D-ribofuranosyl)-7-formylimidazo[1,2-a]purin-
9(4H)-one (ꢀfGuo), and the yield for the reaction carried out at pH 7.4 and 37 °C was about 0.3
mol %. The adduct could not be observed at the detection limit of five adducts per 107 bases in
the hydrolysate of the calf thymus DNA reacted with MX. However, this failure does not rule
out the possibility that lower amounts of the adduct might be involved in the observed
mispairing (adenine incorporated opposite an adducted guanine base) caused by MX in the
Salmonella typhimurium strain TA100.
Ch a r t 1
In tr od u ction
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furan-
one (MX)1 (Chart 1) is one of the most potent direct-acting
mutagens ever tested in the Ames mutagenicity assay
(1-3). MX is formed during chlorine disinfection of
drinking water as a consequence of the reaction of
chlorine with natural humic substances in the raw water
(4-6). MX is present in chlorinated drinking waters at
very low concentrations (a few nanograms per liter to 60
ng/L), but due to its extreme mutagenicity, it has been
estimated to account for a large part (30-50%) of the
Ames mutagenicity in drinking water (7-13). Besides the
mutagenicity in the Ames assay, MX has also been
reported to cause damage to the genetic material in
several in vitro and in vivo assays (14-21). In a very
recent work by Komulainen et al. (22), it was found that
MX is a potent multisite carcinogen in rats.
MX occurs mainly in its cyclic furanone form in acidic
water solutions, but undergoes a tautomeric transition
to the dissociated acyclic form in the pH range of 4-6
(Chart 1). Under neutral conditions, MX exists primarily
in the open acyclic form (3). In water solutions, MX
undergoes isomerization to its geometric isomer, (E)-2-
chloro-3-(dichloromethyl)-4-oxobutenoic acid (E-MX) (23).
Many studies have shown that reactive electrophilic
compounds react with the base units of DNA and form
covalent adducts. The base modifications can lead to gene
mutations during the replication of DNA and contribute
to the initiation of cancer (24, 25). Previous work in our
laboratory has shown that mucochloric acid (MCA) and
3-chloro-4-methyl-5-hydroxy-2-(5H)-furanone (MCF), chlo-
rinated hydroxyfuranones with genotoxic properties and
structures similar to those of MX, react with nucleosides
and DNA and form adducts (26-31). Recently, we
* Author to whom correspondence should be addressed. E-mail:
leif.kronberg@abo.fi. Phone: +358-2-2154138. Fax: +358-2-2154866.
1
Abbreviations: MX, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-
furanone; E-MX, (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid;
CMCF, 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone; MCF,
3-chloro-4-methyl-5-hydroxy-2(5H)-furanone; MCA, 3,4-dichloro-5-hy-
droxy-2(5H)-furanone, mucochloric acid; BMA, bromomalonaldehyde;
ꢀfGuo, 3-(â-D-ribofuranosyl)-7-formylimidazo[1,2-a]purin-9(4H)-one; ꢀfd-
Guo, 3-(2′-deoxy-â-D-ribofuranosyl)-7-formylimidazo[1,2-a]purin-9(4H)-
one; ꢀfG, 7-formylimidazo[1,2-a]purin-9(4H)-one; 5-Me-ꢀfGuo, 3-(â-D-
ribofuranosyl)-7-formyl-5-methylimidazo[1,2-a]purin-9-one; ꢀGuo, 3-(â-
D-ribofuranosyl)imidazo[1,2-a]purin-9(5H)-one (1,N2-ethenoguanosine);
DMF, N,N-dimethylformamide; PEG, poly(ethylene glycol); NOE,
nuclear Overhauser effect; COSY, correlation spectroscopy (H-H);
COLOC, C-H shift correlation NMR spectroscopy via long-range
coupling.
10.1021/tx9801514 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/18/1998

MX Forms an Ethenoformyl Adduct with Guanosine
Chem. Res. Toxicol., Vol. 12, No. 1, 1999 47
approach and are given with an accuracy of (0.3 Hz. Assign-
ment of carbon signals was based on chemical shifts, C-H
correlations, and carbon-proton couplings.
reported that MX reacts with 2′-deoxyadenosine and
DNA, and forms propenal and fluorescent propenoformyl
derivatives (31, 32).
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 samples were introduced by loop injection at a flow rate of
20 µL/min (80/20/1 H2O/CH3CN/acetic acid). PEG 200 was used
as standard for the exact mass determinations. The mass
spectrometer had a resolution of 7000.
The UV spectra of the compounds were recorded with the
diode-array detector as the peaks eluted from the HPLC column.
A Shimadzu UV-160 spectrophotometer was used for the
determination of the molar extinction coefficient (ꢀ).
P r ep a r a tion of 3-(â-D-Ribofu r a n osyl)-7-for m ylim id a zo-
[1,2-a ]p u r in -9(4H)-on e (EfGu o). MX (1 g, 4.6 mmol) was
reacted with guanosine (651 mg, 2.3 mmol) in 400 mL of 0.5 M
phosphate buffer solution (pH 8.2). The pH of the reaction
mixture was checked daily and readjusted with 1 M NaOH when
necessary. The reaction was performed at 37 °C for 7 days and
was followed by HPLC analyses on the C18 analytical column.
The reaction mixture was filtered and then passed through the
preparative C18 column. The column was first eluted with 200
mL of H2O and then with 100 mL batches of 5, 10, and 20%
acetonitrile solutions in water. Fractions of 30 mL were col-
lected. The compound ꢀfGuo eluted from the column with the
5% acetonitrile eluent. The fractions containing the product were
combined and concentrated by rotary evaporation to about 25
mL. Further purification of the compound was carried out using
the semipreparative column. The column was eluted isocrati-
cally with 5% acetonitrile in 0.01 M phosphate buffer solution
(pH 7.1) for 2 min and then with a gradient from 5 to 30%
acetonitrile over the course of 28 min at a flow rate of 4 mL/
min. The collected fraction was then desalted using the prepara-
tive C18 column. The desalted solution was evaporated to
dryness, and the residue was subjected to spectroscopic and
spectrometric studies. The isolated amount of the compound was
3.0 mg.
The mutational spectrum of MX in Salmonella typh-
imurium strain TA100 has shown that MX induces
mainly GC f TA transversions (33). This mutational
specificity indicates that MX modifies the guanine base,
and the mutation results from misincorporation of ade-
nine opposite the modified guanine base. The structural
identification of products from the reactions of MX with
nucleosides may contribute to a better understanding of
the molecular mechanisms of MX-induced mutagenicity
and carcinogenicity. The purpose of this study was to
examine whether MX forms adducts in reactions with
guanosine, cytidine, or thymidine and to determine the
chemical structures of produced adducts. The aim was
also to determine whether such adducts are formed in
calf thymus DNA incubated with MX. The work resulted
in the identification of a stable adduct produced in the
reaction of MX with guanosine. The new guanosine
derivative consists of an etheno bridge to which a formyl
group is attached.
Ma ter ia ls a n d Meth od s
Ca u tion : MX has been found to be a rodent carcinogen and
is one of the strongest known direct-acting mutagens tested in
the Ames mutagenicity assay with S. typhimurium (TA100).
Therefore, caution should be exercised in the handling and the
disposal of the compound.
Ch em ica ls. Guanosine, cytidine, thymidine, 2′-deoxygua-
nosine, calf thymus DNA, DNase from bovine pancreas, nuclease
P1 from Penicillium citrinum, alkaline phosphatase from bovine
intestinal mucosa, and acid phosphatase from white potato were
obtained from Sigma Chemical Co. (St Louis, MO). 3-Chloro-4-
(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was synthe-
sized according to the method of Franze´n and Kronberg (34).
The purity of MX was at least 98%, as estimated by 1H NMR
and GC. 1,N2-Ethenoguanosine was prepared by reacting mu-
cochloric acid with guanosine as described by Kronberg et al.
(26). Bromomalonaldehyde was prepared according to the
method of Trofimenko (35).
The isolated compound had the following spectral character-
istics: UV spectrum [HPLC eluent, 10% acetonitrile in 0.01 M
phosphate buffer (pH 7.1)] UVmax 232 and 360 nm (ꢀ ) 18 500
M-1 cm-1), UVmin 312 nm, with a shoulder between 248 and 265
nm. The positive ion electrospray mass spectrum showed the
following ions m/z (relative abundance, formation): 336 (100,
MH+), 204 (26, MH+ - ribosyl + H). High-resolution mass
spectrometry gave a protonated molecular formula of C13H14N5O6
(MH+ 336.0944, calcd 336.0944).
Ch r om a togr a p h ic Meth od s. Analytical HPLC was per-
formed on
a Kontron Instruments liquid chromatographic
system consisting of a model 322 pump, a 440 diode-array
detector (UV), and a KromaSystem 2000 data handling program
(Kontron Instruments S. P. A., Milan, Italy). The reaction
mixtures were chromatographed on a 5 µm, 4 mm × 125 mm
reversed phase C18 analytical column (Spherisorb ODS2,
Hewlett-Packard, Espoo/Esbo, Finland). The column was eluted
isocratically with 0.01 M phosphate buffer (pH 7.1) for 5 min
and then with a gradient from 0 to 30% acetonitrile over the
course of 25 min at a flow rate of 1 mL/min. The compounds
were isolated from the reaction mixtures by column chroma-
tography on a 4 cm × 4 cm column of preparative C18 bonded
silica grade (40 µm, Bondesil, Analytichem International, Har-
bor City, CA). The compounds were further purified on a semi-
preparative 8 µm, 10 mm × 250 mm (Hyperprep ODS, Hypersil,
Krotek, Tampere/Tammerfors, Finland) 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.
The 1H and 13C NMR spectroscopic data are presented in
Table 1.
P r ep a r a tion of 3-(2′-Deoxy-â-D-r ibofu r a n osyl)-7-for m yl-
im id a zo[1,2-a ]p u r in -9(4H)-on e (Efd Gu o). The 2′-deoxygua-
nosine derivative was prepared in exactly the same way as the
guanosine derivative. The isolated 2′-deoxy derivative was
obtained in about the same yield as ꢀfGuo, and it had the
following spectral characteristics: UV spectrum [HPLC eluent,
11% acetonitrile in 0.01 M phosphate buffer (pH 7.1)] UVmax
232 and 360 nm, UVmin 312 nm, with a shoulder between 248
and 265 nm. The positive ion electrospray mass spectrum
showed the following ions m/z (relative abundance, formation):
320 (27, MH+), 204 (8, MH+ - deoxyribosyl + H). High-
resolution mass spectrometry gave a protonated molecular
formula of C13H14N5O5 (MH+ 320.0990, calcd 320.0995). The 1H
NMR spectral data were as follows: δ 10.61 (s, 1H, CHO), 7.99
(s, 1H, H-6), 7.97 (s, 1H, H-2), 6.29 (dd, 1H, H-1′, J ) 8.3, 5.9
Hz), 5.38 (dd, 1H, 5′-OH, J ) 7.1, 4.7 Hz), 5.25 (d, 1H, 3′-OH, J
) 4.0 Hz), 4.40 (m, 1H, H-3′), 3.87 (dt, 1H, H-4′, J ) 4.1, 2.5
Hz), 3.63 (m, 1H, H-5′), 3.53 (ddd, 1H, H-5′′, J ) 11.9, 7.2, 4.1
Hz), 2.71 (ddd, 1H, H-2′, J ) 13.5, 8.0, 5.3 Hz), 2.20 (ddd, 1H,
H-2′′, J ) 13.1, 6.0, 2.7 Hz). The 13C NMR spectral data were
as follows: δ 178.3 (CHO), 155.0 (C-9), 154.5 (C-4a), 149.3 (C-
1
Sp ectr oscop ic a n d Sp ectr om etr ic Meth od s. The H and
13C NMR spectra were recorded at 30 °C on a J EOL J NM-A500
Fourier transform NMR spectrometer at 500 and 125 MHz,
respectively. The samples were dissolved in Me2SO-d6, and TMS
was used as an internal standard. The 1H NMR signal assign-
ments were based on chemical shifts and H-H and C-H
correlation data. The determination of the shifts and the
coupling constants of the multiplets of the proton signals in the
ribose and deoxyribose units were based on
a first-order

48 Chem. Res. Toxicol., Vol. 12, No. 1, 1999
Munter et al.
Ta ble 1. 1H a n d 13C Ch em ica l Sh ifts (δ)a a n d Sp in -Sp in Cou p lin g Con sta n ts, J H,H a n d J C,H (Her tz), of P r oton s a n d
Ca r bon s in EfGu o
>1
proton
δ
multiplicity
J H,H
carbon
δ
multiplicity
1J C,H
J
C,H
H-2 (1H)
H-6 (1H)
CHO (1H)
7.97
8.00
10.62
s
d
d
C-2
C-6
CHO
C-3a
C-4a
C-7
137.3
140.2
178.3
149.4
154.4
127.0
155.0
116.3
87.8
72.8
70.7
85.6
61.8
dd
dd
d
dd
dd
dd
d
d
d
d
d
211.6
185.7
180.0
3.9
4.1
0.5
0.5
4.7, 3.9
14.4, 0.8
20.1, 14.0
0.8
C-9
C-9a
C-1′
C-2′
C-3′
C-4′
C-5′
10.9
H-1′ (1H)
H-2′ (1H)
H-3′ (1H)
H-4′ (1H)
H-5′ (1H)
H-5′′ (1H)
5′-OH (1H)
2′-OH (1H)
3′-OH (1H)
5.81
4.65
4.14
3.95
3.68
3.55
5.62
5.38
5.08
d
6.3
164.0
147.9
148.5
147.5
140.7
dd
dd
q
dd
dd
br
br
br
6.3, 5.0
5.0, 2.9
3.2
12.2, 3.4
12.1, 3.2
d
t
a
Relative to TMS.
3a), 140.3 (C-6), 136.7 (C-2), 126.9 (C-7), 116.1 (C-9a), 87.7 (C-
4′), 83.6 (C-1′), 71.1 (C-3′), 62.1 (C-5′), 39.3 (C-2′).
4.58 (dd, 1H, H-2′, J ) 5.8, 5.0 Hz), 4.16 (dd, 1H, H-3′, J ) 4.9,
3.4 Hz), 3.95 (q, 1H, H-4′, J ) 3.2 Hz), 3.74 (s, 3H, CH3), 3.66
(dd, 1H, H-5′, J ) 11.7, 4.4 Hz), 3.57 (dd, 1H, H-5′′, J ) 11.9,
4.1 Hz).
R ea ct ion of Br om om a lon a ld eh yd e w it h Gu a n osin e.
Bromomalonaldehyde (5.3 mg, 0.035 mmol) was reacted with
guanosine (5 mg, 0.018 mmol) in 3 mL of 0.5 M phosphate buffer
solutions at pH 9, 8.2, 7.4, 6, and 4.6. The reaction mixtures
were stirred at 37 °C. The pHs of the reaction mixtures were
checked daily, and readjusted with 1 M NaOH if necessary. The
reaction mixtures were analyzed by HPLC using the C18
analytical column.
Clea va ge of th e 2′-Deoxyr ibose Un it fr om Efd Gu o by
Acid Hyd r olysis. Pure ꢀfdGuo (1 mg) was dissolved in 4 mL
of 0.1 M HCl and the mixture heated at 60 °C. The HPLC
analysis of the mixture showed that the cleavage of the
2′-deoxyribose moiety was complete after 35 min. The new
compound, ꢀfG, eluted 2 min earlier than ꢀfdGuo. The compound
was isolated and purified using the semipreparative column.
The following spectral characteristics were recorded: UV spec-
trum [HPLC eluent, 9% acetonitrile in 0.01 M phosphate buffer
(pH 7.1)] UVmax 224, 252, and 360 nm, UVmin 240 and 308 nm.
The positive ion electrospray mass spectrum showed the pro-
tonated molecular ion at 204, with a relative abundance of 100%.
High-resolution mass spectrometry gave a protonated molecular
formula of C8H6N5O2 (MH+ 204.0528, calcd 204.0521). The 1H
NMR spectral data were as follows: δ 10.59 (s, 1H, CHO), 7.99
(s, 1H, H-6), 7.70 (s, 1H, H-2). (The assignment of H-6 was based
on selective decoupling of the formyl proton that led to an
increase in intensity of the signal at δ ) 7.99 ppm due to NOE
enhancement.)
Sm a ll Sca le Rea ction s of MX w ith Gu a n osin e, Cytid in e,
a n d Th ym id in e. MX (9.9 mg, 0.046 mmol) was reacted with
0.023 mmol of guanosine, cytidine, and thymidine in 5 mL of
0.5 M phosphate buffer solutions at pH 9, 8.2, 7.4, 6, and 4.6.
The reaction mixtures were incubated at 37 °C. The pHs of the
reaction mixtures were checked daily and readjusted with 1 M
NaOH if necessary. The reaction mixtures were analyzed by
HPLC using the C18 analytical column.
Meth yla tion of EfGu o [P r ep a r a tion of 3-(â-D-Ribofu r a n -
osyl)-7-for m yl-5-m eth ylim id a zo[1,2-a ]p u r in -9-on e (5-Me-
EfGu o)]. Triethylamine (60 mg, 0.59 mmol) and methyl iodide
(64 mg, 0.45 mmol) were added to a solution of ꢀfGuo (2.7 mg,
0.008 mmol) in 8 mL of DMF. The solution was stirred for 7 h
at 37 °C, and the reaction was followed by HPLC. After the
reaction was complete, DMF was removed by rotary evaporation
and the residue was dissolved in 3 mL of water. The product
was then purified on the semipreparative HPLC column. The
column was eluted isocratically with 5% acetonitrile in water
for 2 min and then with a gradient from 5 to 30% acetonitrile
over the course of 28 min at a flow rate of 4 mL/min. The
collected fraction was rotary evaporated to dryness, and the
residue was analyzed by 1H NMR and electrospray MS. The
isolated compound 5-Me-ꢀfGuo had the following spectral char-
acteristics: UV spectrum [HPLC eluent, 12% acetonitrile in 0.01
M phosphate buffer (pH 7.1)] UVmax 224 and 332 nm, UVmin 270
nm; MS ESI m/z (relative abundance, formation) 350 (56, MH+),
218 (100, MH+ - ribosyl + H). High-resolution mass spectrom-
etry gave a protonated molecular formula of C14H16N5O6 (MH+
350.1109, calcd 350.1100): 1H NMR δ 10.78 (s, 1H, CHO), 8.48
(s, 1H, H-6), 8.28 (s, 1H, H-2), 5.90 (d, 1H, H-1′, J ) 6.1 Hz),
Deter m in a tion of P r od u ct Yield s. Quantitative 1H NMR
analysis, using 1,1,1-trichloroethane as an internal standard,
was performed on aliquots of the ꢀfGuo and ꢀGuo adducts. HPLC
standard solutions were prepared by taking an exact volume of
the NMR samples and diluting it with an appropriate volume
of water. The quantitative determination of the amount of ꢀfGuo
and ꢀGuo in the reaction mixtures was made by comparing the
peak area of the compound in question in the standard solution
with the peak area of the compounds in the reaction mixtures.
The ꢀfGuo and ꢀGuo derivatives were quantified using UV
detection at 360 and 230 nm, respectively. The molar yields were
calculated from the original amount of guanosine in the reaction
mixtures.
Rea ction s of MX w ith Ca lf Th ym u s DNA. MX (50 mg)
was reacted with double-strand calf thymus DNA (10 mg) in
10 mL of 0.5 M phosphate buffer at pH 7.4. The mixture was
stirred and incubated at 37 °C for 4 days. During the first 12 h
of reaction and then twice a day, the pH of the incubation
mixture was monitored and readjusted if necessary. The modi-
fied DNA was recovered by precipitation with cold ethanol. To
1 volume of the incubation mixture were added 0.1 volume of 5
M NaCl and 2 volumes of cold 96% ethanol. This mixture was
centrifugated (10 min, 3000 rpm), and the supernatant was
collected. The supernatant was concentrated about 40 times and
analyzed by HPLC to search for modified purines and pyrim-
idines. The precipitated DNA was washed with 1 volume of 70%
ethanol and then redissolved in 1 volume of water. This
precipitation/washing procedure was performed (at least twice)
until there was no more unreacted MX left in the supernatant
(controlled by HPLC analyses). The enzymatic hydrolysis of the
DNA was carried out following the procedure described by
Martin et al. (36). Briefly, the modified DNA was dissolved (1
mg/mL) in 0.1 M phosphate buffer (pH 7.4) containing 5 mM
MgCl2. DNase I (dissolved at 10 mg of DNase/mL in 0.9% NaCl)
was added to obtain a level of 0.1 mg of DNase/mL. The mixture
was incubated and stirred for 3 h at 37 °C. Nuclease P1
(dissolved at 0.5 mg of nuclease P1/mL in 1 mM ZnCl2) was
added to obtain 20 µg of nuclease/mL as the final concentration.
Finally, alkaline phosphatase (87 units/mL in water) and acid

MX Forms an Ethenoformyl Adduct with Guanosine
Chem. Res. Toxicol., Vol. 12, No. 1, 1999 49
F igu r e 2. Formation of ꢀfGuo under various pH conditions at
37 °C.
F igu r e 1. C18 column HPLC separation of the reaction
mixture of MX and guanosine held at 37 °C and pH 8.2 for 6
days. For analysis conditions, see Materials and Methods.
phosphatase (20 units/mL in water) were added to give final
concentrations of 0.5 and 0.3 unit/mL, respectively. The mixture
was incubated and stirred at 37 °C for 18 h. The mixture of the
hydrolyzed DNA was rotary evaporated to near dryness. The
residue was washed three times with 3 mL of 1/1 ethanol/
methanol. The washes were combined, and insoluble particles
were removed by centrifugation (20 min, 3000 rpm). Finally,
the solution was evaporated to near dryness; an appropriate
amount of water (250 µL for 10 mg of DNA) was added, and
50-100 µL of the solution was injected on the analytical HPLC
column.
Bla n k s a n d HP LC Ch eck s. A blank sample was prepared
by allowing calf thymus DNA to stand in an aqueous solution
at pH 7.4 and 37 °C for 2 days. The precipitation, the enzymatic
hydrolysis, and the HPLC sample preparation were then carried
out exactly as described above.
F igu r e 3. UV absorbance spectrum of ꢀfGuo. The UV spectrum
was recorded with the diode-array detector as the compound
eluted from the HPLC column.
In the positive ion electrospray mass spectrum of the
product, the protonated molecular ion was the most
abundant ion, and it was observed at m/z 336. A second
major fragment peak corresponding to the loss of the
ribosyl unit was present at m/z 204 at a relative abun-
dance of 26%.
1
The H NMR spectrum displayed, besides the signals
of the protons of the ribose moiety which were assigned
using H-H correlation data, one-proton signals at δ )
10.62, 8, and 7.97 ppm (Table 1). The signal at δ ) 10.62
ppm was assigned to the formyl proton on the basis of
the downfield chemical shift and the one-bond C-H
correlation with the carbon signal at δ ) 178.3 ppm. The
signal at δ ) 8 ppm was assigned to the olefinic proton,
H-6, and it displayed a long-range H-H coupling to the
formyl proton. The proton at position 2 in the purine ring
appeared at δ ) 7.97 ppm. The signal showed a H-H
correlation (COSY) with the H-1′ signal in the ribose
moiety at δ ) 5.81 ppm.
Resu lts a n d Discu ssion
The small scale reactions of MX with guanosine,
cytidine, and thymidine were analyzed by HPLC on the
analytical column. No product peaks representing base-
modified nucleosides were observed in the reactions of
MX with thymidine or cytidine. In the reaction of MX
with guanosine, one main product peak with a retention
time longer than that of guanosine was formed (Figure
1). The compound marked ꢀfGuo eluted from the C18
analytical column at 13.5 min. The product was formed
at all the studied pH conditions, but in very low yields
at pH 4.6 and 6. The product was obtained in higher
yields in the reactions carried out under neutral or
slightly basic pH conditions. The yield was about 0.3%
at pH 7.4 and about 0.9% at pH 8.2 and 9 (Figure 2).
To be able to characterize the product by spectroscopic
and spectrometric methods, we performed a large scale
reaction and isolated the product from the reaction
mixture by preparative C18 column chromatography and
HPLC.
The 13C NMR spectrum showed three carbon signals
in addition to the 10 signals arising from the guanosine
unit (Table 1). The signal observed at δ ) 178.3 ppm
displayed a strong one-bond C-H coupling and was
assigned to the formyl carbon. The assignment of the
signal at δ ) 127 ppm to C-7 was confirmed by its strong
two-bond C-H coupling (2J ) 20.1 Hz) to the formyl
proton (37). The signal was further split into a doublet
of doublets due to coupling to H-6 (2J ) 14 Hz) as
confirmed by selective decoupling at δ ) 8 ppm. The
signal at δ ) 140.2 ppm was assigned to C-6 on the basis
of the strong one-bond C-H coupling (1J ) 185.7 Hz) to
H-6 and a weak three-bond coupling to the formyl proton
(3J ) 4.1 Hz). The signal of C-6 also showed C-H
correlation with the formyl proton (COLOC). The signal
at δ ) 137.3 ppm was assigned to C-2 on the basis of the
The UV spectrum of ꢀfGuo showed an absorption
maximum at 360 nm (Figure 3). This bathochromic shift
compared to 1,N2-ethenoguanosine (UVmax ) 284 nm) (26)
is most likely caused by the extended conjugation in the
compound because of the formyl group.

50 Chem. Res. Toxicol., Vol. 12, No. 1, 1999
Munter et al.
Ch a r t 2
Ch a r t 3
strong one-bond coupling (1J ) 211.6 Hz) to the H-2
proton. The carbon C-3a resonance signal appeared at δ
) 149.4 ppm. It showed C-H correlation with the signal
of H-2. The signal at δ ) 154.4 ppm was assigned to C-4a,
and it was split into a doublet due to a three-bond
coupling of 14.4 Hz to H-6 as confirmed by selective
decoupling of H-6. Due to this strong coupling, we assume
that a double bond is placed between C-4a and N-5. The
carbon resonance signal of C-9 appeared at δ ) 155 ppm
as a weakly coupled (J ) 0.8 Hz) doublet. The signal at
δ ) 116.3 ppm was assigned to C-9a on the basis of the
chemical shift and the three-bond coupling (3J ) 10.9 Hz)
to H-2. Further, the corresponding C-H correlation was
observed (COLOC).
To obtain additional support for the assignment of the
position of the formyl group in the etheno bridge (C-6 or
C-7), we methylated ꢀfGuo with methyl iodide and
1
analyzed the product with H NMR spectroscopy. In the
1H NMR spectrum, the methyl protons appeared at δ )
3.74 ppm and showed a NOE and a long-range H-H
correlation with the H-6 proton at δ ) 8.48 ppm. No NOE
or long-range correlations between the formyl proton and
the methyl protons were observed. Therefore, we conclude
that ꢀfGuo was methylated at N-5 and the formyl group
must be bound to C-7 (Chart 2). The location of the
methyl group at N-5 is also reflected in the downfield
shifts of the H-6 (0.48 ppm) and H-2 (0.31 ppm) proton
resonance signals and, in the UV spectrum, in a signifi-
cant blue shift of the long-wavelength maximum from
360 to 332 nm. These spectral changes are most likely
due to the difference in the conjugation of the double
bonds in the two compounds.
ꢀfGuo were formed in the reaction of bromomalonalde-
hyde with guanosine. The compounds were formed under
all the studied pH conditions. The optimal pH for the
formation of ꢀGuo was 7.4, and at this pH, the yield was
about 3% after a reaction time of 7 days. ꢀfGuo was
formed under all the studied pH conditions in about 0.1%
yields. The formation of ꢀfGuo may be explained through
an initial formation of a carbinolamine (Chart 3). Fol-
lowing nucleophilic displacement of bromide by N-1 of
guanosine and dehydration, ꢀfGuo is obtained. In the
reaction of MX with guanosine, we propose a mechanism
in which the dichloromethyl carbon atom of MX is
initially attacked by the exocyclic amino group of gua-
nosine and HCl is displaced (Chart 3). Next, an ethano
bridge is formed by attack of the endocyclic nitrogen on
the double bond. The chlorine atoms are substituted by
hydroxyl groups; glyoxylic acid is cleaved from the
intermediate through a retrograde aldol reaction (39),
and the adduct is obtained by dehydration of the enol.
Rea ction of MX w ith Ca lf Th ym u s DNA. It has
been reported that the mutational specificity of MX could
be explained by the formation of guanosine adducts or
by the occurrence of apurinic sites (33, 40, 41). To
determine whether ꢀfdGuo is formed in DNA, we incu-
The NMR and the UV spectroscopic and the mass
spectrometric data are consistent with the structure of
ꢀfGuo presented in Chart 2.
Previously, Nair et al. (38) prepared ethenoformyl
derivatives of adenosine and cytidine by reacting the
nucleosides with bromomalonaldehyde. In the present
work, we studied the reaction of bromomalonaldehyde
with guanosine and searched for ꢀfGuo. The HPLC
analyses of the reaction mixtures showed that one main
product with a retention time of 14.5 min was formed.
The UV spectrum of the product displayed absorption
maxima at 224 and 284 nm and was identical with the
spectrum of 1,N2-ethenoguanosine (26). In the chromato-
grams, a small peak at exactly the same retention time
as ꢀfGuo was also present. When we spiked the reaction
mixture with pure standards of ꢀGuo and ꢀfGuo, we found
that the compounds in the reaction mixture coeluted with
the standards and gave sharp peaks. Since the UV
spectra of the products were also identical with the
spectra of ꢀGuo and ꢀfGuo, we concluded that ꢀGuo and

MX Forms an Ethenoformyl Adduct with Guanosine
Chem. Res. Toxicol., Vol. 12, No. 1, 1999 51
2(5H)-furanone: 13C NMR chemical shifts as determinants of
mutagenicity. Chem. Res. Toxicol. 4, 35-40.
(3) Meier, J . R., Blazak, W. F., and Knohl, R. B. (1987) Mutagenic
and clastogenic properties of 3-chloro-4-(dichloromethyl)-5-hy-
droxy-2(5H)-furanone: A potent bacterial mutagen in drinking
water. Environ. Mol. Mutagen. 10, 411-424.
(4) Kronberg, L., and Franze´n, R. (1993) Determination of chlorinated
furanones, hydroxyfuranones, and butenedioic acids in chlorine
treated water and in pulp bleaching liquor. Environ. Sci. Technol.
27, 1811-1818.
(5) Kringstad, K. P., Ljungqvist, P. O., De Sousa, F., and Stro¨mberg,
L. (1983) On the formation of mutagens in the chlorination of
humic acid. Environ. Sci. Technol. 17, 553-555.
(6) Fielding, M., and Horth, H. (1986) Formation of mutagens and
chemicals during water treatment chlorination. Water Supply 4,
103-126.
(7) Meier, J . R., Knohl, R. B., Coleman, W. E., Ringhand, H. P.,
Munch, J . W., Kaylor, W. H., Streicher, R. P., and Kopfler, F. C.
(1987) Studies on the potent bacterial mutagen 3-chloro-4-
(dichloromethyl)-5-hydroxy-2(5H)-furanone: Aqueous stability,
XAD recovery and analytical determination in drinking water and
in chlorinated drinking water and in chlorinated humic acid
solutions. Mutat. Res. 189, 363-373.
(8) Smeds, A., Vartiainen, T., Ma¨ki-Paakkanen, J ., and Kronberg,
L. (1997) Concentrations of Ames mutagenic chlorohydroxyfura-
nones and related compounds in drinking waters. Environ. Sci.
Technol. 31, 1033-1039.
(9) Kronberg, L., and Vartiainen, T. (1988) Ames mutagenicity and
concentration of the strong mutagen 3-chloro-4-(dichloromethyl)-
5-hydroxy-2(5H)-furanone and of its geometric isomer E-2-chloro-
3-(dichloromethyl)-4-oxo-butenoic acid in chlorine-treated tap
waters. Mutat. Res. 206, 177-182.
bated DNA with MX and subsequently carried out a
search for the adduct in the DNA hydrolysate. The
chromatograms obtained by HPLC analyses of the hy-
drolysate did not show any peak which could represent
ꢀfdGuo. Apurinic sites may occur as a consequence of
adduct formation followed by breakage of the glycosidic
bond and release of the adducted base from DNA. We
prepared ethenoformylguanine (ꢀfG) by hydrolysis of
ꢀfdGuo and used the compound as a standard to search
for the adducted base in the supernatant, collected during
precipitation of the incubated calf thymus DNA. The
outcome of the HPLC analysis was that the modified
guanine base unit could not be detected in the superna-
tant. These results show that, if the ethenoformyl unit
is incorporated into the guanine base of DNA, the
ethenoformyl guanine derivative is formed in amounts
lower than the detection limit of our HPLC analytical
method. The detection limit of our HPLC analytical
method was about five adducts per 107 bases.
Very recently, Franze´n et al. (42) identified a MX-
guanosine adduct, named 10-formyl-1,N2-benzoquinone
propenoguanosine. We have repeated the experiment
according to the procedure described by Franze´n et al.,
but by using 98% pure MX, we could not detect the
claimed adduct in our HPLC chromatograms.
The mutational spectrum of MX in S. typhimurium
strain TA100 has shown that MX causes primarily GC
f TA transversions at the second position of the target
sequence CCC in the hisG46 allele (33). These mutational
events suggest a mechanism in which MX forms an
adduct with guanine or via depurination of the guanine
base. In a study by Marsteinstredet et al. (40), it was
demonstrated by a DNA sequence analysis of a MX-
treated DNA fragment, that MX reacts preferentially
with the guanine base in vitro to produce chemically
stable DNA adducts. The results of the current study
demonstrate that MX modifies guanosine by forming a
stable, cyclic 1,N2-ethenoformylguanosine adduct. Since
the N-1 and N2 positions of guanine are involved in base
pairing, it seems possible that the adduct might be
involved in the genotoxic effects of MX. However, this
theory would be strengthened if the ꢀfdGuo adduct could
be detected in calf thymus DNA. Therefore, further work
is needed to lower the detection limit of the analysis of
the adduct in DNA to establish whether ꢀfdGuo is formed
at all in DNA.
(10) Horth, H. (1990) Identification of mutagens in water. J . Fr.
Hydrol. 21, 135-145.
(11) Andrews, R. C., Daignault, S. A., Laverdure, C., Williams, D. T.,
and Huck, P. M. (1990) Occurrence of the mutagenic compound
“MX” in drinking water and its removal by activated carbon.
Environ. Technol. 11, 685-694.
(12) Suzuki, N., and Nakanishi, J . (1990) Determination of strong
mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in
drinking water in J apan. Chemosphere 21, 387-392.
(13) Zou, H., Xu, X., Zhang, J ., and Zhu, Z. (1995) The determination
of MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone] in
drinking water in China. Chemosphere 30, 2219-2225.
(14) Brunborg, G., Holme, J . A., So¨derlund, E. J ., Hongslo, J . K.,
Vartiainen, T., Lo¨tjo¨nen, S., and Becher, G. (1991) Genotoxic
effects of the drinking water mutagen 3-chloro-4-(dichloromethyl)-
5-hydroxy-2(5H)-furanone (MX) in mammalian cells in vitro and
in rats in vivo. Mutat. Res. 260, 55-64.
(15) Chang, L. W., Daniel, F. B., and De Angelo, A. B. (1991) DNA
strand breaks induced in cultured human and rodent cells by
chlorohydroxyfuranones-mutagens isolated from drinking water.
Teratog., Carcinog., Mutagen. 11, 103-114.
(16) J ansson, K., Ma¨ki-Paakkanen, J ., Vaittinen, S. L., Vartiainen,
T., Komulainen, H., and Tuomisto, J . (1993) Cytogenetic effects
of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in
rat peripheral lymphocytes in vitro and in vivo. Mutat. Res. 299,
25-28.
(17) J ansson, K., and Hyttinen, J . M. T. (1994) Induction of gene
mutation in mammalian cells by 3-chloro-4-(dichloromethyl)-5-
hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product
in drinking water. Mutat. Res. 322, 129-132.
(18) Furihata, C., Yamashita, M., Kinae, N., and Matsushima, T.
(1992) Genotoxicity and cell proliferative activity of 3-chloro-4-
(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat glandular
stomach. Water Sci. Technol. 25, 341-345.
(19) Daniel, F. B., Olson, G. R., and Stober, J . A. (1991) Induction of
gastrointestinal tract nuclear anomalies in B6C3F1 mice by
3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and 3,4-
(dichloro)-5-hydroxy-2(5H)-furanone, mutagenic by-products of
chlorine disinfection. Environ. Mol. Mutagen. 17, 32-39.
(20) Ma¨ki-Paakkanen, J ., J ansson, K., and Vartiainen, T. (1994)
Induction of mutation, sister-chromatid exchanges, and chromo-
some aberrations by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-
furanone in Chinese hamster ovary cells. Mutat. Res. 310, 117-
123.
(21) Fekadu, K., Parzefall, W., Kronberg, L., Franze´n, R., Schulte-
Hermann, R., and Knasmu¨ller, S. (1994) Induction of genotoxic
effects by chlorohydroxyfuranones, byproducts of water disinfec-
tion, in E. coli K-12 cells recovered from various organs of mice.
Environ. Mol. Mutagen. 24, 317-324.
Ack n ow led gm en t. This work was supported by a
research grant from Åbo Akademi University (T.M.) and
by the European Commission (Contract Nr ERBFM-
BICT961394, F.L.C.). We are grateful to Prof. Osmo
Hormi for fruitful discussions concerning reaction mech-
anisms and to Mr. Markku Reunanen for the mass
spectra.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra and
electrospray mass spectra of ꢀfGuo and the UV spectrum of ꢀfG
(5 pages). Ordering information is given on any current mast-
head page.
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