Chem. Res. Toxicol. 1996, 9, 703-708
703
Identification of Adducts Formed in Reaction of Adenosine with 3-Chloro-4-methyl-5-hydroxy2(5H)-furanone, a Bacterial Mutagen Present in Chlorine Disinfected Drinking Water Tony Munter, Leif Kronberg,* and Rainer Sjo¨holm Department of Organic Chemistry, A° bo Akademi University, Akademigatan 1, FIN-20500 Turku/A° bo, Finland Received November 20, 1995X
3-Chloro-4-methyl-5-hydroxy-2(5H)-furanone, MCF, a genotoxic hydroxyfuranone present in chlorine disinfected drinking water, was reacted with adenosine, guanosine, and cytidine in aqueous solutions. HPLC analyses of the reaction mixtures showed that only in the reaction of MCF and adenosine clearly detectable products were formed. The two major products were isolated by C18 column chromatography and characterized by UV absorbance, 1H and 13C NMR spectroscopy, and mass spectrometry. The products were identified as 4-(N6-adenosinyl)-3formyl-3-butenoic acid (I) and 5-(N6-adenosinyl)-3-chloro-4-methyl-2(5H)-furanone (II). The yield of I and II in reactions performed at pH 7.4 and 37 °C was 0.8% and 0.5%, respectively. Reaction of adenosine with 13C-3-labeled MCF was employed to elucidate the mechanism of formation of I. It was found that the product was formed by a nucleophilic attack of the exocyclic amino group in adenosine on the carbon in the 4-methyl group of MCF. These adducts appear to be novel and not structurally related to those previously identified in the reaction of adenosine with mucochloric acid, another genotoxic hydroxyfuranone.
Introduction Reaction of chlorine with humic material during chlorine disinfection of drinking water leads to formation of genotoxic compounds (1-3). The presence of mutagenic compounds in chlorinated drinking water has raised concern over the possible health effects of long-term consumption of chlorine disinfected water. Several studies have suggested that an increase in the risk of bladder, kidney, and stomach cancer is associated with the consumption of chlorinated tap water (4, 5). In work aimed at the identification of main mutagens in drinking water, the extremely potent direct acting bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy2(5H)-furanone (MX)1 (Scheme 1), was identified (6, 7). The mutagenicity of MX in Salmonella typhimurium tester strain TA100 has been reported to range from 4000 to 13 000 revertant colonies/nmol (7-12). Recent studies have shown that, besides MX, several other structurally related chlorohydroxyfuranones are present in chlorinated drinking water (13-15). Among these compounds are mucochloric acid (MCA; 3,4-dichloro-5-hydroxy-2(5H)furanone) and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) (Scheme 1). Also, MCA and MCF are direct acting mutagens, inducing about 1-10 revertant colonies/ nmol (14, 16, 17). In Scheme 1 the compounds are presented in the ring form (as hydroxyfuranones) and in the open chain form (as oxobutenoic acids). At acid pH conditions, the compounds exist primarily in the ring form, and at neutral pH they adopt the open chain form (18, 19). * Author for correspondence. E-mail:
[email protected]; phone: +358-21-2654186; FAX: +358-21-2654866. X Abstract published in Advance ACS Abstracts, April 15, 1996. 1 Abbreviations: MX, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone; MCF, 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone; MCA, mucochloric acid, 3,4-dichloro-5-hydroxy-2(5H)-furanone; DMF, N,Ndimethylformamide.
S0893-228x(95)00192-5 CCC: $12.00
Scheme 1
Chemical reactions of genotoxic compounds with the base units of DNA may cause gene mutations and contribute to cancer initiation (20). The characterization of reaction products of nucleosides and genotoxic agents provides information on the kinds of adducts that could be produced in DNA. Previous work in our laboratory has been concerned with the study of MCA nucleoside reaction products. The compound has been shown to form “etheno”, “ethenocarbaldehyde”, and “dimeric” derivatives of nucleosides (21-23). In the current work, we have studied the reactions of MCF with nucleosides. We report on the isolation and structural elucidation of two novel adducts, formed upon reaction of MCF with adenosine (I and II in Chart 1). We also present plausible mechanisms for the formation of the modified adenosine derivatives.
Materials and Methods Caution: MCF has been tested positive in the Ames mutagenicity assay with S. typhimurium (TA100) without metabolic activation. Therefore, caution should be exercised in the handling and disposal of the compound. HPLC analyses were carried out using dual Shimadzu LC9A pumps equipped with a variable wavelength Shimadzu SPD6A UV spectrophotometric detector (Shimadzu Europe, Germany). The reaction mixtures were chromatographed on a 5 µm, 4- × 125-mm reversed phase C18 analytical column
© 1996 American Chemical Society
704 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Munter et al.
Chart 1
(Spherisorb ODS2, Phase Separation, U.K.). The column was eluted isocratically for 5 min with 5% acetonitrile in 0.01 M potassium dihydrogen phosphate (pH 4.6) and then with a gradient from 5% to 30% in 25 min at a flow rate of 1 mL/min. Preparative isolation of the products was performed by column chromatography on a 2.5- × 10-cm column of preparative C18 bonded silica grade (40 µm, Bondesil, Analytichem International, Harbor City, CA). The 1H and 13C NMR spectra were recorded at 30 °C on a Jeol JNM-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 determination of the shifts and the coupling constants of the multiplets of the proton signals in the ribose units were based on a first order approach. Assignment of carbon signals was based on chemical shifts and carbon-proton couplings. The assignment of H-2 and H-8 in the adenine moiety was made by recording selectively proton-decoupled 13C NMR spectra. The direct inlet chemical ionization (DCI) mass spectra were recorded on a VG 7070E mass spectrometer. Methane and ammonia were used as ionization gases. The electrospray mass spectrum and the LC-MS analysis (electrospray) were carried out on a VG Autospec mass spectrometer. The LC separation was performed on a Zorbax SB C18 column (150- × 4.6-mm). The column was eluted isocratically for 5 min with 95% of mobile phase A (water containing 0.05% formic acid and 0.05% ammonium acetate) and 5% of mobile phase B (acetonitrile/water, 80/20, containing 0.05% formic acid and 0.05% ammonium acetate) and then with a gradient fom 5% B to 40% B in 25 min at a flow rate of 1 mL/min. About 1/20 of the eluate was allowed to enter the ion source. The UV spectra of the isolated products were recorded with a Shimadzu UV-160 spectrophotometer (Shimadzu Europa, Germany). MCF and 13C-3-labeled MCF were synthesized and purified according to the method of Franze´n and Kronberg (24) and crystallized from dichloromethane/n-hexane at -20 °C (mp 54-56 °C). The ring-chain tautomerism of MCF was studied by recording UV spectra of MCF dissolved in water at pH conditions ranging from pH 4.6 to 8.0. 4-(N6-Adenosinyl)-3-formyl-3-butenoic Acid (I). MCF (150 mg, 1.0 mmol) and adenosine (135 mg, 0.5 mmol) were dissolved in 100 mL of 0.5 M aqueous phosphate buffer (KH2PO4/Na2HPO4 in a ratio adjusted to pH 6.0), and the resulting solution was stirred for 6 days at 60 °C. The product was isolated from the reaction mixture by column chromatography on the C18 preparative column. The column was washed with 100 mL of 0%, 5%, 10%, and 20% acetonitrile solutions in 0.01 M KH2PO4 (pH 4.6). Fractions of 30 mL were collected. The fractions containing the pure product (10% washes) were combined and concentrated to approximately 20 mL. This solution was desalted by the use of the same preparative C18 column. The desalted solution was rotary evaporated to dryness and the residue was subjected to spectrometric studies. The reaction of 13C-3-labeled MCF with adenosine was carried out the same way as described above. In the reaction 27% 13C-3labeled MCF was used. The isolated compound (I) had the following spectral properties: UV spectrum (H2O) UVmax 329 ( 43 500 M-1 cm-1), 243, 223 nm, UVmin 273, 236, 214 nm. The following ions were
Figure 1. C18 column HPLC chromatogram of the reaction mixture of MCF and adenosine (A) held at 37 °C and pH 6.0 for 2 days. For analysis conditions, see Materials and Methods. observed in the ammonia DCI mass spectrum [m/z (relative abundance, formation)]: 351 (2, MH+ - CHO); 268 (38, adenosine + H)+; 204 (21, MH+ - CO2 - ribosyl + H); 176 (100, MH+ - CO2 - CO - ribosyl + H). Positive ion electrospray mass spectrum: 380 (100, MH+); 248 (33, MH+ - ribosyl + H). The 1H and 13C NMR spectroscopic data are presented in Table 1. 5-(N6-Adenosinyl)-3-chloro-4-methyl-2(5H)-furanone (II). MCF (150 mg, 1.0 mmol) and adenosine (135 mg, 0.5 mmol) were dissolved in 10 mL of N,N-dimethylformamide (DMF) and reacted for 6 days at 60 °C. The reaction mixture was diluted with water (final volume 200 mL). This solution was passed through the preparative C18 column. The column was eluted with 100 mL of 0%, 5%, 10%, and 20% acetonitrile solutions in water. Fractions of 30 mL were collected. The fractions containing the pure product (20% acetonitrile washes) were combined and rotary evaporated to dryness. The dried residue was subjected to spectrometric studies. The isolated compound (II) had the following spectral properties: UV spectrum (H2O) UVmax 264 ( 19 500 M-1 cm-1 ), UVmin 238 nm. The following ions were observed in the methane DCI mass spectrum [m/z (relative abundance, formation)]: 400/398 (7/22, MH+); 362 (6, MH+ - HCl); 296/294 (4/13, MH+ - ribosyl + HCO); 268/266 (43/100, MH+ - ribosyl + H); 230 (61, MH+ HCl -ribosyl + H); 186 (45, MH+ - HCl - CO2 - ribosyl + H). Positive ion electrospray mass spectrum: 400/398 (22/55, MH+); 268/266 (34/100, MH+ - ribosyl + H). The 1H and 13C NMR spectroscopic data of the isolated product are presented in Table 2. Small Scale Reactions of MCF with Adenosine, Guanosine, and Cytidine. MCF, 0.014 mmol (2 mg), was reacted with 0.007 mmol of adenosine, guanosine, and cytidine in 2 mL of 0.5 M phosphate buffer at pH 7.4 and 6.0. The same reactions were also carried out in 2 mL of DMF. The reactions were performed at 37 and 60 °C. The formation of products was followed by HPLC analyses of aliquots of the reaction mixtures. Determination of Product Yields. Quantitative 1H NMR analysis, using 1,1,1-trichloroethane as an internal standard, was performed on aliquots of the adducts. Then standard solutions were prepared for HPLC analysis by taking an exact volume of the NMR sample and diluting it with an appropriate volume of water. The quantitative determination of adducts in the reaction mixtures was made by comparing the peak area of the adducts in the standard solutions with the area of the adduct peaks in the reaction mixtures. Adduct I and adduct II were quantified using UV detection at 325 and 254 nm, respectively. The molar yields of the adducts were calculated from the original amount of adenosine in the reaction mixture.
Results and Discussion The small scale aqueous reactions of MCF with adenosine, guanosine, and cytidine were followed by HPLC
Bacterial Mutagen MCF Reacts with Adenosine
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 705
Figure 2. Formation of I and II at various reaction conditions: (0) ) pH 6.0, 60 °C; (O) ) pH 6.0, 37 °C; (() ) pH 7.4, 37 °C; (9) ) 60 °C; (2) ) 37 °C.
analyses with UV detection at 254 and 325 nm. In the reactions of MCF with guanosine and cytidine, hardly any products could be observed. On the other hand, in MCF reactions with adenosine at 37 °C, two distinct product peaks were observed at longer retention times than adenosine (Figure 1). The compound marked I eluted at 13.5 min, and compound II at 21.0 min. The compounds with shorter retention times than adenosine were observed also in the chromatogram of an aqueous solution containing only MCF and were likely to be formed as a consequence of MCF degradation. Product I was found to be formed in higher yield at pH 6.0 (7% after 8 days of incubation) than at pH 7.4 (0.8%) (Figure 2). Also, increasing the temperature from 37 to 60 °C resulted in a higher yield (15%) of the product. The maximum yield of II was about 0.5%, and this yield was obtained in reactions carried out at pH 7.4 and 37 °C (Figure 2). When DMF was used as solvent for the reactions of MCF and adenosine, only one major product was formed. Following isolation of the product and spiking of the aqueous MCF and adenosine reaction mixture with the compound, we observed that the compound coeluted with II. The UV spectra of the compounds were identical and, as a fine structure, exhibited weak shoulders between 228-230, 248-250, and 270-272 nm. The shoulders were easily observed in the derivatized UV spectra (not shown). Since also the electrospray MS spectrum of II in the aqueous reaction mixture (recorded by LC-MS) was in all essential features identical to the electrospray MS of the isolated compound in DMF, we conclude that the compound formed in DMF is structurally identical to II. The yield of II was higher in DMF (3%) than in water. In order to determine the structures of I and II, large scale reactions were performed in water and in DMF. The products were isolated and purified on the preparative C18 column. The structures of I and II were assigned on the basis of data obtained by UV and NMR spectroscopy, and mass spectrometry (Chart 1). The UV spectrum of the modified adenosine derivative I showed an intense absorption maximum at 329 nm (Figure 3). This long-wavelength maximum is most likely due to the resonance interactions of the lone pair electrons of the exocyclic nitrogen atom with the R,βunsaturated aldehyde moiety (25). In the electrospray mass spectrum of the product, the protonated molecular ion (MH+) was observed at m/z 380. The fragment peak at m/z 248 was formed by cleavage of the ribosyl unit from MH+.2 No isotope mass peaks due to the presence of chlorine in the molecule were observed. The ammonia DCI mass spectrum did not 2 When the ribosyl unit is cleaved, a proton becomes attached to N-9 in the adenine moiety.
Figure 3. UV absorbance spectra of adenosine (Ado), I, and II.
show a protonated molecular ion peak. A small ion peak could be observed at m/z 351, formed by loss of the aldehyde group from MH+. The fragment peaks at m/z 204 and 176 were formed from MH+ by cleavage of ribosyl together with carbon dioxide, and ribosyl together with carbon dioxide and carbon monoxide, respectively. The fragment peak observed at m/z 268 corresponded to protonated adenosine. The 1H NMR spectrum of I displayed three signals besides the signals from the adenosine unit (Table 1). The aldehyde proton appeared at δ ) 9.31 ppm as a triplet due to coupling to the methylene protons (J ) 1.5 Hz). The olefinic proton, Ha, was observed as a broad one proton signal at δ ) 8.68 ppm. The broadening of the signal was most likely due to the anisotropy of the neighboring nitrogen atom. The two-proton singlet at δ ) 3.34 ppm was assigned to the methylene protons (Hc). The carboxyl and amino protons could not be observed, because of exchange with residual water in the sample. The 13C NMR spectrum showed five carbon resonance signals in addition to the 10 signals arising from the adenosine unit (Table 1). The signal observed at δ ) 190.8 ppm displayed a strong one-bond C-H coupling and was assigned to the formyl carbon. In addition, three-bond C-H couplings were observed to Ha, as well as to the methylene protons. The coupling to Ha (3J ) 7.2 Hz) indicated a cis relation of the formyl carbon and Ha (26). The carbons Ca and Cb, in the double bond, were observed at δ ) 147.3 ppm and δ ) 117.6 ppm, respectively. The signal of Ca was slightly broadened due to the anisotropy of the neighboring nitrogen atom. The Ca carbon displayed a one-bond coupling of 1J ) 171.2 Hz, while carbon Cb displayed a two-bond coupling of 2J ) 24.4 Hz to the formyl proton, and smaller couplings to Ha (2J ) 6.5 Hz) and the methylene protons (2J ) 3.0 Hz). The methylene carbon appeared at δ ) 30.1 ppm as a triplet. The presence of a carboxyl group was indicated by a resonance signal at δ ) 172.4 ppm. This signal was split to a triplet with a coupling of 2J ) 7.8
706 Chem. Res. Toxicol., Vol. 9, No. 4, 1996 Table 1. 1H and protona H-8 H-2 N-H
13C
Munter et al.
Chemical Shifts (δ) and Spin-Spin Coupling Constants, JH,H and JC,H (Hz) of Protons and Carbons in I multiplicity
δ 8.69 (1H) 8.55 (1H) n.o.c
JH,H
s s
Ha
8.68 (1H)
br s
Hc COOH CHO
3.34 (2H) n.o.c 9.31 (1H)
s
H-1′ H-2′ H-3′ H-4′ H-5′ H-5′′
6.01 (1H) 4.61 (1H) 4.20 (1H) 3.99 (1H) 3.70 (1H) 3.59 (1H)
d t dd q dd dd
OHd OH
5.5 (1H) 5.2 (2H)
br br
t
1.5 5.7 5.3 4.7; 3.9 3.7 12.0; 3.8 12.1; 3.9
carbonb
1J C,H
δ
multiplicity
C-8 C-2
142.3 151.8
dd d
C-4 C-5 C-6
151.2 120.8 149.7
ddd d dd
Ca Cb Cc COOH CHO
147.3 117.6 30.1 172.4 190.8
d ddt t t ddt
171.2
d d d d t
165.3 149.5 149.5 148.7 141.6
C-1′ C-2′ C-3′ C-4′ C-5′
87.7 73.7 70.2 85.6 61.2
215.2 204.1
13C
171.2 24.4; 6.5; 3.0 128.0
d
7.8 7.2; 3.5
No effort was made to
Chemical Shifts (δ) and Spin-Spin Coupling Constants, JH,H and JC,H (Hz) of Protons and Carbons in II
protona
δ
multiplicity
H-8 H-2 N-H
8.55 (1H) 8.42 (1H) 9.05 (1H)
s s br s
H5f H6f
4.1 12.4; 4.9; 3.6 11.9 11.4; 3.6
a H-1′-H-5′′ ) protons in the ribosyl unit. b C-1′-C-5′ ) carbons in the ribosyl unit. c n.o. ) not observed. assign the signals to the specific hydroxyl groups in the ribosyl unit.
Table 2. 1H and
>1JC,H
7.32 (1H) 2.07 (3H)
JH,H
br s s
H-1′ H-2′ H-3′ H-4′ H-5′ H-5′′
5.97 (1H) 4.60 (1H) 4.17 (1H) 3.99 (1H) 3.69 (1H) 3.57 (1H)
d t dd q dd dd
OHd OH
5.5 (1H) 5.2 (2H)
br br
5.9 5.5 5.0; 3.5 3.7 12.0; 3.7 12.1; 3.9
carbonb
δ
C-8 C-2
141.3 151.8
dd d
C-4 C-5 C-6
150.0 120.0 156.5
br ddd br
C5f C6f C2f C3f C4f
84.1 12.2 166.3 119.8 153.2
br d q quintc dq br d
175.1 130.3
C-1′ C-2′ C-3′ C-4′ C-5′
87.7 73.6 70.3 85.6 61.3
d d d d t
165.5 147.4 149.5 148.5 140.7
multiplicity
1J C,H
214.1 202.8
>1JC,H 4.1
11.9; 4.6; 3.0
1.3 6.1; 3.0 9.8
a H-1′-H-5′′ ) protons in the ribosyl unit. b C-1′-C-5′ ) carbons in the ribosyl unit. c The signal appeared as a quintet because of equal couplings to the methyl and methine protons. d No effort was made to assign the signals to the specific hydroxyl groups in the ribosyl unit.
Hz to the two methylene protons. In the adenosine moiety, the addition of an unsaturated carbon atom to the exocyclic amino group was reflected in an upfield shift (6.7 ppm) of carbon C-6 relative to C-6 in adenosine at δ ) 156.4 ppm. In addition, smaller downfield shifts were noted of carbons C-4 (2.3 ppm), C-8 (2.4 ppm), and C-5 (1.0 ppm), relative to the respective carbons in unmodified adenosine. The spectral data are consistent with structure I (Chart 1). The UV spectrum of compound II resembled the UV spectrum of adenosine and exhibited only a slight bathochromic shift of the absorption maximum (UVmax 264 nm) relative to adenosine (UVmax 260 nm) (Figure 3). The protonated molecular ion (MH+) at m/z 398 was observed in the electrospray and the methane DCI mass spectra. The presence of one chlorine atom in the structure was evident from the observation of a fragment ion two mass units higher than MH+ in an abundance of 33% of the MH+ ion. The chlorine isotope cluster could
also be observed at 266 (MH+ - ribosyl + H) and at m/z 294 (MH+ - ribosyl + HCO; only the CI mass spectrum). In addition, cleavage of HCl and of HCl and the ribosyl unit from MH+ produced the fragment ions at m/z 362 and 230, respectively, in the CI mass spectrum.2 The fragment ion at m/z 186 was formed by loss of the ribosyl unit together with hydrogen chloride and carbon dioxide from the protonated molecular ion. The 1H NMR spectrum of II displayed five signals in addition to the signals of the ribosyl protons (Table 2). It was obvious that the addition reaction had taken place at the exocyclic nitrogen of adenosine since only one amino proton could be found in the spectrum. This signal appeared as a broad singlet at δ ) 9.05 ppm. Upon addition of D2O to the sample, the signal disappeared. The methine proton, H5f, in the furanone ring, appeared as a broad singlet at δ ) 7.32 ppm. The reason for the broadening of this signal is most likely due to uneven electron distribution of the neighboring nitrogen atom
Bacterial Mutagen MCF Reacts with Adenosine
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 707 Scheme 2a
a
(*) )
13C-labeled
carbon.
(anisotropy). The signal of the three protons of the methyl group, H6f, was observed at δ ) 2.07 ppm. The 13C NMR spectrum of II displayed five carbon signals in addition to the 10 signals from adenosine (Table 2). The carbonyl carbon was observed at δ ) 166.3 ppm. The signal appeared as a slightly distorted quintet (J ) 1.3 Hz) in the proton coupled spectrum because of almost equal couplings to H5f and the protons of the methyl group. The signals at δ ) 153.2 and δ ) 119.8 ppm were assigned to the double-bonded carbons C4f and C3f, respectively. The C4f carbon signal was split in a broad doublet (J ) 9.8 Hz) due to coupling to the methine proton (H5f). The broadening of the signal is most likely due to weak, unresolved couplings to the methyl protons. The C3f carbon appeared as doublet of a quartet due to coupling to the methine proton (J ) 3.0 Hz) and the methyl protons (J ) 6.1 Hz). The methine carbon C5f, bonded to the exocyclic nitrogen atom of adenosine, gave a very broad doublet (J ) 175.1 Hz) signal at δ ) 84.1 ppm. The methyl carbon, C6f, appeared as a quartet (J ) 130.3 Hz) at δ ) 12.2 ppm. There were no large differencies in shifts (e1.4 ppm) of the base and sugar carbons between II and unmodified adenosine. Also, the shifts of the furanone ring in II and of unmodified MCF were close to each other, with the only exception of the C-5 carbon, whose resonance signal appeared in II 14 ppm upfield from that in MCF (27). Collectively, the spectral data of the compound are consistent with structure II (Chart 1). In order to be able to suggest a plausible mechanism for formation of I, first we had to determine whether MCF undergoes the same kind of ring-chain tautomerism as MCA and MX (19, 28). In the UV spectra of MCF in aqueous solutions at various pH conditions, a shift was observed in UV maximum from 223 nm at pH 6.0. Thus, at pH 6.0 the compound adopts the open form, primarily. Further information on the formation of I was obtained by reacting 13C-3-labeled MCF with adenosine. The 13C NMR spectrum of labeled I showed a significantly more intensive signal for the methylene carbon (Cc), than for any other carbon present in the spectrum. These findings provided a plausible explanation for the formation of the product. Initially, a proton
transfer takes place from the methyl carbon in the open form of MCF to the labeled carbon atom (Scheme 2). The addition should be acid catalyzed (as indicated in Scheme 2), and this explains why the yield of I was found to be higher in reactions performed at pH 6.0 than at pH 7.4 (Figure 2). Next, the exocyclic amino group of adenosine attacks the former methyl carbon via a conjugate addition reaction, and the enol A is obtained. Subsequent displacement of HCl and proton transfer will yield the product I. This proton transfer is energetically favorable, since the carbon-carbon double bond in I will be in resonance both with the unshared pair of electrons of the nitrogen atom and with the carbonyl group. When the reaction was performed in DMF, the only product detected was II. Previously, it has been reported that mucochloric acid and mucobromic acid, when reacted with primary amines in absolute ethanol, form 5-aminofuranones, similar to II (28). Although NMR studies of MCF have shown that in organic solvents the compound, like other CHFs, adopts the ring form, primarily (2729), trace amounts of the open form could be present and II would be formed by attack of the amino group of adenosine on the exposed aldehyde group, yielding a mixed acetal, that subsequently undergoes ring closure (Scheme 2). In aqueous solutions the same mechanism applies, and the higher yield of II at pH 7.4 than at pH 6.0 (Figure 2) may be due to the higher amount of the open form of MCF at higher pH. The ring-chain tautomerism of II was studied by recording the UV spectra of II dissolved in water at pH conditions ranging from pH 4.0 to 9.0. The UV spectra recorded at these conditions were all almost identical, and thus it is obvious that II does not tautomerize as readily as the hydroxyfuranones. The chromatographic properties of II on the C18 columns were not affected by change in eluent composition; II had approximately the same retention time when phosphate buffer at pH 4.6 or water was used with acetonitrile as eluent. This shows that II does not contain a free ionizable carboxyl group, and therefore, the furanone ring must be present also in aqueous solutions. 3 S. Knasmu ¨ ller, E. Zo¨hrer, L. Kronberg, M. Kundi, and R. SchulteHermann, submitted for publication.
708 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
The current work demonstrates that MCF modifies the base moiety of adenosine, by forming two structurally different adducts. It should be noted that the hydroxyfuranones MCA and MCF do not form structurally related adducts with adenosine. The reason for this is that the compounds have different groups attached to C-4; in MCF a methyl group is bound at C-4, while in MCA an easily displaced chlorine is found at C-4. The formation of the previously identified A, cA, cA,A, and oA,A adducts of MCA requires displacement of the chlorine at C-4 (21-23). In MCF, the methyl group at C-4 cannot be displaced, and the reaction does not proceed further from the coupling of the adenosine amino group to the aldehyde group of MCF, and subsequently II is obtained. The MCF product I is formed when the C-4 methyl group is attacked by adenosine, and obviously, an analogous product cannot be obtained from MCA. The difference in reaction of MCA and MCF with adenosine and possibly also with other nucleosides might be reflected in difference in the mechanism by which the compounds induce mutagenicity. Recently, Knasmu¨ller et al. studied the mutation spectra of MCA and MCF in S. typhimurium, and, in fact, found marked dissimilarities in the spectra of the compounds (30).3
Acknowledgment. This work was supported by a research grant from the Maj and Tor Nessling Foundation in Finland and by the Academy of Finland, Research Council for Environment and Natural Resources.
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