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Synthesis and stability of 2′-deoxyguanosine 3′-monophosphate adducts of dimethyl sulfate, ethylene oxide and styrene oxide
Chem.-Biol Interaction, 75 (1990)281- 292 Elsevier Scientific Publishers Ireland Ltd.
28]
SYNTHESIS AND STABILITY OF 2'-DEOXYGUANOSINE 3'-MON(~ PHOSPHATE ADDUCTS OF DIMETHYL SULFATE, ETHYLENE OXIDE AND STYRENE OXIDE
KARI HEMMINKI, ANNELI ALHONEN-RAATESALMI, PERTTI KOIVISTO and PAVEL VODICKA* Institute of Occupational Healt~ Topeliuksenkatu ~1 a A, 00~50 Helsinki [FinlandJ (Received December 19th, 1989) (Revision received March 15th, 1990) (Accepted March 16th, 1990)
SUMMARY
Deoxyguanosine 3'-monophosphate (dGMP) was alkylated at the 7-position by dimethyl sulfate, ethylene oxide and styrene oxide in aqueous media and glacial acetic acid, respectively, to yield reasonable quantities of the products, which were purified by HPLC. dGMP adducts are needed as standards for the ~P-postlabelling assay. The stability of the adducts was studied at 37 ° and neutral pH. The half-lives of disappearance of 7-methyl~iGMP and the/J-isomers of the styrene oxide adducts were about 250 min; 7-hydroxyethyl-dGMP and the a-isomers of the styrene oxide adducts had respective half-lives of 340 and 440 min. In all cases the main degradation product was the corresponding guanine adduct. The results demonstrate considerable lability of the 7-alkylation products of dGMP which has to be taken into consideration in devising the 82p-postlabelling assay.
Key words: Depurination -- 7-Methyl-dGMP - 7-(2-Hydroxyethyl)-dGMP Styrene oxide-dGMP adducts
INTRODUCTION
The 32p-postlabelling assay has been applied as a sensitive method to detect chemicals bound to DNA [1-5]. It has been most useful to detect the DNA-binding products of large non-polar carcinogens or of complex mixtures containing such constituents, including modifications of DNA in unexposed *Permanent address: Institute of Hygiene and Epidemiology, Srobarova 48, 10048 Prague 10, Czechoslovakia. 0009-2797/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
282 humans and experimental animals [6-8]. Examples of the adducts that have been chemically identified include species derived from polycyclic aromati( hydrocarbons [9,10], nitropyrenes [11], aldehydes and related compounds forming cyclic adducts [12] and styrene oxide [13,14]. The method has been applied to a limited extent to small, polar adducts: examples of such applica tions include 5-methylcytosine, O6-methylguanine [15], 8-hydroxyguanine [15 and thymine glycol [16]. An important group of DNA-adducts, 7-alkylation products of guanine, have found a very limited application so far, probably because such adducts are polar and chemically labile. Yet we have shown elsewhere that 7-methyl guanine adducts are quantitatively phosphorylated by T 4 polynucleotide kinase at sub-femtomolar quantities [17-19], and even their stable, ring-opened forms are phosphorylated but less efficiently. In order to develop seP-post labelling further towards various 7-alkylation products of guanine we des cribe here the syntheses of 7-methylated, 7-hydroxyethylated and 7-hydroxy phenylethylated adducts of 2'-deoxyguanosine 3'-monophosphate (dGMP) an¢ of 2'-deoxyguanosine 3'-,5'-diphosphate (dGdP). We also study the stabilit) of these adducts in conditions used for postlabelling to provide data foI methods development. MATERIALS AND METHODS
Synthesis and stability of 7-methyl-dGMP dGMP and dGdP (Sigma and Pharmacia), 2 mg/ml, were reacted with dimethyl sulfate (Fluka), 500 mM, in 0.5 M sodium phosphate buffer (pH 6.5 at room temperature for 1 h (dGMP) or for 30 min (dGdP); pH was main tained by adding 1 M NaOH. The methylated products were purified by HPLC in a Varian 5000 HPLC equipment with a reverse phase C-18, 4.6 mm × 250 mm Shandon ODS-Hypersil column, particle size 5 ~m. Sample volume was 0.5 ml; eluent was isocratic 50 mM ammonium formate buffer (pH 5.4) the pumping rate was 0.8 ml/min. The HPLC fractions were collected and characterized by UV absorption spectroscopy (Beckman DU 64) in neutra] (6.5), alkaline (13.0) and acidic (1.0) pH. The stability of the methylated products was tested in 66 mM Tris--HC] buffer (pH 7.4) at 37 °C. Samples were taken after 0, 30 min, 1 h, 3 h, 6 h and 24 h time intervals and analysed by HPLC as above.
Synthesis and stability of 7-(2-hydroxyethyl)-dGMP dGMP, 2.5 mg/ml, was incubated with 50 ~l of ethylene oxide (Fluka) in dry acetic acid in a screw cap test tube at a temperature below 10°C for about 20 h. Before use, glacial acetic acid (Merck) was dried by solidifying about 80% of it in an ice-salt-bath and decanting the mother liquor. The procedure was repeated twice. The reaction was stopped by evaporation oJ acetic acid and the excess of ethylene oxide by blowing under N 2. After evaporation, distilled water was added for HPLC purification.
283
HPLC equipment was an LKB 2150 HPLC pump with a controller and a spectral detector, fitted to a C-18 Spherisorb $ 5 0 D S 2 (column 4.6 x 150 ram, particle size 5 ~m) and a Shimadzu C-R3A integrator. The eluent was 50 mM ammonium formate buffer (pH 5.4) and pumping rate 1.0 ml/min. HPLC gradient program increased from 0°/0 methanol to 25% in 15 rain. The fractions collected were stored at - 2 0 °C and characterized as described above. The stability of the products was studied in 66 mM Tris--HCl buffer (pH 7.4) at 37°C, the samples for HPLC analysis (see above) were taken after 0, 1, 3, 6 and 24 h time intervals.
Synthesis and stability of 7-(2-hydroxy-l-phenylethyl)- and 7-(2-hydroxy-2phenylethyl)-dGMP dGMP, 1 mg/ml, was incubated with 86.7 mM styrene oxide (Aldrich, more than 98% pure) either in 10 mM sodium phosphate buffer (pH 6.0), containing 1 M NaC1, at 37 °C overnight or in glacial acetic acid (Merck) at room temperature for 1 h. After termination of the incubation, acetic acid was evaporated by blowing under N 2 and 500 ~l of 50 mM ammonium formate buffer (pH 5.1) was added. The excess of styrene oxide was removed by ether extraction. The reaction mixtures were purified by HPLC using a Varian 5500 HPLC equipment with a reverse phase C-18 4.6 mm × 150 mm Spherisorb ODS 2 column, particle size 5 ~m. Sample volume was 0.5 ml, the eluent was 50 mM ammonium formate buffer (pH 5.1) and the pumping rate was 1.0 ml/min. The methanol gradient was increased from 0% to 60% in 40 rain. The HPLC fractions were collected and characterized by UV absorption spectrometer (Beckman DU 64) in neutral (6.5), alkaline (13.0) and acidic (1.0) pH. The stability studies were carried out at 37°C in three different incubation media: 10 mM sodium phosphate buffer (pH 6.0) containing 1 M NaC1, 20 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl 2, or 66 mM Tris - H C I buffer (pH 7.4). Samples were drawn after 0, 1, 3, 6 and 24 or 26 h time intervals and analyzed as above. As an end-point, the aliquot of material was boiled for 10 rain and analyzed by HPLC showing the complete depurination. Half-lives of the products were obtained graphically from semi-logarithmic plots of time versus In A, where A is absorbance at 254 nm. RESULTS
Synthesis of 7-alkylated dGMPs The structures of the synthesized standards are shown in Fig. 1. The preparation of 7-methylated-dGMP and 7-methylated-dGdP was described earlier [17]. The products showed appropriate UV-spectra and they depurihated to 7-methylguanine upon boiling. The product yields were 50% for 7methyl-dGMP and 70% for 7-methyl-dGdP.
284
0
HO~H2 CH 2
0
R
0
HOCH 2 ~:H CsHs
R
CH 3
R
0
OHI CH2CHCsH5
R
Fig. 1. Structures of the synthesized standards: 7-(2-hydroxyethyD-dGMP (top left), 7-methyl dGMP (top right), 7-(2-hydroxy-l-phenylethyl)-dGMP (bottom left), 7(2-hydroxy-2-phenylethyl) dGMP (bottom right). R = deoxyribose 3'-monophosphate.
The HPLC analysis of the reaction mixture of ethylene oxide with dGMP in glacial acetic acid revealed that 7-(2-hydroxyethyl)-dGMP was one of the major products formed (Fig. 2, fraction a). The total yield of this product amounted to about 30%. The product was characterized by UV spectroscopy providing the typical 7-alkyl-dGMP spectra under various pHs [17] and it depurinated to 7-alkylguanine upon boiling. When styrene oxide reacted with dGMP in acetic acid, four isomeric products were isolated by HPLC (Fig. 3). The products were characterized by UV spectroscopy. Depurination (10 min of boiling) of the individual fractions gave rise to the corresponding a- and ~-7-alkylguanines as described elsewhere [20]. Fractions /~1 and fJ2 of Fig. 3 are the diastereomeric 7-(2-hydroxy-2-phenylethyl)-dGMPs while fractions a 1 and /32 are the diastereomeric 7-(2-hydroxy-l-phenylethyl)dGMPs. The yield of 7-alkylated products was substantially lower (5.6%) as compared to ethylene oxide; the individual diastereomers were formed in roughly similar proportions (f31 = 1.2%, ofI ~ - 1.2%,/32 = 1.8%, a 2 = 1.4°/o). Attempts were undertaken to prepare 7-alkyl-dGMP products by reacting styrene oxide with dGMP in aqueous medium (10 mM sodium phosphate buffer, pH 6.0, containing 1 M NaC1). Under such conditions the depurination was efficient (almost completed after a 24 h of incubation) signalled by the appearance of 7-alkylguanines.
285
lid 0,1
e
1'o TIME (min)
Fig. 2. HPLC purification of 7-(2-hydroxyethyl)-dGMP. e, dGMP; a, 7-(2-hydroxyethyl)-dGMP. The reaction was in glacial acetic acid below 10 ° for 20 h.
e
0( 1
,q. lid 04
\ 10
I
I
I
20
30
40
TIME (min)
Fig. 3. HPLC purification of 7-alkyl-dGMPs formed by styrene oxide, e, dGMP; ~,, 7-(2-hydroxy-2phenylethyl)-dGMP; a,, 7-(2-hydroxy-l-phenylethyl)-dGMP; /]~, 7-(2-hydroxy-2-phenylethyl-dGMP; ~2, 7-(2-hydroxyl-l-phenylethyl)-dGMP.
286
a
f i
,~ Lt~
A
!
B
¢q
a
I
I
I
10
20 TIME (mm)
30
40
Fig. 4. The depurination of7-methyl-dGMPin 66 mM Tris--HClbuffer(pH 7.4) at37°C as sna lyzed by HPLCintime0(A) and after6h(B).a, 7-methyl-dGMP;f,7-methylguanine.
The stability of 7-alkylated dGMPs and dGdPs The half-life of 7-methyl-dGMP was determined by HPLC and the main product formed was apparently 7-methylguanine (Fig. 4). 7-Methyl-dCdP depurinated slower (Fig. 5); in addition to the final product, 7-methylguanine, some intermediate 7-methyl-3'-dGMP and 7-methyl-5'-dGMP were also seen. The half-lives of disappearance of 7°methyl-dGMP and 7-methyl-dGdP were 230 and 380 rain, respectively (Fig. 6). 7-(2-Hydroxyethyl)-dGMP was transformed exclusively to 7-(2-hydroxyethyl)guanine (Fig. 7). The kinetics of depurination are shown in Fig. 8. The half-life of disappearance was 340 min. The rate of disappearance of the styrene-oxide-dGMP adducts was studied by HPLC. Figure 9 shows the concentrations of alkyl-dGMP and alkylguanine at two time points for one of the a and one of the/~ isomers, respectively. Essentially, the products are quantitatively converted to the corresponding guanine derivatives. The kinetics of disappearance of the starting material and appearance of the guanine derivatives are shown in Fig. 10. The/~-derivatives depurinate at a faster rate than the corresponding a-derivatives; the respective half-lives were 280 min (/~) and 440 min (a). The rates of depurination of two diastereomeric a- and ]3-7-alkyl-dGMPs formed by styrene oxide were determined under three different conditions (Table I). Several differences are apparent. Firstly, under all conditions
287
a
f .A
!
Lo
f a
L
h A
ii
2'o
1'0
4o
3O
TIME (min)
Fig. 5. The depurination of 7-methyl-dGdP in 66 mM T r i s - H C 1 buffer (pH 7.4) as analyzed by HPLC in time 0 (A) and after 6 h (B). a, 7-methyl-3'-dGdP; f, 7-methylguanine; h, 7-methyl-3'dGMP and 7-methyl-5'-dGMP.
loo ~ ~ _
~ _~4...____=__= ==-=_-~o---
/
,¢
10
/ 2 = 6h 2 4 '
,
04
! I
10 t
I
t
0
5
10
I
I
t
20
25
T I M E (h)
Fig. 6. Rate of disappearance of 7-methyl-dGMP ( . - . ) and 7-methyl-dGdP (O O) in 66 mM T r i s - H C l buffer (pH 7.4) at 37°C. The appearance of 7-methylguanine from 7-methyl-dGMP (A . . . . A) and 7-methyl-dGdP (o . . . . . e ) is also shown.
288 a
A
,q. tO ¢q
5
10 TIME (ram)
5
10
Fig. 7. The depurination of 7-(2-hydroxyethyl)-dGMP in 66 mM Trzs--HCI buffer (pH 7.4) as analyzed by HPLC in time 0 (A) and after 6 h (B). a, 7-(2-hydroxyethyl)-dGMP; f, 7-(2-hydroxyethyl) guanine.
100
,,"'" X - _- --~" - - '...... X - X -..... , .-X-- ~
~
A
ae
v
~
.
~
T
1/2 = 5h 40
/
10
L~ <
I I I I I I
I I
I
I
I
I
0
5
10
I
I
I
20
25
TIME (h) Fig. 8. Rate of depurmatlon of 7-(2-hydroxyethyl)-dGMP ( O - - O ) in 66 mM Trls--HCI buffer (pH 7.4) at 37°C and the appearance of 7-(2-hydroxyethyl) guamne ( × . . . . × ).
289 A 0(
B
to 04 ,(
~n 1 10
20
,,
30
4'0
TIME (rain) Fig. 9. The depurination of 7-alkyl-dGMPs formed by styrene oxide ,n 66 mM T n s - H C I buffer (pH 7.4) as analyzed by HPLC in time 0 { ) and after 6 h (. . . . ), a-isomer (A), /]-isomer (B)./], 7-(2-hydroxy-2-phenylethyl)-dGMP; a, 7-(2-hydroxy-l-phenylethyl)-dGMP;/]', 7-(2-hydroxy-2-phenylethyl) guanine; a', 7-(2-hydroxy-l-phenylethyl) guanine.
oo
~..-_--~--6
1 I
I
I
0
5
10
I
I
I
20
25
TIME {h)
Fig. 10. Rate of depurination of 7-alkyl-dGMPs formed by styrene oxide in 66 mM T r i s - H C l buffer (pH 7.4) at 37°C. (*) 7-(2-hydroxy-2-phenylethyl)-dGMP (/]); (O) 7-(2-hydroxy-l-phenylethyl~ dGMP (a); (&) 7-(2-hydroxy-2-phenylethyl) guanine (/]); (@) 7-(2-hydroxy-l-phenylethyl) guanine (a).
290 TABLE I THE STABILITY OF 7-ALKYLATED dGMPs AND 7-METHYLATED dGdP Temperature at 37oc. The values are expressed as means of at least two determinations. Compound
T1r2 disappearance (min)
7-Methyl-dGMP 7-Methyl-dGdP 7-Hydroxyethyl-dGMP 7-(2-Hydroxy-l-phenylethyl)-dGMP, % 7-(2-Hydroxy-2-phenylet hyl}-dGMP, f3~ 7-(2-Hydroxy-l-phenylet hyl)-dGMP, a 2 7-(2-Hydroxy-2-phenylet hyl)-dGMP,/~2
230 a 380 340 n.d. n.d. 440 280
n.d. b n.d. n.d. 260 160 270 150
n.d. ¢ n.d. n.d. 430 270 440 260
"66 mM T r i s - H C l buffer (pH 7.4). bn.d. = not determined; 10 mM sodium phosphate buffer (pH 6.0) containing 1 M NaCl. ~20 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl 2.
tested, the diastereomers (a 1 and %, fJ~ and f~2) depurinated at a similar rate, but essential difference was found between the positional isomers (i.e. in all cases/3-derivatives depurinated approximately 1 . 6 - 1 . 8 times faster than the a-derivatives). Secondly, there were differences among the incubation conditions tested. Although the difference between a- and f3-products was always constant, the rate of depurination was found to be substantially faster (1.7 times) in 10 mM sodium phosphate (pH 6.0) containing 1 M NaC1, than that in 66 mM T r i s - H C l buffer (pH 7.4) and in 20 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl 2. On the other hand, no difference was observed between 66 mM Tris--HCl buffer (pH 7.4) and 20 mM sodium succinate buffer (pH 6.0), containing 8 mM CaC12 {Table I). DISCUSSION
The present work describes synthesis of large quantities of 7-alkylated dGMP derivatives which can be used as substrates for the 32p-postlabelling assay. Ethylene oxide and styrene oxide derivatives can be produced in glacial acetic acid, conditions previously used for reactions with bases and nucleosides [21]. With dGMP and dGdP, our earlier experiments resulted in depurinated products, but when dry acetic acid is used in mild incubation conditions, large amounts of intact adducts could be isolated. Furthermore, aqueous reactions with ethylene oxide and styrene oxide yielded negligible quantities of 7-alkylation products of dGMP underlining the feasibility of the glacial acetic acid method, dGMP adducts have also been synthesized using chemically blocked nucleotides [14,15], but such methods are not suitable in the applied form for the 7-alkyl-dGMP derivatives because alkali-treatment is used in the final deblocking phase and this would lead to imidazole ringopening. The results of the kinetic studies are summarized in Table I. The first observation for all the compounds tested is that they almost exclusively
291 depurinated. All the adducts were tested in identical condition in 66 mM Tris--HCl (pH 7.4) at 37°C. 7-Methyl-dGMP and the ~ isomer of styrene oxide-derived dGMP had similar half-lives, i.e. about 250 rain. Interestingly, 7-methyl-dGdP was more stable than 7-methyl-dGMP, which may relate to the presence of the phosphate groups in both the 3'- and the 5'-position, thus resembling polynucleotide. We have shown elsewhere that depurination from polynucleotides (single-stranded DNA) is 20 times slower than that from deoxynucleosides [20,22]. Depurination from deoxynucleosides resembles that from deoxynucleoside 3'-monophosphates. In styrene-oxide modified dGMPs the/~ isomers depurinated almost twice as fast as the a isomers, analogous to the corresponding deoxyguanosine derivatives [20]. Ethylene oxide-modified dGMP depurinated at an intermediate rate. The present results show that 7-alkylated dGMPs are prone to depurination in the conditions used to digest DNA to nucleotides for postlabelling (e.g. 20 mM sodium succinate, pH 6, containing 8 mM CaCI2). The halflives of the monophosphate adducts ranged from 4 to 7 h. Judging from work with 7-alkyldeoxyguanosine, the present adducts are relatively stable 7alkylguanine derivatives and for many adducts shorter half-lives could be expected [22]. This indicates that unduly lengthy manipulations in the digestion of DNA and subsequent phosphorylation by T 4 kinase need to be avoided. ACKNOWLEDGEMENTS
The work was supported by the Medical Research Council and the Work Environment Fund of Finland. REFERENCES 1 2 3
4 5
6
7
K. Randerath, M.V. Reddy and R.C. Gupta, s2P-labeling test for DNA damage, Proc. Natl. Acad. Sci. USA, 78 (1981) 6126-6129. R.C. Gupta, M.V. Reddy and K. Randerath, ~P-postlabeling analysis of non-radioactive aromatic earcinogen-DNA adducts, Carcinogenesis, 3 (1982) 1081-1092. M.V. Reddy and K. Randerath, Nuclease Pl-meditated enhancement of sensitivity of 32p_ postlabelling test for structurally diverse DNA adducts. Carcinogenesis, 7 (1986) 15431551. R.C. Gupta and K. Earley, 82P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures. Carcinogenesis, 9 (1988) 1687-1693. K. Randerath, E. Randerath, T.F. Danna, K.L. van Golen and K.L. Putman, A new sensitive s2P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates, Carcinogenesis, 10 (1989) 1231 - 1239. K. Hemminki and K. Randerath, Detection of genetic interaction of chemzcals by biochemical methods: determination of DNA protein adducts, in: B.A. Fowler (Ed.), Mechanisms of Cell Injury: Implications for Human Health, Wiley, New York, 1987, pp. 209-227. D.H. Phillips, A. Newer and P.L. Grover, Aromatic DNA adducts in human bone marrow and peripheral blood leukocytes, Carcinogenesis, 7 (1986) 2071- 2075.
292 8
9
10 11 12 13 14
15
16 17 18 19 20 21 22
H. Bartsch, K. Hemminki and I.K. O'Neill (Eds.), Methods for Detecting DNA Damaging Agents in Humans: Apphcations in Cancer Epidemiology and Prevention, IARC Scientific Publication No. 89, International Agency for Research on Cancer, Lyon, 1988. H. Schmeiser, A. Dipple, M.E. Schurdak, E. Randerath and K. Randerath, Comparison of ~P-postlabeling and high pressure liquid chromatographic analyses for 7,12-dimethylbenz(a)anthracene-DNA adducts, Carcinogenesis, 9 (1988) 633-638. N.J. Gorelick and G.N. Wogan, Fluoranthene-DNA adducts: identification and quantification by an HPLC-S~P-postlabeling method, Carcinogenesis, 10 (1989) 1567-1577. A.K. Roy, K. EI-Bayoumy and S.S. Hecht, 32p-postlabeling analysis of 1-nitropyrene-DNA adducts in female Spraque-Dawley rats, Carcinogenesis, 10 (1989) 195-198. W.P. Watson and A.E. Crane, HPLC-Z2p-postlabelling analysis of 1,N6-ethenodeoxyadenosme and 3,N%thenodeoxycytidme, Mutagenesis, 4 (1989) 75-77. S.F. Liu, S.M. Rappaport, J. Rasmussen and W.J. BodeI1, Detection of styrene oxide-DNA adducts by ~P-postlabeling, Carcinogenesis, 9 (1988) 1401-1404. K. Pongracz, S. Kaur, A.L. Burhngame and W.J. Bodell, 08 subshtuted-2'-deoxyguanosine-3 Jphosphate adduets detected by 3~p-post-labeling of styrene oxide treated DNA, Carcinogenesis, 10 (1989) 1009-1013. V.L. Wilson, A.K. Basu, J.M. Essigmann, R.A. Smith and C.C. Harris, 06-Alkyldeoxyguan osine detection by ~P-postlabelhng and nucleotide chromatographic analysis, Cancer Res., 48 (1988) 2156-2161. M.E. Hegi, P. Sagelsdorff and W.K. Lutz, Detection by 32P-postlabeling of thymidine glycol in y-irradiated DNA, Carcinogenesis, 10 (1989) 43-47. K. Hemminki, Ring-opened 7-methylguanine nucleotides are resistant to nuclease P~ digestion and good substrates to polynucleotide kinase, Carcinogenesis, 10 (1989) 1761-1763. K. Hemminki and R. Mustonen, ~2P-postlabelling of 7-methylguanine adducts in DNA, Teratog. Carcinog. Mutag., in press. K. Hemminki, K. Peltonen and R. Mustonen, 32P-postlabelling of 7-methyl-dGMP, ringopened 7-methyl-dGMP and platinated dGpdG, Chem.-Biol. Interact., 74 (1990) 45--54. P. Vodicka and K. Hemminki, Identification of alkylation products of styrene oxide in single- and double-stranded DNA, Carcinogenesis, 9 (1988) 1657-1660. P. Brookes and P.D Lawley, The alkylation of guanosine and guanylic acid, J. Chem. Soc., (1961) 3923- 3928. K. Hemminh, K. Peltonen and P. Vodicka, Depurination from DNA of 7-methylguanine, 7-(2aminoethyl)-guanine and ring-opened 7-methylguamnes, Chem.-Biol. Interact., 70 (1989) 289 -- 303.
28]
SYNTHESIS AND STABILITY OF 2'-DEOXYGUANOSINE 3'-MON(~ PHOSPHATE ADDUCTS OF DIMETHYL SULFATE, ETHYLENE OXIDE AND STYRENE OXIDE
KARI HEMMINKI, ANNELI ALHONEN-RAATESALMI, PERTTI KOIVISTO and PAVEL VODICKA* Institute of Occupational Healt~ Topeliuksenkatu ~1 a A, 00~50 Helsinki [FinlandJ (Received December 19th, 1989) (Revision received March 15th, 1990) (Accepted March 16th, 1990)
SUMMARY
Deoxyguanosine 3'-monophosphate (dGMP) was alkylated at the 7-position by dimethyl sulfate, ethylene oxide and styrene oxide in aqueous media and glacial acetic acid, respectively, to yield reasonable quantities of the products, which were purified by HPLC. dGMP adducts are needed as standards for the ~P-postlabelling assay. The stability of the adducts was studied at 37 ° and neutral pH. The half-lives of disappearance of 7-methyl~iGMP and the/J-isomers of the styrene oxide adducts were about 250 min; 7-hydroxyethyl-dGMP and the a-isomers of the styrene oxide adducts had respective half-lives of 340 and 440 min. In all cases the main degradation product was the corresponding guanine adduct. The results demonstrate considerable lability of the 7-alkylation products of dGMP which has to be taken into consideration in devising the 82p-postlabelling assay.
Key words: Depurination -- 7-Methyl-dGMP - 7-(2-Hydroxyethyl)-dGMP Styrene oxide-dGMP adducts
INTRODUCTION
The 32p-postlabelling assay has been applied as a sensitive method to detect chemicals bound to DNA [1-5]. It has been most useful to detect the DNA-binding products of large non-polar carcinogens or of complex mixtures containing such constituents, including modifications of DNA in unexposed *Permanent address: Institute of Hygiene and Epidemiology, Srobarova 48, 10048 Prague 10, Czechoslovakia. 0009-2797/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
282 humans and experimental animals [6-8]. Examples of the adducts that have been chemically identified include species derived from polycyclic aromati( hydrocarbons [9,10], nitropyrenes [11], aldehydes and related compounds forming cyclic adducts [12] and styrene oxide [13,14]. The method has been applied to a limited extent to small, polar adducts: examples of such applica tions include 5-methylcytosine, O6-methylguanine [15], 8-hydroxyguanine [15 and thymine glycol [16]. An important group of DNA-adducts, 7-alkylation products of guanine, have found a very limited application so far, probably because such adducts are polar and chemically labile. Yet we have shown elsewhere that 7-methyl guanine adducts are quantitatively phosphorylated by T 4 polynucleotide kinase at sub-femtomolar quantities [17-19], and even their stable, ring-opened forms are phosphorylated but less efficiently. In order to develop seP-post labelling further towards various 7-alkylation products of guanine we des cribe here the syntheses of 7-methylated, 7-hydroxyethylated and 7-hydroxy phenylethylated adducts of 2'-deoxyguanosine 3'-monophosphate (dGMP) an¢ of 2'-deoxyguanosine 3'-,5'-diphosphate (dGdP). We also study the stabilit) of these adducts in conditions used for postlabelling to provide data foI methods development. MATERIALS AND METHODS
Synthesis and stability of 7-methyl-dGMP dGMP and dGdP (Sigma and Pharmacia), 2 mg/ml, were reacted with dimethyl sulfate (Fluka), 500 mM, in 0.5 M sodium phosphate buffer (pH 6.5 at room temperature for 1 h (dGMP) or for 30 min (dGdP); pH was main tained by adding 1 M NaOH. The methylated products were purified by HPLC in a Varian 5000 HPLC equipment with a reverse phase C-18, 4.6 mm × 250 mm Shandon ODS-Hypersil column, particle size 5 ~m. Sample volume was 0.5 ml; eluent was isocratic 50 mM ammonium formate buffer (pH 5.4) the pumping rate was 0.8 ml/min. The HPLC fractions were collected and characterized by UV absorption spectroscopy (Beckman DU 64) in neutra] (6.5), alkaline (13.0) and acidic (1.0) pH. The stability of the methylated products was tested in 66 mM Tris--HC] buffer (pH 7.4) at 37 °C. Samples were taken after 0, 30 min, 1 h, 3 h, 6 h and 24 h time intervals and analysed by HPLC as above.
Synthesis and stability of 7-(2-hydroxyethyl)-dGMP dGMP, 2.5 mg/ml, was incubated with 50 ~l of ethylene oxide (Fluka) in dry acetic acid in a screw cap test tube at a temperature below 10°C for about 20 h. Before use, glacial acetic acid (Merck) was dried by solidifying about 80% of it in an ice-salt-bath and decanting the mother liquor. The procedure was repeated twice. The reaction was stopped by evaporation oJ acetic acid and the excess of ethylene oxide by blowing under N 2. After evaporation, distilled water was added for HPLC purification.
283
HPLC equipment was an LKB 2150 HPLC pump with a controller and a spectral detector, fitted to a C-18 Spherisorb $ 5 0 D S 2 (column 4.6 x 150 ram, particle size 5 ~m) and a Shimadzu C-R3A integrator. The eluent was 50 mM ammonium formate buffer (pH 5.4) and pumping rate 1.0 ml/min. HPLC gradient program increased from 0°/0 methanol to 25% in 15 rain. The fractions collected were stored at - 2 0 °C and characterized as described above. The stability of the products was studied in 66 mM Tris--HCl buffer (pH 7.4) at 37°C, the samples for HPLC analysis (see above) were taken after 0, 1, 3, 6 and 24 h time intervals.
Synthesis and stability of 7-(2-hydroxy-l-phenylethyl)- and 7-(2-hydroxy-2phenylethyl)-dGMP dGMP, 1 mg/ml, was incubated with 86.7 mM styrene oxide (Aldrich, more than 98% pure) either in 10 mM sodium phosphate buffer (pH 6.0), containing 1 M NaC1, at 37 °C overnight or in glacial acetic acid (Merck) at room temperature for 1 h. After termination of the incubation, acetic acid was evaporated by blowing under N 2 and 500 ~l of 50 mM ammonium formate buffer (pH 5.1) was added. The excess of styrene oxide was removed by ether extraction. The reaction mixtures were purified by HPLC using a Varian 5500 HPLC equipment with a reverse phase C-18 4.6 mm × 150 mm Spherisorb ODS 2 column, particle size 5 ~m. Sample volume was 0.5 ml, the eluent was 50 mM ammonium formate buffer (pH 5.1) and the pumping rate was 1.0 ml/min. The methanol gradient was increased from 0% to 60% in 40 rain. The HPLC fractions were collected and characterized by UV absorption spectrometer (Beckman DU 64) in neutral (6.5), alkaline (13.0) and acidic (1.0) pH. The stability studies were carried out at 37°C in three different incubation media: 10 mM sodium phosphate buffer (pH 6.0) containing 1 M NaC1, 20 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl 2, or 66 mM Tris - H C I buffer (pH 7.4). Samples were drawn after 0, 1, 3, 6 and 24 or 26 h time intervals and analyzed as above. As an end-point, the aliquot of material was boiled for 10 rain and analyzed by HPLC showing the complete depurination. Half-lives of the products were obtained graphically from semi-logarithmic plots of time versus In A, where A is absorbance at 254 nm. RESULTS
Synthesis of 7-alkylated dGMPs The structures of the synthesized standards are shown in Fig. 1. The preparation of 7-methylated-dGMP and 7-methylated-dGdP was described earlier [17]. The products showed appropriate UV-spectra and they depurihated to 7-methylguanine upon boiling. The product yields were 50% for 7methyl-dGMP and 70% for 7-methyl-dGdP.
284
0
HO~H2 CH 2
0
R
0
HOCH 2 ~:H CsHs
R
CH 3
R
0
OHI CH2CHCsH5
R
Fig. 1. Structures of the synthesized standards: 7-(2-hydroxyethyD-dGMP (top left), 7-methyl dGMP (top right), 7-(2-hydroxy-l-phenylethyl)-dGMP (bottom left), 7(2-hydroxy-2-phenylethyl) dGMP (bottom right). R = deoxyribose 3'-monophosphate.
The HPLC analysis of the reaction mixture of ethylene oxide with dGMP in glacial acetic acid revealed that 7-(2-hydroxyethyl)-dGMP was one of the major products formed (Fig. 2, fraction a). The total yield of this product amounted to about 30%. The product was characterized by UV spectroscopy providing the typical 7-alkyl-dGMP spectra under various pHs [17] and it depurinated to 7-alkylguanine upon boiling. When styrene oxide reacted with dGMP in acetic acid, four isomeric products were isolated by HPLC (Fig. 3). The products were characterized by UV spectroscopy. Depurination (10 min of boiling) of the individual fractions gave rise to the corresponding a- and ~-7-alkylguanines as described elsewhere [20]. Fractions /~1 and fJ2 of Fig. 3 are the diastereomeric 7-(2-hydroxy-2-phenylethyl)-dGMPs while fractions a 1 and /32 are the diastereomeric 7-(2-hydroxy-l-phenylethyl)dGMPs. The yield of 7-alkylated products was substantially lower (5.6%) as compared to ethylene oxide; the individual diastereomers were formed in roughly similar proportions (f31 = 1.2%, ofI ~ - 1.2%,/32 = 1.8%, a 2 = 1.4°/o). Attempts were undertaken to prepare 7-alkyl-dGMP products by reacting styrene oxide with dGMP in aqueous medium (10 mM sodium phosphate buffer, pH 6.0, containing 1 M NaC1). Under such conditions the depurination was efficient (almost completed after a 24 h of incubation) signalled by the appearance of 7-alkylguanines.
285
lid 0,1
e
1'o TIME (min)
Fig. 2. HPLC purification of 7-(2-hydroxyethyl)-dGMP. e, dGMP; a, 7-(2-hydroxyethyl)-dGMP. The reaction was in glacial acetic acid below 10 ° for 20 h.
e
0( 1
,q. lid 04
\ 10
I
I
I
20
30
40
TIME (min)
Fig. 3. HPLC purification of 7-alkyl-dGMPs formed by styrene oxide, e, dGMP; ~,, 7-(2-hydroxy-2phenylethyl)-dGMP; a,, 7-(2-hydroxy-l-phenylethyl)-dGMP; /]~, 7-(2-hydroxy-2-phenylethyl-dGMP; ~2, 7-(2-hydroxyl-l-phenylethyl)-dGMP.
286
a
f i
,~ Lt~
A
!
B
¢q
a
I
I
I
10
20 TIME (mm)
30
40
Fig. 4. The depurination of7-methyl-dGMPin 66 mM Tris--HClbuffer(pH 7.4) at37°C as sna lyzed by HPLCintime0(A) and after6h(B).a, 7-methyl-dGMP;f,7-methylguanine.
The stability of 7-alkylated dGMPs and dGdPs The half-life of 7-methyl-dGMP was determined by HPLC and the main product formed was apparently 7-methylguanine (Fig. 4). 7-Methyl-dCdP depurinated slower (Fig. 5); in addition to the final product, 7-methylguanine, some intermediate 7-methyl-3'-dGMP and 7-methyl-5'-dGMP were also seen. The half-lives of disappearance of 7°methyl-dGMP and 7-methyl-dGdP were 230 and 380 rain, respectively (Fig. 6). 7-(2-Hydroxyethyl)-dGMP was transformed exclusively to 7-(2-hydroxyethyl)guanine (Fig. 7). The kinetics of depurination are shown in Fig. 8. The half-life of disappearance was 340 min. The rate of disappearance of the styrene-oxide-dGMP adducts was studied by HPLC. Figure 9 shows the concentrations of alkyl-dGMP and alkylguanine at two time points for one of the a and one of the/~ isomers, respectively. Essentially, the products are quantitatively converted to the corresponding guanine derivatives. The kinetics of disappearance of the starting material and appearance of the guanine derivatives are shown in Fig. 10. The/~-derivatives depurinate at a faster rate than the corresponding a-derivatives; the respective half-lives were 280 min (/~) and 440 min (a). The rates of depurination of two diastereomeric a- and ]3-7-alkyl-dGMPs formed by styrene oxide were determined under three different conditions (Table I). Several differences are apparent. Firstly, under all conditions
287
a
f .A
!
Lo
f a
L
h A
ii
2'o
1'0
4o
3O
TIME (min)
Fig. 5. The depurination of 7-methyl-dGdP in 66 mM T r i s - H C 1 buffer (pH 7.4) as analyzed by HPLC in time 0 (A) and after 6 h (B). a, 7-methyl-3'-dGdP; f, 7-methylguanine; h, 7-methyl-3'dGMP and 7-methyl-5'-dGMP.
loo ~ ~ _
~ _~4...____=__= ==-=_-~o---
/
,¢
10
/ 2 = 6h 2 4 '
,
04
! I
10 t
I
t
0
5
10
I
I
t
20
25
T I M E (h)
Fig. 6. Rate of disappearance of 7-methyl-dGMP ( . - . ) and 7-methyl-dGdP (O O) in 66 mM T r i s - H C l buffer (pH 7.4) at 37°C. The appearance of 7-methylguanine from 7-methyl-dGMP (A . . . . A) and 7-methyl-dGdP (o . . . . . e ) is also shown.
288 a
A
,q. tO ¢q
5
10 TIME (ram)
5
10
Fig. 7. The depurination of 7-(2-hydroxyethyl)-dGMP in 66 mM Trzs--HCI buffer (pH 7.4) as analyzed by HPLC in time 0 (A) and after 6 h (B). a, 7-(2-hydroxyethyl)-dGMP; f, 7-(2-hydroxyethyl) guanine.
100
,,"'" X - _- --~" - - '...... X - X -..... , .-X-- ~
~
A
ae
v
~
.
~
T
1/2 = 5h 40
/
10
L~ <
I I I I I I
I I
I
I
I
I
0
5
10
I
I
I
20
25
TIME (h) Fig. 8. Rate of depurmatlon of 7-(2-hydroxyethyl)-dGMP ( O - - O ) in 66 mM Trls--HCI buffer (pH 7.4) at 37°C and the appearance of 7-(2-hydroxyethyl) guamne ( × . . . . × ).
289 A 0(
B
to 04 ,(
~n 1 10
20
,,
30
4'0
TIME (rain) Fig. 9. The depurination of 7-alkyl-dGMPs formed by styrene oxide ,n 66 mM T n s - H C I buffer (pH 7.4) as analyzed by HPLC in time 0 { ) and after 6 h (. . . . ), a-isomer (A), /]-isomer (B)./], 7-(2-hydroxy-2-phenylethyl)-dGMP; a, 7-(2-hydroxy-l-phenylethyl)-dGMP;/]', 7-(2-hydroxy-2-phenylethyl) guanine; a', 7-(2-hydroxy-l-phenylethyl) guanine.
oo
~..-_--~--6
1 I
I
I
0
5
10
I
I
I
20
25
TIME {h)
Fig. 10. Rate of depurination of 7-alkyl-dGMPs formed by styrene oxide in 66 mM T r i s - H C l buffer (pH 7.4) at 37°C. (*) 7-(2-hydroxy-2-phenylethyl)-dGMP (/]); (O) 7-(2-hydroxy-l-phenylethyl~ dGMP (a); (&) 7-(2-hydroxy-2-phenylethyl) guanine (/]); (@) 7-(2-hydroxy-l-phenylethyl) guanine (a).
290 TABLE I THE STABILITY OF 7-ALKYLATED dGMPs AND 7-METHYLATED dGdP Temperature at 37oc. The values are expressed as means of at least two determinations. Compound
T1r2 disappearance (min)
7-Methyl-dGMP 7-Methyl-dGdP 7-Hydroxyethyl-dGMP 7-(2-Hydroxy-l-phenylethyl)-dGMP, % 7-(2-Hydroxy-2-phenylet hyl}-dGMP, f3~ 7-(2-Hydroxy-l-phenylet hyl)-dGMP, a 2 7-(2-Hydroxy-2-phenylet hyl)-dGMP,/~2
230 a 380 340 n.d. n.d. 440 280
n.d. b n.d. n.d. 260 160 270 150
n.d. ¢ n.d. n.d. 430 270 440 260
"66 mM T r i s - H C l buffer (pH 7.4). bn.d. = not determined; 10 mM sodium phosphate buffer (pH 6.0) containing 1 M NaCl. ~20 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl 2.
tested, the diastereomers (a 1 and %, fJ~ and f~2) depurinated at a similar rate, but essential difference was found between the positional isomers (i.e. in all cases/3-derivatives depurinated approximately 1 . 6 - 1 . 8 times faster than the a-derivatives). Secondly, there were differences among the incubation conditions tested. Although the difference between a- and f3-products was always constant, the rate of depurination was found to be substantially faster (1.7 times) in 10 mM sodium phosphate (pH 6.0) containing 1 M NaC1, than that in 66 mM T r i s - H C l buffer (pH 7.4) and in 20 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl 2. On the other hand, no difference was observed between 66 mM Tris--HCl buffer (pH 7.4) and 20 mM sodium succinate buffer (pH 6.0), containing 8 mM CaC12 {Table I). DISCUSSION
The present work describes synthesis of large quantities of 7-alkylated dGMP derivatives which can be used as substrates for the 32p-postlabelling assay. Ethylene oxide and styrene oxide derivatives can be produced in glacial acetic acid, conditions previously used for reactions with bases and nucleosides [21]. With dGMP and dGdP, our earlier experiments resulted in depurinated products, but when dry acetic acid is used in mild incubation conditions, large amounts of intact adducts could be isolated. Furthermore, aqueous reactions with ethylene oxide and styrene oxide yielded negligible quantities of 7-alkylation products of dGMP underlining the feasibility of the glacial acetic acid method, dGMP adducts have also been synthesized using chemically blocked nucleotides [14,15], but such methods are not suitable in the applied form for the 7-alkyl-dGMP derivatives because alkali-treatment is used in the final deblocking phase and this would lead to imidazole ringopening. The results of the kinetic studies are summarized in Table I. The first observation for all the compounds tested is that they almost exclusively
291 depurinated. All the adducts were tested in identical condition in 66 mM Tris--HCl (pH 7.4) at 37°C. 7-Methyl-dGMP and the ~ isomer of styrene oxide-derived dGMP had similar half-lives, i.e. about 250 rain. Interestingly, 7-methyl-dGdP was more stable than 7-methyl-dGMP, which may relate to the presence of the phosphate groups in both the 3'- and the 5'-position, thus resembling polynucleotide. We have shown elsewhere that depurination from polynucleotides (single-stranded DNA) is 20 times slower than that from deoxynucleosides [20,22]. Depurination from deoxynucleosides resembles that from deoxynucleoside 3'-monophosphates. In styrene-oxide modified dGMPs the/~ isomers depurinated almost twice as fast as the a isomers, analogous to the corresponding deoxyguanosine derivatives [20]. Ethylene oxide-modified dGMP depurinated at an intermediate rate. The present results show that 7-alkylated dGMPs are prone to depurination in the conditions used to digest DNA to nucleotides for postlabelling (e.g. 20 mM sodium succinate, pH 6, containing 8 mM CaCI2). The halflives of the monophosphate adducts ranged from 4 to 7 h. Judging from work with 7-alkyldeoxyguanosine, the present adducts are relatively stable 7alkylguanine derivatives and for many adducts shorter half-lives could be expected [22]. This indicates that unduly lengthy manipulations in the digestion of DNA and subsequent phosphorylation by T 4 kinase need to be avoided. ACKNOWLEDGEMENTS
The work was supported by the Medical Research Council and the Work Environment Fund of Finland. REFERENCES 1 2 3
4 5
6
7
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9
10 11 12 13 14
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16 17 18 19 20 21 22
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