Chem. Res. Toxicol. 1995, 8, 316-320
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Alkylation of DNA by the Nitrogen Mustard Bis(2-chloroethy1)methylaminet Martin R. Osborne,*t* Derry E. V. Wilman,§ and Philip D. Lawleyt Section of Molecular Carcinogenesis and CRC Centre for Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Received September 26, 1994@
Alkylation of DNA by the nitrogen mustard bis(2-chloroethy1)methylamine(mechlorethamine; HN2) gave four principal products, derived by mono-alkylation of guanine a t N-7 and adenine at N-3 and by cross-linking of guanine to guanine or guanine to adenine a t these positions. These products were isolated by hydrolysis from DNA a t neutral pH, followed by ion-exchange chromatography on SP-Sephadex and reversed phase chromatography on ODs. They were characterized by identification with products from the reaction of nitrogen mustard with adenine or deoxyguanylic acid, and by their U V , mass, and proton magnetic resonance spectra.
Introduction The nitrogen mustard bis(2-chloroethy1)methylamine (I, Figure 1; also known as HN2, mustine, mechlorethamine, chloromethine, or 2-chloro-N-(2-chloroethyl)-Nmethylethanamine) is a reactive alkyating agent which (as its hydrochloride) has been used in cancer chemotherapy for 50 years (11. It is thought to act by covalent bonding to DNA and especially by bifunctional reaction to cause interstrand cross-linking (2). Despite the long experience of its use and knowledge of its biological properties, the chemistry of reaction of this substance with DNA has been imperfectly understood, and not all of the products have been characterized. Brookes and Lawley showed that the principal site of reaction in DNA is the 7-position of guanine (3) and synthesized one of the products, the cross-linked adduct bis(2-(guanin-7-yl)ethy1)methylamine(GMG, Figure 11, by reaction of I with guanylic acid, followed by degradation with hydrochloric acid (4). Kallama and Hemminki (5, 6) prepared the mono-adduct N-(2-chloroethyl)-N-(2-(7-guanosiny1)ethyl)methylamine (riboside of GMC1, Figure 1)by reaction of I with guanosine in trifluoroethanol and confirmed its structure by U V and proton magnetic resonance spectroscopy and by observing loss of tritium when the synthesis was done using [8-3Hlguanosine. But the occurrence of other adducts in DNA and their relative abundance have not been reported. Here we describe methods for the separation of adducts of bis(2-chloroethy1)methylaminewith guanine and adenine and characterization of further reaction products.
Materials and Methods High molecular weight DNA from salmon testes, "type 111", and mechlorethamine (nitrogen mustard, I) hydrochloride were obtained from Sigma Chemical Co. (Poole, U.K.). Caution:
* To whom correspondence should be addressed.
+ Abbreviations: GMC1, N-(2-chloroethyl)-N-(2-(7-guaninyl)ethyl)methylamine; GMOH, N-(2-hydroxyethyl)-N-(2-(7-guaninyl)ethyl)methylamine; GMG, bis(2-(guanin-7-yl)ethyl)methylamine; AMC1, N-(2chloroethyl)-N-(2-(3-adeninyl)ethyl)methylamine; AMOH, N42-hydroxyethyl)-N-(2-(3-adeninyl)ethyl)methylamine;GMA, N-(2-(3-adeninyl)ethyl)-N-(2-(7-guaninyl)ethyl)methylamine; see Figure 1. Section of Molecular Carcinogenesis. 6 CRC Centre for Cancer Therapeutics. Abstract published in Advance ACS Abstracts, February 1, 1995.
*
@
GMCl (X-CI)
I
GMOH (X-OH)
GMG
AMOH
CH,
GMA
GMM
Figure 1. Structure of nitrogen mustard (I) and products isolated from DNA alkylated with it.
nitrogen mustard is toxic and carcinogenic and can be absorbed through the skin; exercise due care in handling. Liquid chromatography was carried out using Pharmacia FPLC or Waters HPLC equipment. Elution was monitored with a Pharmacia UV-1 or Waters 440 absorbance detector. The systems used were as follows. (i)Cation Exchange Chromatography on SP-Sephadex C25 (Pharmacia, St. Albans, U.K.). The column, 200 x 13 mm, was eluted a t 2 m u m i n with buffers making a pH gradient: (A) 0.15 M acetic acid + 0.05 M sodium acetate (pH 4.3) and (B) 0.01 M Na2HP04 0.01 M Na3P04 (pH 11.7). (ii) Reversed Phase Chromatography. The column, 250 x 4.6 mm Nucleosil5 ODS (Fisons, Loughborough, U.K.), was eluted with a linear gradient from 0.05 M ammonium formate in water (pH 6) to 30%or 40% methanol in water, in 40 min a t 1 mumin. (iii) Ion-Pair Chromatography. The column, a p-Bondapak cartridge (Type 8MBC18,lO pm) and Z-module from Waters (Watford, U.K.), was eluted initially with 25 mM tetrabutylammonium hydroxide adjusted to pH 4.4 or 6.6 with acetic acid (isocratic for 5 min after injection) and, as the final solvent, the same buffer containing 95% (v/v) methanol (linear gradient from 5 to 50 min at 1 m u m i n , collecting 1mL fractions). Mass spectroscopywas carried out using a Finnigan TSQ 700 triple-quadrupole mass spectrometer fitted with an electrospray ion source as described before (7)or by injecting the compound in glycerol solution into a VG7070H spectrometer (fast atom
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0893-228x/95/2708-0316$09.00/0 0 1995 American Chemical Society
Nitrogen Mustard and DNA
Chem. Res. Toxicol., Vol. 8, No. 2, 1995 317
bombardment). Proton magnetic resonance spectra of the compounds in deuterium oxide solution were obtained using a Bruker AC250 spectrometer. Reaction with dGMP. Nitrogen mustard (0.25 g; I, HCl salt) and 0.25 g of sodium 5'-deoxyguanylate were dissolved in 10 mL of dimethyl sulfoxide and 10 mL of 0.12 M sodium acetate, and the pH was adjusted to 6 by adding 1 M HC1 (ca 0.6 mL). The mixture was kept at 37 "C for 28 days, to allow complete reaction and loss of chlorine from the reagent. The precipitate was spun down, and the yellow supernatant was fractionated by chromatography on SP-Sephadex, in 10 lots of 2 mL, using the gradient A to 80% B in 20 min and then to 100% B in a further 40 min. Three fractions were collected: Gl(37-42 min), a mixture of the bis-adduct GMG and unidentified substances; G2 (47-52 min), the mono-adduct N42hydnryethyl)-N-(2-(7-guaninyl)ethyl)methylamhe(GMOH); and G3 (55-58 min). These were each desalted and concentrated using Waters Sep-Pak ODS cartridges prior to analysis. Reaction with Deoxyguanosine. Nitrogen mustard (11 mg; I, HCl salt) and 4.6 mg of deoxyguanosine in 1 mL of 0.25 M sodium acetate were incubated at 37 "C for 3 h. Sodium chloride (56 mg) was added, and the mixture heated to 95 "C for 30 min to remove deoxyribose from the products. They were separated as for d G M P the monoadduct GMCl was eluted a t 35-40 min. It is unstable in aqueous solution (see Results). Reaction with Adenine. Adenine (35 mg) was dissolved in 35 mL of hot water, and then 0.35 mL of 0.5 M sodium acetate, 70 mg of mustard (I, HC1 salt), and 0.16 mL of 1 M NaOH (to bring the pH to 6.5) were added. The mixture was kept a t 37 "C for 22 h, adding NaOH a s necessary to keep the pH above 6. The mixture was acidified with 0.3 mL of acetic acid and separated in 3 mL portions on SP-Sephadex, as for dGMP. This removed the starting materials (adenine was eluted at 30 min), but the products were poorly resolved; most were eluted in a broad band at 50-60 min. Reaction with DNA DNA (150 mg) was dissolved in 25 mL of water, the solution was treated with 150 mg of mustard (I, HC1 salt), and 5 mL of 0.5 M sodium acetate and 0.2 mL of 1 M NaOH were added to adjust the pH to 7.0. The mixture was kept a t 37 "C for 4 h, adding solid NaOAc at 0.7 h to restore the pH t o 7. Aliquots of 5 mL were heated to 95 "C for 30 min, acidified with 50 pL of acetic acid 2 mL of water, and fractionated on SP-Sephadex. The column was eluted with solvent A for 20 min, which eluted unreacted materials, and then with a pH gradient as for dGMP. Estimation of Chloroethyl Groups in DNA. Samples (0.5 mL) of a mustard DNA reaction mixture (as above) were treated with 1.3 mL of 3% NH40Ac in ethanol to precipitate the DNA. The DNA was washed, dried, and redissolved in 0.5 mL of water. The unreacted chloroethyl groups were estimated by reaction with p(nitrobenzy1)pyridine (20 mg of 4-(p-nitrobenzy1)pyridine in 1 mL of 40% ethanol-water containing 0.05 M Tris-HC1, pH 7.4; 60 "C, 5 h), adding 0.5 mL of triethylamine, and measuring the blue color (600 nm).
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Results Treatment of DNA with nitrogen mustard in vitro resulted in a number of products, which were released when the DNA was subsequently heated or treated with acid. To separate these products in sufficient quantity for characterization, we adopted two successive chromatographic steps. The products were first freed of starting materials and partly resolved by cation exchange on SP-Sephadex, and then the fractions from this were purified by reversed phase chromatography on an ODS column. Figure 2 shows the elution of alkylpurines from DNA reacted with nitrogen mustard, as detected by their W absorption. Band I1 contained little material and was not further investigated. The components of the other bands were separated and characterized as follows.
f
-
P In N
a
c 6
g P
a
-20
20
40
time,
60
minutes
Figure 2. Separation on SP-Sephadex of alkylated purines from DNA treated with nitrogen mustard, as detected by their
U V absorbance. Unreacted DNA came straight through the column (peak I). 0.0
--
D C
t
!!
-E
-
-0.5
c
-m -1.0
0
20
40
time,
60 hours
BO
Figure 3. Upper line: rate of hydrolysis of the mustardguanine adduct GMCl (Figure 1)to GMOH a t 37 "C a t pH 7. The mixture was analyzed by chromatography on ODs, and the ratio r = GMCU(GMCl+ GMOH) was determined from the area under the respective peaks on the chromatogram. The data are plotted as log((r - r..)/(ro - r 3 ) . Lower line: rate of hydrolysis at 37 "C of chloroethyl groups in DNA alkylated with nitrogen mustard, as estimated by the colored product obtained with p(nitrobenzy1)pyridine. The data are plotted a s log((A - A-)/ (A0 - A J ) , where A = absorbance a t 600 nm, against tims (after allowing 1h for maximum reaction).
Band I11 contained the bis(guanine) adduct GMG and unhydrolyzed monoadduct GMC1. These were separable by chromatography on ODs. This was not without difficulty, because GMG tends to precipitate from solution a t neutral pH and, if desalted using a Sep-Pak ODS cartridge, was recovered only in moderate yield. GMG thus obtained was chromatographically identical to that obtained from dGMP, and to the substance characterized by Brookes and Lawley (4). The appearance of GMCl was unexpected, as one might have expected this compound to have been all hydrolyzed to GMOH when the DNA was heated. We prepared GMCl from deoxyguanosine (see Methods) in order to determine its stability in aqueous solution. The product was incubated a t 37 "C in 0.02 M sodium acetatef0.05 M sodium phosphate (pH 7.0), and a t intervals aliquots were analyzed by chromatography on ODs. Disappearance of GMCl (Figure 3, upper line) followed first order kinetics, with a half-life of 16 h a t pH 7 (33 h a t pH 6). The same product in DNA is hydrolyzed faster. We estimated the
318 Chem. Res. Toxicol., Vol. 8, No. 2, 1995
Osborne et al.
adenine with nitrogen mustard (see Methods). The mixture of products from this reaction was separated by reversed phase chromatography into six components, Al-A6. The major product, Al, accounted for 58% of this mixture. It was identical in UV spectrum and chromatographic behavior to the principal component of band VI from DNA. The UV spectrum had Am= 274 nm and Amin 245 nm a t neutral pH, and ,A 275 in acid (pH 11, with a spectroscopic pK, (determined by the change in A,,o) of about 5.7. These properties are close to those of 3--_230, methyladenine (273.5 nm; 243 nm; 274 nm; 5.7, respec250 270 290 310 tively). The high Amax in acid shows that A1 was not a w m v d e n p l h , nm 1-, N6- or 9-alkyladenine, and the pKa is higher than that Figure 4. Comparison of the UV spectrum of the cross-linked of 7-methyladenine (pK, ca. 3.5). The mass spectrum adduct GMA with theoretical spectra obtained by adding the spectra of (i)7-methylguanine+ 3-methyladenine or (ii)7-meth(fast atom bombardment in glycerol) showed peaks at ylguanine + 7-methyladenine.All spectra at pH 1. m l z 237 (MH+,loo%), 238 (22%), 187 (22%),149 (22%), 136 ((adenine>H+,25%) and 102 ((hydroxyethy1)methyrate of conversion in DNA by analyzing alkylated DNA laziridinium, 83%). The proton magnetic resonance for chloroethyl groups, which could be determined by spectrum had singlets a t 6 = 8.41 (2-adenine), 8.07 (8their reaction with p(nitrobenzy1)pyridine. The results adenine), and 2.42 (NCH3) and triplets a t 4.49 ((ade(Figure 3, lower line) show loss of chlorine, with a halfnine)CHz; J = 6.61, 3.66 (CHzOD; J = 6.01, 3.12 (6.61, life of about 1.8 h. This corresponds to loss from both and 2.72 (6.0). After heating the solution to 75 "C for 7 GMCl and a smaller amount of the corresponding addays, the only change was a reduction in the band a t 6 enine adduct N-(2-chloroethyl)-N-(2-(3-adeninyl)ethyl)- = 8.41. The exchange of this 2-proton indicates substitumethylamine (AMC1). Kallama and Hemminki (6)estition at the 1- or 3-position of adenine. mated the half-life of the free nucleoside N-(2-chloroethyl)The minor products of alkylation of adenine, A2-A6, N-(2-(7-deoxyguanosinyl)ethyl)methylamineas 0.65 h in were not found among the products of alkylation of DNA. 0.01 M phosphate buffer (pH 7.4) a t 27 "C. A2 (13%of the total) had its UV absorption maximum In both the rate of initial reaction with DNA and the a t 262 nm, a spectroscopic pKa a little over 3, and a mass hydrolysis of the second chlorine of the mustard, the spectrum like that of A1 and was probably the 9-alkylaliphatic nitrogen mustard reacts faster than the aroadenine. A3-A6 were not identified; none of them matic analogue melphalan (7). This accords with the appeared to be a 7-alkyladenine, which might have been results for alkylation of DNA in human cells, where the expected as another product of the alkylation of adenine. maximum number of cross-links was reached within the Band VI1 contained products which absorbed strongly first hour of treatment with nitrogen mustard ( 2 ) . to the cation exchange column, suggesting that they were Bands IV and V were poorly separated from each other positively charged a t neutral pH. The mixture was on SP-Sephadex but easily separated by pooling the resolved on ODS into five components. One of these material and rechromatographing it on ODs. IV was the (VIIa) was identical to a substance similarly isolated from cross-linked adduct GMA (Figure l), as identified by the fraction G3 from the reaction of nitrogen mustard with following evidence. The mass spectrum showed the dGMP. This was clearly a 7-alkylguanine, from its UV molecular ion a t mlz 370 (MH+, 100%;C15H19N110 + H+), spectrum; Am= 284, Amin 261 nm a t pH 7; Amax 253 a t pH and also 371 (17%) and 237 (30%); MH+ gave daughter 1. The mass spectrum (by electrospray injection) showed ions at 235 (83%; guaninylethylaziridinium) and 178 a molecular ion mlz 354 (C15HzaN703+)with daughter (20%; (vinylguanine>H+). The UV spectrum showed A, ions a t 279 and 102. This indicates a combination of two at 274 (pH 1)or 276 (pH 4.4); it was almost identical with molecules of nitrogen mustard with one guanine. One that calculated by adding the spectra of 7-methylguanine possible structure, a 1,7-dialkylguanine, was ruled out and 3-methyladenine and was different from that obby the observation of a change of spectrum a t pH 9.0, tained by adding those of 7-methylguanine and 7-methshowing that the ionizable proton a t N-1 was still yladenine (Figure 4; similar results a t pH 4.4). The present. A hypothetical structure for VIIa is shown in proton magnetic spectrum showed singlets a t 6 = 7.98 Figure 1,"GMM". For an alkylating agent to react with and 7.94 (2-adenine, 8-adenine) and 7.70 (8-guanine)-cf. itself is unusual, but the existence of dimeric forms of 3-methyladenine (2-H a t 8.3 and 8-H a t 8.1) and 7-ethnitrogen mustard has been postulated t o explain the ylguanine (8-H at 7.9); triplets a t 4.37 and 4.17 (purine kinetics of its hydrolysis a t pH 8 (8). GMM could be CHz), 3.04 and 2.95 (MeNCHZ), all J = 5.5 cps; and a formed by reaction of such a dimer with DNA or by singlet a t 2.47 (NCH3). reaction of GMCl or GMOH with a second molecule of Band V was GMOH, as shown by its spectrum (Amm = nitrogen mustard. 285 nm; 7-ethylguanine has A, = 284) and its identity The other components of VI1 were not identified. The to the principal adduct prepared from dGMP and nitromost abundant of these, VIIb, also had the UV spectrum gen mustard (G2, Methods). The mass spectrum of G2 of a 7-alkylguanine (Amm 285 and 245 nm a t pH 7, 254 (fast atom bombardment in glycerol) showed the following nm a t pH 1);the principal peaks in the mass spectrum ions: m / z 253 (MH+,100%;C10H&602 H+),207 (60%; were a t mlz 454,354, and 142, again suggesting multiple M - CHzCHzOH), 275 (27%; MNa+), 254 (19%),and 186 reaction of the mustard with guanine. (19%). The properties of the principal products isolated from Band VI was the 3-alkyladenine N-(2-hydroxyethyl)nitrogen mustard treated DNA, and their relative proN-(2-(3-adeninyl)ethyl)methylamine (AMOH; Figure 1). portions, are summarized in Table 1. This compound was also obtained by alkylation of
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Chem. Res. Toxicol., Vol. 8, No. 2, 1995 319
Nitrogen Mustard and DNA GMCl tR(1)” tR(2) tR(3) tR(4) A, nm yieldb
Table 1. Properties of the Principal Nitrogen Mustard-Purine Adducts GMOH GMG GMM AMOH GMA A2 VIIb
39 37
48 19 7
285
285 63
13
38 39 18 27 285 23
57 36 11 284 8
53 33 9 25 274 25
45 38 11 27 275 8
53 37
57 26 8
262 0
285 9
gua
ade
20 10 9 11 246
31 20 16 18 261
a tR = retention time, minutes: (1)on SP-Sephadex, timed from the start of the pH gradient; (2) on ODS, using a 0-30% methanol gradient over 40 min; (3)on ODS with a n ion-pairing agent, pH 4.4; (4) the same as (3) a t pH 6.6. The retention times of guanine and adenine are given for comparison. Yield: 0. D. units obtained from 100 mg of DNA 100 mg of I. The quantities of GMCl and GMOH are combined as GMOH.
*
Discussion We have shown that the main products released on heating nitrogen mustard treated DNA are derived by alkylation of guanine and adenine. There are probably further reaction products which are not released from the DNA by heating, such as phosphotriesters and alkylcytosines; but our experience with the aromatic nitrogen mustard melphalan (7) suggests that such products account for less than 10% of the total reaction with DNA. The principal sites of attack of nitrogen mustard on DNA are the N-7 of guanine and N-3 of adenine, in a ratio of about 86:14. In this respect the mustard resembles the simple alkylating agent dimethyl sulfate, where the proportion of alkylation a t adenine is 17% (9). The proportion of alkylation a t adenine is a little lower than that found with melphalan (30%;7). Both the guanine and adenine adducts are lost spontaneously from the DNA and should give rise to apurinic sites. Thus a DNA fragment treated with nitrogen mustard suffered depurination a t guanine sites; the amount of reaction at a given site was determined by its neighbors in the DNA sequence (10). However, reaction a t adenine did not appear to generate apurinic sites (11). The reason for this is unclear. It may be that reaction at adenine is considerable a t high ratios of mustard to DNA, such as we have used here, and negligible a t the lower dose used in the sequence specificity studies. This seems unlikely, because (a) simple alkylating agents like dimethyl sulfate react to a considerable extent with adenine, even a t low dosage, and (b) our experiments with melphalan showed a similar proportion of reaction a t adenine a t high and low doses (7) and in cells in culture (unpublished work). The alternative explanations, that reaction a t adenine only occurs a t rare DNA sequence contexts or that the adduct can be released as AMOH without leaving a normal abasic site, are also implausible. The formation of the guanine-guanine cross-linked product GMG was reported by Brookes and Lawley over 30 years ago and is to be expected, as the 7-positions of nearby guanines are suitably spaced to allow such crosslinkage to occur. This could theoretically be between adjacent guanines on opposite strands, in 5’-GC-3’ sequences ( 3 ) ,but has been found to occur more readily when the guanines are separated by one base, in 5’-GNC3’ sequences (12, 13). The five atom bridge between the two guanines in GMG is not quite long enough to connect such nonadjacent guanines in DNA without some distortion of the helical structure (14). Cross-linking of adenine to guanine, however, is unexpected. This is because the sites of alkylation are in different “grooves” of the DNA double helix, N-3 of adenine being in the minor groove and N-7 of guanine in the major groove. Therefore, adenine-guanine cross-
+
linking would require considerable distortion of the double helical conformation of DNA. It may be that the observed cross-linking occurred during hydrolysis of the DNA, by reaction through the chloroethyl groups of bases released from the DNA before reaction of the “second arm” of the mustard group. To test this possibility, we treated DNA with nitrogen mustard for a long period (7 days a t 37 “C)before heating and analysis as before. The proportion of the cross-linked guanine-adenine product (GMA) in the products, 4% of the total, was the same as that found afier 3 h of reaction. It would seem, therefore, that the formation of GMA takes place in native DNA. Our methods cannot prove that the cross-link exists intact in alkylated DNA, as established for GMG crosslinks in oligonucleotides (14). It is possible that some AMCl becomes released from the DNA during incubation and attacks another DNA molecule to yield GMA. But we presume that whatever the mechanism, if it happens a t 37 “C in vitro, it must also occur in the living cell. The corresponding adduct from DNA treated with melphalan (7) has also been detected in DNA isolated from cells treated with melphalan (unpublished results). The occurrence of further alkylation products such as GMM (Figure 1) arises from the high concentration of reagent which we used in these experiments in order to obtain large quantities of adducts for analysis. They are probably not relevant to the reaction of the mustard with DNA in the cell, where the concentration is much lower. To investigate this further, 14C-labelednitrogen mustard should be synthesized and the products of its reaction with cellular DNA analyzed.
Acknowledgment. We would like to thank G. K. Poon and M. H. Baker for their assistance in obtaining mass spectra and D. H. Phillips, in whose laboratory the work was carried out. The work was supported by a grant from the Cancer Research Campaign.
References (1) Rhoads, C. P. (1946) Nitrogen mustards in the treatment of neoplastic disease. J. Am. Med. Assoc. 131, 656-658. (2) Hansson, J., Lewensohn, R., Ringborg, U.,and Nilsson, B. (1987) Formation and removal of DNA cross-links induced by melphalan and nitrogen mustard in relation to drug-induced cytotoxicity in human melanoma cells. Cancer Res. 47, 2631-2637. (3) Brookes, P., and Lawley, P. D. (1961a) The reaction of mono- and di-functional alkylating agents with nucleic acids.Biochem. J.80, 496-503. (4) Brookes, P., and Lawley, P. D.(1961b)The alkylation of guanosine and guanylic acid. J . Chem. SOC. 1961, 3923-3928. (5) Kallama, S., and Hemminki, K. (1984) Alkylation of guanosine by phosphoramide mustard, chloromethine hydrochloride and chlorambucil. Acta Pharmucol. Toxicol. 54, 214-220. (6) Kallama, S., and Hemminki, K. (1986) Stabilities of 7-alkylguanosines and 7-deoxyguanosines formed by phosphoramide mustard and nitrogen mustard. Chem.-Bid. Interact. 57, 85-96.
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320 Chem. Res. Toxicol., Vol. 8, No. 2, 1995 (7) Osbome, M. R., and Lawley, P. D. (1993) Alkylation of DNA by melphalan with special reference to adenine derivatives and adenine-guanine cross-linking. Chem.-Biol. Interact. 89, 49-60. (8) Golumbic, C., Fruton J. S., and Bergmann, M. (1946) Chemical
reactions of the nitrogen mustard gases. I. The transformations of methyl-bis(/3-chloroethy1)aminein water. J . Org. Chem. 11, 518-535. (9) Lawley, P. D., and Warren, W. (1976) Removal of minor methylation products 7-methyladenine and 3-methylguanine from DNA of Escherichia coli treated with dimethyl sulphate. Chem.-Biol. Interact. 12, 211-220. (10) Kohn, K W., Hartley, J. A., and Mattes, W. B. (1987) Mechanisms of DNA sequence selective alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res. 16,10531-10549. (11) Pieper, R. O., and Erickson, L. C. (1990) DNA adenine adducts induced by nitrogen mustards and their role in transcription termination in vitro. Carcinogenesis 11, 1739-1746.
(12) Ojwang, J. O., Grueneberg, D. A., and Loechler, E. L. (1989) Synthesis of a duplex oligonucleotide containing a nitrogen mustard interstrand DNA-DNA cross-link. Cancer Res. 49,65296537. (13) Hopkins, P. B., Millard, J. T., Woo, J., Weidner, M. F., Kirchner, J. J., Sigurdson, S. T., and Raucher, S. (1991) Sequence preferences of DNA interstrand cross-linking agents: importance of minimal DNA structural reorganization in the cross-linking reactions of mechlorethamine, cisplatin and mitomycin C. Tetrahedron 47,2475-2489. (14) Rink,S. M., Solomon, M. S., Taylor, M. J., Rajur, S. B., McLaughlin, L. W., and Hopkins, P. B. (1993) Covalent structure of a nitrogen mustard-induced DNA interstrand cross-link: an N7to-N'I linkage of deoxyguanosineresidues at the duplex sequence 5'-d(GNC). J. Am. Chem. Soc. 116,2551-2557.
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