The Design of Agents To Control DNA Methylation Adducts. Enhanced Major Groove Methylation of DNA by an N-Methyl-N-nitrosourea Functionalized Phenyl Neutral Red Intercalator

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Abstract
An N-methyl-N-nitrosourea (MNU) moiety [CH3N(NO)C(O)NH−] linked to the C4‘-position of the 5-substituted phenyl ring of phenyl neutral red (PNR), 2-methyl-3-amino-5-[p-[[2-[(N-nitroso-N-methylcarbamoyl)amino]ethyl]carbamoyl]phenyl]-7-(dimethylamino)phenazenium chloride (MNU-PNR), has been synthesized as an approach to design a molecule that will deliver alkylating agents with some preference to guanine (Gua) in the major groove of DNA. The PNR nucleus was chosen because previous studies suggested the following: (1) PNR binds with a slight preference for G/C rich sequences; and (2) PNR intercalates into DNA from the major groove with the 5-phenyl ring pointing out into the major groove (Müller, W., Bünemann, H., and Dattagupta, N. (1975) Eur. J. Biochem.54, 279−291). It is demonstrated that MNU-PNR yields 2.6 and 6.0 times more N7-methylguanine (7-MeGua) than MNU at low salt (10 mM Tris buffer) and high salt (10 mM Tris buffer + 200 mM NaCl), respectively. It is also shown that the ratio of 7-MeGua (a major groove adduct) to N3-methyladenine (a minor groove adduct) is approximately 5 times higher for MNU-PNR than for MNU. The yield of the 7-MeGua adduct is decreased by the coaddition of a nonmethylating analogue of MNU-PNR or NaCl, but increased in the presence of the minor groove intercalator, ethidium bromide. Using a 32P-end-labeled restriction fragment, the enhanced methylation by MNU-PNR at 7-Gua is confirmed, and it is demonstrated that the sequence-dependent formation of 7-MeGua from MNU-PNR is the same as that seen with MNU. UV, circular dichroism, and viscosity studies are consistent with MNU-PNR binding to DNA via an intercalation-based process.

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