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

An N-methyl-N-nitrosourea (MNU) moiety [CH3N(N=O)C(=O)NH-] linked to t he C4'-position of the 5-substituted phenyl ring of phenyl neutral red (PNR), hyl]carbamoyl]phenyl]-7-(dimethylamino)phenazenium chloride (M NU-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 previou s studies suggested the following: (1) PNR binds with a slight prefere nce 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 groov e (Muller, W., Bunemann, H., and Dattagupta, N. (1975) fur. J. Biochem . 54, 279-291). It is demonstrated that MNU-PNR yields 2.6 and 6.0 tim es more N7-methylguanine (7-MeGua) than MNU at low salt (10 mM Tris bu ffer) and high salt (10 mM Tris buffer + 200 mM NaCl), respectively. I t is also shown that the ratio of 7-MeGua (a major groove adduct) to N 3-methyladenine (a minor groove adduct) is approximately 5 times highe r for MNU-PNR than for MNU. The yield of the 7-MeGua adduct is decreas ed by the coaddition of a nonmethylating analogue of MNU-PNR or NaCl, but increased in the presence of the minor groove intercalator, ethidi um bromide. Using a P-32-end-labeled restriction fragment, the enhance d 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 stud ies are consistent with MNU-PNR binding to DNA via an intercalation-ba sed process.