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 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]ethy l] 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 dichrosism, and viscosity studies are consistent with MNU-PNR binding to DNA via an intercalation-based process.
Mehta
P
P
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198, USA.
Church
K
K
Williams
J
J
Chen
F X
FX
Encell
L
L
Shuker
D E
DE
Gold
B
B
D000225:Adenine / Q000031:analogs & derivatives / Q000032:analysis; D002942:Circular Dichroism; D018736:DNA Adducts / Q000737:chemistry*; D019175:DNA Methylation; D015195:Drug Design; D004996:Ethidium; D006147:Guanine / Q000031:analogs & derivatives / Q000032:analysis; D007364:Intercalating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D008770:Methylnitrosourea / Q000737:chemistry*; D009499:Neutral Red / Q000031:analogs & derivatives* / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D018736:DNA Adducts; D007364:Intercalating Agents; C102822:phenyl neutral red; D009499:Neutral Red; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D008770:Methylnitrosourea; D004996:Ethidium; D000225:Adenine