Title page for ETD etd-06282001-064000


Type of Document Dissertation
Author Milkevitch, Matthew
URN etd-06282001-064000
Title Mixed-Metal Ruthenium-Platinum Polyazine Supermolecules: Synthesis, Characterization and Exploration of DNA Binding
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Brewer, Karen J. Committee Chair
Winkel, Brenda S. J. Committee Co-Chair
Anderson, Mark R. Committee Member
Hanson, Brian E. Committee Member
Long, Gary L. Committee Member
Keywords
  • Cisplatin
  • Gel Densitometry
  • Electrochemistry
  • Supermolecules
  • Gel Electrophoresis
  • Spectroscopy
  • DNA Binding
Date of Defense 2000-07-14
Availability unrestricted
Abstract
The goal of this research was to design, prepare and study a new class of supermolecules coupling ruthenium and platinum, which would display covalent binding to DNA. Drawing upon the well-established efficacy of cis-diamminedichloroplatinum(II) (cisplatin) and the DNA-binding properties of select ruthenium polyazine complexes, the approach was to bind the cis-PtIICl2 active site of cisplatin to ruthenium light absorbers using the dpq and dpb bridging ligands (where dpq = 2,3-bis(2-pyridyl)quinoxaline, dpb = 2,3-bis(2-pyridyl) benzoquinoxaline). These complexes are potentially bifunctional, capable of DNA intercalation through the bridging ligand and covalent binding to DNA through the cis-PtCl2 site. Synthetic methods were developed to prepare the mixed-metal, bimetallic complexes [(bpy)2Ru(BL)PtCl2](CF3SO3)2 and [(phen)2Ru(BL)PtCl2](CF3SO3)2 (where bpy = 2,2¢-bipyridine, phen = 1,10-phenanthroline) in high purity and good overall yields. The DNA-binding ability of these complexes was probed by reaction with linearized plasmid DNA and subsequent analysis by native and denaturing gel electrophoresis. The known DNA binders, cisplatin and trans-{[PtCl(NH3)2]2(m-H2N(CH2)6NH2)}(NO3)2 (1,1/t,t), were examined under equivalent conditions and used as positive controls. Native gel electrophoresis was used to show that these complexes strongly bind DNA, retarding the migration of DNA through the gel in a fashion inversely proportional to the ratio of DNA base pairs (bp) to metal complex (mc). Analysis by denaturing gel electrophoresis determined that the Ru-Pt complexes bind to DNA in a fashion similar to cisplatin, forming primarily intrastrand adducts. However, these systems also appear to form interstrand adducts at a 10-fold lower metal concentration than cisplatin.

In addition to affecting the migration rate, the bimetallic complexes also significantly reduced the fluorescence of DNA-intercalated ethidium bromide for the Ru-Pt reacted samples at low-DNA bp: mc ratios. This was not observed for the cisplatin and 1,1/t,t treated samples. This observation was quantitated by gel densitometry. Precipitation of the DNA by cisplatin, 1,1/t,t and all four Ru-Pt complexes was determined not to be the cause of reduced ethidium bromide fluorescence intensity. Homogenous solution fluorescence quenching studies have revealed that the Ru-Pt complexes quench the emission of ethidium bromide even in the absence of DNA, whereas cisplatin and 1,1/t,t do not.

In order to compare the effects on DNA migration produced by cisplatin, 1,1/t,t and the Ru-Pt complexes, Rf values were calculated. This analysis has revealed that all four Ru-Pt complexes retard DNA migration to approximately the same degree. Calculation of theoretical DNA migration distances, based upon the molecular weight change of DNA caused by metal-complex binding, have revealed that the observed affect on DNA migration cannot be accounted for by an increase in molecular weight alone. This indicates that changes in charge and three-dimensional shape of the DNA upon binding of the Ru-Pt complexes may also contribute.

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  Appendices.pdf 1.31 Mb 00:06:04 00:03:07 00:02:44 00:01:22 00:00:06
  Dissertch1-4.pdf 4.10 Mb 00:18:59 00:09:46 00:08:32 00:04:16 00:00:21
  Frontmatter.pdf 173.54 Kb 00:00:48 00:00:24 00:00:21 00:00:10 < 00:00:01
  Vitae.pdf 44.02 Kb 00:00:12 00:00:06 00:00:05 00:00:02 < 00:00:01

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