I. Ground and Excited State Studies of Persistent 1,1-Diazenes. II. Design of Sequence Specific DNA Cleaving Molecules (2004)

CHAPTER I Direct studies of the ground and excited state properties of kinetically persistent l,l-dialkyldiazenes are reported. The electronic absorption spectrum of N-(2,2,5,5-tetramethylpyrrolidyl)nitrene 3 reveals a structured absorption band for the n,π* transition: λmax = 497 nm, λ0,0 = 572 nm (λmax = 20 ± 3) in CH2Cl2; λ0,0 = 552 nm in i-PrOH. The infrared spectrum shows a strong absorption at 1638 cm-1, providing evidence that the 1,1-diazene N-N stretch has considerable double bond character. The major products resulting from pyrolysis of 3 at 0°C are isobutylene 21, tetramethylcyclobutane 20, and tetrazene 24, consistent with unimolecular and bimolecular decomposition pathways. The activation parameters for unimolecular fragmentation are: log A = 12.4 ± 0.4, Ea = 19.0 ± 0.6 kcal mole-1 in Et2,O; log A = 12.1 ± 0.3; Ea = 19.1 ± 0.4 kcal mole-1 in THF; log A = 10.9 ± 0.3, Ea = 16.8 ± 0.5 kcal mole-1 in hexane. The activation parameters for the bimolecular dimerization of 3 could not be measured, however log A = 3.8 ± 0.7 and Ea = 6.4 ± 0.9 kcal mole-1 for the dimerization of N-(2,2,6,6-tetramethylpiperidyl)nitrene 2 in CDCl3. The fluorescence spectrum of 3 has a 0-0 band at 607 nm which is the maximum. The spacing between the peaks at 607 and 672 nm is consistent with the N=N stretch of S0 obtained from the infrared spectrum. The fluorescent quantum yields are ΦF = 2 x 10-3 (MTHF, -78°C), 7 x 10-8 (CFCl3, -196°C) and 1 x 10-3 (EPA, -196°C). The fluorescent lifetime of 3, τF is 5 x 10-9 sec (CFCl3, -78°C) and 2.3 x 10-8 sec (CFCl3, -196°C). Direct irradiation of 3 in the visible (-78°C) affords 54% 21, 44% 20, and tetrazene 24, again consistent with unimolecular and bimolecular decomposition pathways. Triplet sensitized photolysis affords a high cleavage/closure ratio: 74% 21 and 24% 20. An approximate quantum yield for decomposition of 3 on direct irradiation (-78°C) is ΦD = 1.1 x 10-2. D,1-N(2,5-diethyl-2,5-dimethylpyrrolidyl)nitrene 26 has a structured visible absorption with λmax at 507 nm. The maximum of the fluorescence spectrum is 620 nm, ΦF = 9 x 10-3 (MTHF, -196°C). Direct and sensitized irradiation of 26 in the visible (-78°C) affords closure, cleavage, and disproportionation products as well as tetrazene. The retention of configuration in the cyclobutane products was 98% and 68% for direct and sensitized decompositions, respectively. This spin correlation effect indicates that kISC &lt; &lt; kN2 (S1), consistent with the large calculated S1-T1 gap in 1,1-diazenes. Comparison of these results with those in the literature for the isomeric 1,2-diazene provides information on the nature of intersystem crossing in nonconjugated diradicals. The deactivation rates for S1 of 1,1-diazene 3 have been determined: kIC = 2 x 108 sec-1 (-78°C); kF = 3 x 105 sec-1; kDIM ~ 8 x 107 M-1 s-1 (-78°C); kN2 ~ 3 x 105 sec-1 (-78°C); ET ≤ 31 kcal mole-1. The photochemistry of the six-membered ring cyclic 1,1-diazene 2 was also examined. CHAPTER II The ability of restriction endonucleases to cleave double helical DNA on opposite strands at or near 4-6 base pair recognition sequences makes possible DNA sequencing, gene isolation and recombinant DNA technology. Studies aimed at defining those elements necessary for the design of synthetic double strand DNA cleaving molecules with defined target sequences and binding site sizes are described. The covalent modification of sequence specific DNA binding molecules with a chelated metal capable of redox chemistry generates bifunctional DNA cleaving molecules. Attachment of EDTA to the amino or carboxy terminus of the N-methylpyrrole oligopeptide distamycin A affords distamycin-EDTA (DE) and EDTA-distamycin (ED), respectively. DE•Fe(II) and ED•Fe(II) (10-6M) sequence specifically single strand cleave pBR-322 plasmid DNA (10-5 bp) in the presence of O2 and dithiothreitol (DTT). Cleavage occurs over three to five base pairs adjacent to a five base pair 5'-XXTTT-3' (X = A or T) binding site and is consistent with oxidative degradation of the deoxyribose. Analysis of the DNA cleavage patterns generated by DE•Fe(II) and ED•Fe(II) (high resolution denaturing gel electrophoresis) provides information on the location, size, and orientations of distamycin binding sites on DNA. This direct method for defining small molecule binding sites on DNA, "DNA affinity cleaving", is compared to methidiumpropyl-EDTA•Fe(II) (MPE•Fe(II)) footprinting. A model for distamycin binding is proposed. Attachment of EDTA to the amino terminus of a penta-N-methylpyrrole peptide yields P5E. In the presence of O2 and DTT, P5E•Fe(II) (10-8M bp) converts supercoiled pBR-322 DNA (10-3M) to 40% open circular DNA, indicating that cleavage is catalytic, with a minimum of nine turnovers. P5E•Fe(II) sequence specifically double strand cleaves linear pBR-322 DNA (4362 base pairs) into discrete fragments. The cleavage sites are located at 4.3, 4.2, 3.3, 3.2 kb (major) and 2.6, 2.4, 2.0, 1.8 kb (minor). P5E•Fe(II) binds preferentially to 6-7 base pair poly(dA)•poly(dT) sites. EBD and BED consist of two N-methylpyrrole tripeptide units coupled at the amino termini via a flexible linker with EDTA tethered to one or both carboxy termini, respectively. BED•2Fe(II) and EBD•Fe(II) cleave pBR-322 (10-5M bp) DNA at nanomolar concentrations. These reagents cleave linear pBR-322 plasmid DNA at 4.3 and 3.3 kb to afford discrete fragments. BED•Fe(II) and EBD•Fe(II) bind as dimers to 8-9 base pair A+T sites and monomers to 5-6 base pair A+T sites.