225th National Meeting of the American Chemical Society

New Orleans , March 23-27, 2003

CHED 895

Flanking sequences modulate diepoxide cross-linking efficiencies at the 5’-GNC site

Gregory A. Sawyer and Julie T. Millard, Department of Chemistry, Colby College, 5757 Colby College, Waterville, ME 04901.

Diepoxybutane, diepoxyoctane, and mechlorethamine are cytotoxic DNA cross-linking agents that vary in their carcinogenic versus chemotherapeutic potentials. Interstrand cross-linking occurs between the N7 positions of deoxyguanosine residues on opposite strands of the DNA duplex. Each synthetic DNA oligomer used in this study contains four 5’-N1GN2CN3 sites (within a 32 base sequence), and we are currently systematically varying the bases in the N1 and N2 positions to determine the resulting cross-linking efficiencies of each cytotoxic agent. Each duplex is 5’-end labeled and incubated with cross-linking agent. Interstrand cross-links are purified through denaturing polyacrylamide gel electrophoresis and then subjected to piperidine cleavage. The amount of cleavage at each deoxyguanosine residue is determined by sequencing gel analysis and represents the cross-linking efficiency at that site. We have determined that cross-linking efficiency varies with the identity of N1 and N2.

CHED 484

Mild one-pot conversion of primary alcohols to a,b-unsaturated ketones: Application to natural product synthesis

Traci J Speed and Dasan M. Thamattoor. Department of Chemistry, Colby College, 5750 Mayflower Hill, Colby College, Waterville, ME 04901

The oxidation of alcohols followed by in situ Wittig reaction has been a subject of much interest in the synthetic organic chemistry community. Indeed, a number of protocols have been developed to carry out such conversions with a variety of oxidants and stabilized Wittig reagents. There is, however, much less known about the utility of this one-pot, two-step process to prepare a,b-unsaturated ketones from alcohols. Recently we have had some success in carrying out the Swern oxidation of primary alcohols and trapping the resulting aldehydes with stabilized Wittig reagents bearing keto groups. The olefin is produced predominantly in the E form. A practical application of this expeditious procedure is exemplified by the synthesis of the naturally occurring marine cytotoxins Montipoynes A and B.

CHED 901

Hydrogen bonding in discrete base pairs containing 6-thioguanine

Stephen U. Dunham and Andrew H. Moraco, Department of Chemistry, Colby College, 5763 Mayflower Hill, Waterville, ME 04901

In 1966 the anti-metabolite chemotherapy drug 6-thioguanine (6SG) was approved by the FDA. After being incorporated into the cell, it is has been hypothesized that the increased size and decreased electronegativity of the thio-group may lead to cytotoxicity. This may result from altering hydrogen bonds between nucleic acid duplexes causing disruption of the conformation of the DNA, thus inhibiting replication and transcription. Through NMR spectroscopy, we have gained insight into the enigmatic mechanism of this age old chemotherapy agent by examining the rotation dynamics of the amino groups involved in hydrogen bonding of the discrete base paired complexes. The 1:1 complexes of G:C, 6SG:C, G:U and 6SG:U were prepared via NMR titration of the protected nucleoside 5’-tert-butyldimethylsilyl-2’3’-O-isopropylidene guanosine(it-G), cytosine (it-C), uridine (it-U) or 6-thioguanosine (it-6SG). The it-6SG sulfur to cytosine amino proton hydrogen bond exhibited a clear decrease in stability relative to its control it-G:it-C. Additionally, the it-6SG:it-C samples did not form tetramers at low temperatures as was observed for the it-G:it-C solutions. Finally, despite evidence that 6SG will also cause mispairing in DNA replication due to an ability to code for thymine or cytosine, there was little proof of hydrogen bonding between it-6SG and it-U.

CHED 945

Quantitative PCR analysis of diepoxybutane damage within the chicken beta-globin domain

André M. Pilon and Julie T. Millard. Department of Chemistry, Colby College, 5750 Mayflower Hill, Waterville, ME 04901

In industrial polymer and synthetic rubber production facilities, workers are exposed to 1,3-butadiene. This compound is converted in vivo to 1,2,3,4-diepoxybutane (DEB) and has been linked to increased incidences of cancer in these individuals. Carcinogenesis has been attributed to formation of DNA interstrand cross-links. Previous studies have demonstrated that DEB cross-links deoxyguanosine residues within 5’-GNC sequences in synthetic DNA, in restriction fragments, and in defined sequence nucleosomes. We are using PCR to examine DEB cross-linking frequencies within nuclear genes, found within “open” regions of chromatin, as compared to regions of unexpressed sequence that reside in tightly packed, “closed” chromatin. Initial studies are being performed in chicken liver homogenates. Once PCR conditions have been optimized, we will perform time course studies in cultured chicken cells to determine the rates of lesion repair in open versus closed DNA.

CHED 944

Quantitative PCR analysis of diepoxybutane damage within rat liver mitochondrial DNA

Justin E. Juskewitch and Julie T. Millard. Department of Chemistry, Colby College, 5700 Mayflower Hill Drive, Waterville, ME 94901

Diepoxides are byproducts of mammalian metabolism of hydrocarbons that can lead to adverse health effects upon binding to DNA. For example, diepoxybutane (DEB), linked to the high incidence of certain cancers amongst workers exposed to butadiene in industrial settings, cross-links duplex DNA at 5’-GNC sites in vitro. We are using PCR in conjunction with polyacrylamide gel electrophoresis to determine reaction sites and frequency in vivo. Quantitative PCR indicates a linear dose response of DEB damage within a 307 base pair fragment of the cytochrome b gene in rat mtDNA extracted from liver cells. Linear amplification products will be analyzed on sequencing gels in order to determine the sites targeted within this fragment. Our goal is to provide insight into the insult on liver mitochondria of those exposed to industrial butadiene

CHED 447

Experimental and theoretical study of a ß-methoxycarbene

Stanislav Presolski and Dasan M. Thamattoor. Department of Chemistry, Colby College, 5750 Mayflower Hill, Waterville, ME 04901

The effect of bystander groups on the reactivity of carbenes/carbenoids has been a great source of interest to chemists. While carbenes containing several bystander groups have been investigated experimentally and computationally, many of these intermediates were generated from conventional nitrogenous sources such as diazo compounds and diazirines. Unfortunately, excited states of the nitrogenous sources can themselves lead to products that may be erroneously attributed to carbenes. In this study we describe our progress in generating and studying a ß-methoxycarbene, namely 2-methoxy-2-methylpropylidene (2), from a nonnitrogenous phenanthrene-based precursor (1). Computational insights into the structure and reactivity of this carbene, obtained using hybrid density functional theory (B3LYP/6-31G*), will be also discussed.