ISOLATIONAND INVESTIGATION` OF THE ROLE OF HMG1 IN DIEPOXIDE CROSS-LINK RECOGNITION.  Tiffany F. Frazar and Julie T. Millard. Department of Chemistry, Colby College, Waterville ME 04901.

 

The most well characterized group of nonhistone chromosomal proteins are the high mobility group (HMG) proteins, which are involved in nonspecific DNA binding and packaging of DNA.  HMG1 binds preferentially to distorted DNA, including adducts formed by the anti-tumor drug cisplatin. While HMG1 has been shown to recognize both cisplatin DNA intrastrand and interstrand cross-links, it does not recognize lesions induced by the trans isomer of cisplatin, which is clinically inactive. Thus, cisplatin’s chemotheropeutic abilities are thought to involve cross-link recognition by HMG1, blockage of nucleotide excision repair, and cell death. Diepoxyalkanes such as diepoxybutane may also induce structural alterations in DNA upon cross-linking. In order to determine if HMG1 binds to diepoxide cross-linked DNA, HMG1 was first isolated from chicken erythrocytes using salt extraction techniques and chromatography.  Gel-shift assays are being performed to determine the extent of HMG1 binding to diepoxide cross-linked DNA.

 

 

DIEPOXIDE INTERSTRAND CROSS-LINKING OF 5S DNA. Junko Goda and Julie T. Millard. Department of Chemistry, Colby College, Waterville ME 04901.

 

While DNA alkylating agents are some of the most clinically useful antitumor agents, they are also widely implicated in carcinogenecity. Bifunctional alkylators such as diepoxybutane are frequently more carcinogenic than their monofunctional analogues, a fact which has been attributed to their formation of DNA cross-links. While cross-linking within DNA oligomers has been explored extensively, there is less information available about these interactions with longer DNA. We have initiated studies on the diepoxide-induced interstrand cross-linking of the 5S DNA of Xenopus borealis in both the free and nucleosomal states. We are using polyacrylamide gel electrophoresis to determine whether  the 5’-GNC consensus sequence for the formation of interstrand cross-links previously found in DNA oligomers is preserved in longer DNA.

 

 

FLANKING SEQUENCE EFFECTS ON DNA INTERSTRAND CROSS-LINKING PREFERENCES OF DIEPOXIDES. Subhashini Srivatsan and Julie T. Millard. Dept. of Chemistry, Colby College, Waterville, ME 04901.

 

DNA interstrand cross-linking agents result in chromosomal aberrations, thereby causing cancer. However, some of these DNA cross-linking agents can be used as antitumor therapeutics. For example, despite sharing the same 5’-GNC target sequence for interstrand cross-linking, the nitrogen mustards are effective antitumor agents, whereas diepoxyalkanes merely cause cancer. Therefore, the ultimate biological effect of a DNA cross-linking agent cannot be attributed simply to its preferred core target sequence. We are using polyacrylamide gel electrophoresis (PAGE) to study how flanking sequences around the core 5’-GNC target impact the cross-linking efficiency of diepoxyalkanes relative to mustards. We are using synthetic DNA oligomers containing the central sequence [N1GN2CN3] containing each of the four possible bases for N1, N2, and N3. Upon reaction with cross-linking agent, interstrand cross-linked product can be isolated via denaturing PAGE and quantitated via phosphorimagery.

 

 

Metal ion stabilization of G-tetrads containing 6-thioguanosine.  Stephen U. Dunham and Julia C. Drees, Department of Chemistry, Colby College, 5763 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, s_dunham@colby.edu, jcdrees@colby.edu

 

As alternative structures to the double helix, G-tetrads are thought to occur in DNA with many repeating deoxyguanosines (dG). These tetrads have one dG on each corner of a plane and can stack into tetraplexes that can be aptamers and may be found in telomeres. G-tetrads are destabilized by incorporation of 6-thiodeoxyguanosine (6SdG). Addition of metal ions that bind sulfur may stabilize these 6SdG-tetrads. We have carried out a survey of metal ion titrations into 6SG. UV/Visible spectrophotometry indicates that G-tetrads containing two 6SG bases can be stabilized by metal ion coordination. These findings are applied to experiments with oligonucleotides containing 6SdG to determine if tetraplexes can be stabilized.

 

 

Stability of DNA duplexes containing 6-thioguanine .  Stephen U. Dunham and Rodwell Mabaera, Department of Chemistry, Colby College, 5763 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, s_dunham@colby.edu, r_mabaer@colby.edu

 

Incorporation of the modified base 6-thioguanine (6SG) into double-stranded DNA during replication has been implicated in the biological activity of 6SG as an anticancer agent. To understand further the mode of action of this agent, thermodynamic stability of DNA duplexes containing the modified base are compared with duplexes containing unmodified guanine. Nonself-complementary eleven-nucleotide oligomers (11mers) in which the central dG residue was replaced with 6SdG were synthesized by phosphoramidite methods and purified by ion-exchange HPLC. The 11mers were annealed with their complementary strands in which the central residue was either dC or T. Duplex melting equilibrium profiles, obtained through temperature dependent UV-Visible Spectroscopy, were used to determine melting temperature, enthalpy and entropy of melting. Results obtained indicate lower melting temperature for the duplexes that contain 6SdG suggesting destabilization of the 6SdG-dC base pair compared to the normal dG-dC base pair. Duplexes containing 6SdG-T or dG-T base pairs were less stable than those containing either 6SdG-dC or dG-dC.

 

 

Structural analysis of 6-thioguanine in duplex DNA by NMR spectroscopy  Stephen U. Dunham and Heather E. Olson, Department of Chemistry, Colby College, 5763 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, s_dunham@colby.edu, morrison@colby.edu

 

            6-thioguanine (6SG) is an antimetabolite chemotherapy drug approved by the FDA in 1966 and used primarily to treat leukemia.  Despite its long history as a therapeutic agent, its biochemical mode of action is still not completely understood.  Studies suggest that 6SG is converted to d6SGTP and incorporated into DNA, leading to death of rapidly dividing cells.  It has been shown that d6SG disrupts the structure of duplex DNA, but a high-resolution X-ray or NMR structure of d6SG in duplex DNA is unavailable at this time. In this study, we have synthesized a sequence of DNA and an identical sequence with one guanine replaced by d6SG by using automated phosphoramidite chemistry.  Oligonucleotides were purified using high pressure liquid chromatography (HPLC), characterized by HPLC of enzymatic digestions, and annealed to complementary strands to form duplex DNA.  Using NMR spectroscopy, we have analyzed the structural differences between native and d6SG modified DNA duplexes.


 

 

 


Structural determination of an MRI contrast agent

Stephen U. Dunham and Greg Shelton, Department of Chemistry, Colby College, 5763 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, s_dunham@colby.edu, gsshelton@colby.edu

 

Often, the clarity of magnetic resonance imaging (MRI) can be improved through the use of contrast agents. These agents, due to their paramagnetic properties, enhance the differentiation between anatomical structures as seen in the obtained images. Contrast agents typically consist of chelated gadolinium ions and can be administered to an MRI subject in order to shorten the T1 and T2 relaxation times of nearby water molecules. This change in water relaxation time subsequently improves the contrast of the image. The purpose of this study is to characterize isomers of lanthanide diethylenetriaminepentaacetic acid (DTPA) complexes and determine their structures in solution. Isomers of various lanthanide DTPA complexes are first separated using HPLC and then analyzed using NMR spectroscopy. In addition to 1-D proton and carbon spectra, 2-D COSY and NOESY spectra will be used to assign resonances and determine interatomic distances. Computer models will be refined using these NMR data as restraints in molecular mechanics and dynamics calculations.

 

 

 

Structural analysis of a DNA duplex containing 6-thiodeoxyguanosine by ESR and NMR spectroscopy  Stephen U. Dunham and Baskut O. Tuncak, Department of Chemistry, Colby College, 5763 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, s_dunham@colby.edu, botuncak@colby.edu

 

A thioderivative of the nucleic acid base guanine, 6-thioguanine (6SG), is a metabolic inhibitor and possesses anti-tumor activity. Substituting for deoxyguanosine, 6SdG can be readily incorporated into nucleic acids.  It has been proposed that biological activity of 6SG is the result of alterations in the conformation of duplex DNA. We are interested in determining changes in structure and dynamics of DNA modified by 6SdG. Automated synthesis has been used to obtain 11-base pair DNA sequences with incorporation of 6SdG and/or convertible nucleosides.  Modification of these convertible nucleosides with either 4-aminoTEMPO or 15NH4OH can be used to incorporate specific probes for electron paramagnetic spin resonance (ESR) or nuclear magnetic resonance (NMR) spectroscopy.  Following DNA purification by HPLC, NMR and ESR spectroscopy will be used to characterize structure and dynamics of 6SdG in duplex DNA.


 

 

 

 


ACID PROMOTED ELECTROPHILIC AROMATIC SUBSTITUTION REACTIONS IN THE FORMATION OF 1,2-DIQUATERNARY CENTERS.  AN APPROACH  TO  THE CONCISE SYNTHESIS OF (±)-CUPARENE  AND RELATED NATURAL PRODUCTS.   Nicholas Bizier1, Amanda Brooks2, Bradford P. Mundy1, and Thomas Poon2. (1) Chemistry Department, Colby College, Mayflower Hill, Waterville, Maine 04901, fax: 207-872-3804, bpmundy@colby.edu, (2) Joint Science Department, W.M. Keck Science Center, 925 N. Mills Ave., Claremont, CA 91711, fax: 909-621-8588, tpoon@jsd.claremont.edu.

 

Two routes are being developed towards the synthesis of (±)-cuparene, a naturally occurring sesquiterpene first isolated from conifers of the family Cupressaceae by Enzell and Erdtman in 1958.  The first involves treatment of 2,2-dimethylcyclopentanone with methylmagnesium bromide in toluene followed by the addition of a Lewis or protic acid.  A similar route from 1,2-dimethylcyclopentene oxide supports the formation of a carbocation directly from the Grignard salt of 1,2,2-trimethylcyclopentanol followed by electrophilic aromatic substitution on toluene.  Model reactions as well as discussions of the cuparene synthesis will be discussed.  The efficacy of various Lewis and protic acids in this approach will be described..

 

 

 

 

 

Synthetic Approaches to Hexaoxa[6]peristylane and Its Derivatives

Douglas A. Otte, Robin A. Farlow, Sarah B. Lovitz and Dasan M. Thamattoor

Department of Chemistry, Waterville, ME 04901

 

            The structural beauty and interesting bonding properties of polycyclic cage compounds have been a source of fascination to chemists. In this work, progress toward the synthesis of large oxabowl systems, such as hexaoxa[6]peristylane (2, R = H) and its derivatives (2, R = phenyl, tert-butyl, and methyl), is described.  Thus, the adduct obtained from the cycloaddition of maleic anhydride and cycloocatatetraene is converted into key dicarbonyl intermediates such as 1. Subsequently, the ozonolysis of 1 is carried out in order to generate 2. Molecular modeling studies of 2, performed at the semi-empirical and ab initio levels, seem to indicate substantial puckering of the cage resulting in reduced symmetry.

 

 

 

An Unusual Oxidative Rearrangement Promoted by DDQ

Ali H. Ghaffari, Douglas A. Otte, and Dasan M. Thamattoor

Department of Chemistry, Colby College

Waterville, ME 04901

 

The DDQ oxidation of 9-cyclopropyl-9-methyl-1,4,5,8-tetrahydro-4a,8a-methanonaphthalene (1, R = cyclopropyl) leads to the rearranged alkene 1-(1-cycloropylvinyl)naphthalene (2, R = cyclopropyl). The proposed mechanism for the rearrangement involves partial oxidation of one of the six-membered rings in 1, followed by a Berson-Willcott 1,5-sigmatropic shift of the one-carbon bridge. Subsequent removal of a hydride from the methyl group by DDQ prompts the opening of the three-membered ring, and the resulting tertiary cation is deprotonated and aromatized to 2. Tests for these mechanistic postulates, as well as molecular orbital calculations on the suspected intermediates, are described. Investigations into the scope of this rearrangement with other substrates (1, R = hydrogen, methyl, tert-butyl, and phenyl), are also discussed.

 

 

 

 

 

 

 

Intramolecular Reactions of a b -Hydroxycarbene

Robin A. Farlow and Dasan M. Thamattoor

Department of Chemistry, Colby College

Waterville, ME 04901

 

A b-hydroxycarbene has been generated, for the first time, by photolysis of the tertiary alcohol precursor exo-2-(1a,9b-dihydro-1H-cyclopropa[l]phenanthren-1yl)-propan-2-ol (1). The synthesis of 1 was accomplished by cyclopropanation of the 9,10-double bond in phenanthrene with ethyl diazoacetate, followed by transformation of the resulting ester into the tertiary alcohol. Photolysis of 1 in deuterated benzene afforded phenanthrene and 2-butanone along with other unidentified products. The mechanism for the formation of 2-butanone is explained in terms of a methyl shift in the putative b-hydroxycarbene intermediate 2-hydroxy-2-methylethylidene (2). The presence of other potential products from 2 such as 1-methylcyclopropanol and 2,2-dimethyloxirane, which could be produced by intramolecular O-H and C-H insertions respectively, are pending verification. Molecular orbital calculations performed on 2, using HF and B3LYP methods, are also discussed.

 

 

 

 

 

 

 

Computational studies of the reaction of OH radical with Cl-, SO4= and CO3=. Deborah L. Schwartz, Margaret H. Hennessy, and D. Whitney King, Department of Chemistry, Colby College, Box 6877 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, dlschwar@colby.edu

 

The hydroxide radical is a powerful oxidizing agent in the environment. The hydroxide radical's reactivity is often moderated by the presence of anionic species such as chloride, sulfate and carbonate, which ultimately form the less reactive radical species Cl., SO4.- and CO3.-. A mechanistic understanding of these reactions requires structural information for the OH. -anion intermediates. Past computational investigations of the chloride intermediate yielded the surprising result that Cl- + OH.ó ClHO.-, where the bridging atom is hydrogen. This work reports expanded calculations using Density Functional Theory on this chloride intermediate and comparable CO3= and SO4= species.

 

 

Photochemical degradation of 2,4-D and Atrazine in well-defined media. D. Asher Ghertner, Tim Bertram, Chaz Langelier, and D. Whitney King, Department of Chemistry, Colby College, 7798 Mayflower Hill, Waterville, ME 04901, daghertn@colby.edu

 

Photochemical degradation has been proposed as a remediation strategy for the common herbicides 2,4-D and Atrazine. In the presence of iron and hydrogen peroxide these herbicides decay by both thermal and light induced oxidation. Past studies have focused primarily on sun light as an energy source. This work provides a mechanistic description of herbicide degradation incorporating intermediate degradation products produced in the dark and under well-defined light conditions.

 

 

Free radical intermediates in the Fe(II)-luminol chemiluminescence system: Application to ultra-trace analysis of Fe(II). Sara B. Lovitz1, Caibin Xiao2, and D. Whitney King1. (1) Department of Chemistry, Colby College, 5755 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, sblovitz@colby.edu, (2) Betz Dearborn

 

The Fe(II) catalized oxidation of luminol has been a well accepted chemiluminescence method for the trace and ultra-trace analysis of Fe(II) in natural waters. Investigation into the mechanism of the reaction has shown that CO2(g) significantly increases the chemiluminescence signal by an order of magnitude. The mechanism of this reaction will be discussed, and the potential for CO2(g) mediated signal enhancement and/or positive interference will be quantified.

 

Determination of hydrogen peroxide in ocean water using a chemiluminescent flow-injection analysis system.  Christopher R. Morgan, Amity Burr, Stephen M. Theberge, and D. Whitney King, Department of Chemistry, Colby College, 5755 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, crmorgan@colby.edu

 

Cooper et al. have shown that 10-methyl-9-(p-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate can be used as a chemiluminescent method to accurately quantify hydrogen peroxide in natural waters. We have adapted this chemistry for flow-injection analysis; significantly improving detection limits and sample throughput. The FIA system was field tested against an established enzymatic method in which surface water was analyzed on a transect from Narragansett, RI to the Delaware River estuary. These samples represent a salinity range from brackish to open ocean water. The two techniques were statistically indistinguishable (n=126).

 

Flow injection analysis of superoxide in aqueous solution: A quantitative determination using the chemiluminescent probe MCLA.  Nathan E. Boland, and D. Whitney King, Department of Chemistry, Colby College, 5755 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, neboland@colby.edu

 

Superoxide is present at low concentrations (nm) in environmental aqueous solutions. As of yet there are no simple methods for measuring superoxide in these systems. MCLA reacts with superoxide to produce light. A flow injection system using MCLA as a chemiluminescent probe is used to quantitatively measure steady state superoxide concentrations and reaction rates.

 

Mechanistic investigation of copper-1,10-phenanthroline chemiluminescence

Anne M. Isham1, Caibin Xiao2, and D. Whitney King1. (1) Department of Chemistry, Colby College, 7031 Mayflower Hill, Waterville, ME 04901, amisham@colby.edu, (2) Betz Dearborn

 

Application of 1,10-phenanthroline as a chemiluminescence reagent for copper has been well established. The proposed mechanism of the reaction was the oxidation of the copper-1,10-phenanthroline complex by hydrogen peroxide. Recent studies in our lab show carbon dioxide is a negative interferent in this system. These results are inconsistent with the simple oxidative mechanism. A new mechanism is proposed.