223rd National Meeting of the American Chemical Society

Orlando , April 7-11, 2002

ANYL 120

Quanitification of superoxide in natural water systems using flow injection analysis with chemiluminescence detection

Lindsey G. Malbon, and D. Whitney King, Department of Chemistry, Colby College, Mayflower Hill, Waterville, ME 04901

The analysis of superoxide in natural water systems is of considerable interest due to the role that this species plays in the redox cycling of metals and the oxidation of organic compounds. Clean, quantifiable superoxide standards were produced via the photolysis of a benzophenone in an aqueous 2-propanol solution. A flow injection analysis (FIA) system was used to characterize the reaction of the chemiluminescent reagent MCLA [2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2- a]pyrazin-3-one, hydrochloride] with known concentrations of superoxide (1-50 nM) over the pH range of 4.5-9. Using the FIA system, the production rate and steady state concentrations of superoxide in sunlight-irradiated lake water have been determined.

CHED 110

Crime Scene Investigation: Identification of "forensic" samples via mitochondrial DNA in the undergraduate biochemistry laboratory

Julie T. Millard, and Andre Pilon, Department of Chemistry, Colby College, 5757 Mayflower Hill, Waterville, ME 04901, jtmillar@colby.edu

A recent forensic approach to identify unknown biological samples is sequencing of mitochondrial DNA (mtDNA). MtDNA is most useful for identification of human remains when the only reference material available is from maternal relatives or when the samples are badly degraded. We describe a laboratory exercise suitable for an undergraduate biochemistry course that can be easily modified for non-science majors. The polymerase chain reaction is used to amplify the hypervariable HV1 subregion of human mtDNA to generate a fragment of 440 base pairs from a variety of "crime scene" samples: e.g., teeth, hair, nails, cigarettes, and chewing gum. Successful amplification is verified via agarose gel electrophoresis and then samples are subjected to cycle sequencing. Sequence comparisions are made via the computer program CLUSTALW (www.ebi.ac.uk/clustalw) available at the Cold Spring Harbor Laboratory?s Dolan DNA Learning Center website (http://vector.cshl.org/), which also facilitates creation of a similarity tree from the sequence data.

CHED 265

Theoretical and laboratory studies of the peroxycarbonate radical, CO4-, implications for luminol-based chemiluminescence

Shawn Burnell, and D. Whitney King, Department of Chemistry, Colby College, 5755 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, spburnel@colby.edu

Recent investigations of luminol and 1,10-phenanthroline chemiluminescence have demonstrated that these reactions are strongly influenced by dissolved carbon dioxide; attributed to the formation of the peroxycarbonate radical intermediate. The reaction between CO2 and O2- to form CO4- has been well documented in aprotic solvents, but never in water. Theoretical calculations of the reaction CO2 + O2-= CO4- at the MP2/6-31G level are consistent with a strongly favored reaction. Laboratory studies using UV/Vis spectroscopy of O2- provide consistent reaction rates and complex stability.

CHED 313

DNA binding studies of cis and trans-RuCl2 (DMSO)4

Shari U. Dunham, and Thomas F. Curran, Department of Chemistry, Colby College, 5762 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, sudunham@colby.edu, tfcurran@colby.edu

Both the cis and trans isomers of RuCl2(DMSO)4 have previously been shown to inhibit the growth of tumor cells. Although these compounds are in clinical trials for treatment of certain kinds of cancers, the mechanisms by which they kill cancer cells are not yet known. The anticancer capabilities of these compounds, however, are proposed to involve their covalent interactions with DNA. The studies described here intend to determine the location of covalent binding of both isomers on DNA and quantitate amounts of each covalent adduct to establish the in vitro DNA-binding profile of these potential drugs. Each ruthenium compound was incubated in aqueous solution with either nucleosides, short duplex oligonucleotides, or calf-thymus DNA under various conditions. DNA adducts were then analyzed by ICP, enzymatic digestion, HPLC and ESI-MS. Preliminary data will be presented and results of DNA binding studies will be discussed.

CHED 319

Effect of 6-thioguanine and methyl-6-thioguanine on stability of DNA duplexes

Stephen U. Dunham, and Rodwell Mabaera, Department of Chemistry, Colby College, 5763 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, sdunham@colby.edu, r_mabaer@colby.edu

Incorporation of 2'-deoxy-6-thioguanosine 5'-triphosphate (6SdGTP) into DNA during replication has been implicated in the biological activity of thiopurines as antileukemic agents. The presence of either 6-thioguanosine (6SG) or the metabolite methyl-6-thioguanosine (Me6SG) residues in DNA duplexes leads to notable structural alterations. To understand these alterations, we studied six variations of 11mer oligonucleotide duplexes in which the central residue in one strand was G, 6SG or Me6SG paired with either C or T in the complimentary strand. Temperature dependent ultra violet-visible spectroscopy was used to determine melting temperatures for the duplexes. Results indicate lower melting temperatures for duplexes that contain 6SG or Me6SG compared to the control G duplex. Duplexes containing 6SG-T or G-T base pairs had lower melting temperatures than either 66SG-C or G-C. Van't Hoff thermodynamic parameters suggest that 6SG destabilization is primarily entropy driven, consistent with theoretical calculations that show hydration changes in DNA containing 6SG.

CHED 350

LC-MS identification of epichlorohydrin adducts in DNA

Kathryn M. Kosuda, Julie T. Millard, and Thomas W. Shattuck, Department of Chemistry, Colby College, Waterville, ME 04901, kmkosuda@colby.edu

The cross-linking activity of epichlorohydrin (ECH) is responsible for its usefulness in the production of plastics, coatings, epoxy resins, solvents, and other common items. This three-carbon monoepoxide, a suspect carcinogen in humans, has been shown to cause cell damage in humans who have been exposed occupationally and to cause tumors in rats and mice. Evidence suggests that ECH binds to DNA although the products of such reactions remain undetermined. Previous reports on free bases have shown that ECH forms adducts most prominently with the N7 of guanine, O6 of guanine, and N6 of adenine. In this project, LC-MS will be explored as a method of determining the products of epichlorohydrin's reaction with a synthetic DNA dodecamer of known sequence. Capillary LC using a reversed-phase polystyrene-divinyl benzene column will be used to separate the reaction products, while negative ion electrospray mass spectrometry will be used to determine the structure of the resulting adducts.

CHED 363

Purification of the HMG1 protein and investigation of its interactions with diepoxide cross-linked DNA

Gayle Pageau, and Julie T. Millard, Department of Chemistry, Colby College, 7330 Mayflower Hill, Colby College, Waterville, ME 04901, gjpageau@colby.edu, jtmillar@colby.edu

HMG1 is a nonhistone chromosomal protein that binds preferentially to some types of globally modified DNA. HMG1 has been implicated in the cellular response to the anti-cancer agent cisplatin, functioning to block excision repair of specific distorted platinated DNA lesions and resulting in enhanced cytotoxicity. We are investigating whether other DNA-binding agents also produce lesions that are recognized by HMG1. Specifically, we are using polyacrylamide gel shift assays to monitor the potential HMG1 binding of diepoxybutane interstrand cross-links, which have been suggested to induce DNA bending. We have isolated the protein from chicken erythrocytes using NaCl extraction techniques and are testing its reactivity with a cross-linked restriction fragment of circa 100 base pairs length. We will also monitor the binding of engineered HMG domain proteins obtained from expression vectors to cross-linked DNA oligomers. These studies may allow the determination of the generality of HMG1 recognition of cross-linked DNA.

CHED 372

Role of bases flanking the 5'-GNC site on diepoxide DNA interstrand cross-linking efficiency

Elizabeth D. Frederick, and Julie T. Millard, Department of Chemistry, Colby College, 5757 Mayflower Hill, Waterville, ME 04901, edfreder@colby.edu

Diepoxides form interstrand cross-links between deoxyguanosine residues on opposite strands of duplex DNA preferentially at 5'-GNC sequences. We are examining the effects of the bases flanking this consensus sequence on the efficiency of cross-linking by diepoxybutane (DEB) and related compounds. DEB is believed to the active form of the antitumor prodrug treosulfan, used in the treatment of advanced ovarian cancer, and interstrand cross-linking is thought to be the biologically relevant event in this anticancer activity. We are using synthetic DNA oligomers containing four potential sites for cross-linking differing in the identity of either N1, N2, or N3 in the 5'-N1GN2CN3 site. Following purification of interstrand cross-links, quantification of piperidine cleavage products via denaturing polyacrylamide gel electrophoresis and phosphorimagery allows determination of the relative amount of cross-linking at each of the four sites. We have that cross-linking efficiency varies with the identity of N1, N2, and N3.

CHED 523

Investigations of intramolecular copper(I)-arene interactions using an NS2-macrocyclic ligand with different pendant aryl arms

Rebecca R. Conry1, Paul J. Lee1, and A. Alex Tipton2. (1) Department of Chemistry, Colby College, 5764 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, rrconry@colby.edu, pjlee@colby.edu, (2) Department of Chemistry, University of Nevada

Copper(I)-arene interactions are relatively rare; in fact, only a handful of solid-state systems exhibiting h2-Cu-arene bonds have been reported. Copper-arene solution binding is even rarer. We only recently reported the first unequivocal solution binding for a complex with a naphthyl-appended NS2- macrocyclic ligand (Organometallics, 1998, 17, 3146-3148) that also binds the naphthalene in the solid state (Inorg. Chem., 1999, 38, 2833-2843). We have now extended our studies to include the synthesis of new additional aryl-appended ligands and Cu(I) complexes of those ligands. For example, the complexes of the ligand N-[2-phenylethyl]-1-aza-4,8-dithiacyclodecane will be discussed along with other ligand derivatives that probe steric and electronic influences on the copper-arene interaction in this system.

CHED 581

Towards the synthesis of bioinorganic model complexes with a new tetradentate sulfur ligand based on two biphenyl groups

Rebecca R. Conry, and Alexis A. Bond, Department of Chemistry, Colby College, 5764 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, rrconry@colby.edu, aabond@colby.edu

Molybdenum and nickel complexes with the biphenyl-2-2'-dithiolate ligand have been previously synthesized and reported by our group (JBIC, 2001, 6, 359-366; Inorg. Chem., 2000, 39, 1674-1679). However, these complexes are readily oxidized, resulting in complex decomposition via the formation of the ligand disulfide derivative. Thus, we are synthesizing a new tetradentate ligand, that features two linked biphenyl-S2 fragments followed by Mo, W, and Ni complexes with this ligand. Our synthetic strategies and results will be discussed. These complexes will then be used to elucidate the physical and chemical properties of metal ions in a similar environment to those seen the relevant metalloenzymes.

CHED 725

Generation and chemistry of the parent a-acetoxycarbene

Jonathan R. Mann, and Dasan M. Thamattoor, Department of Chemistry, Colby College, 5750 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, jrmann@colby.edu

Baeyer-Villiger oxidation of the known ketone (1), available in two steps from phenanthrene, led to the ester (2) which is a photochemical precursor of the parent a-acetoxycarbene intermediate (3). Photolysis of 2 in cyclohexene afforded an isomeric mixture of the endo- and exo- acetates (4). The formation of 4 may be explained as an intermolecular trapping reaction of the carbene (3). Other aspects of the inter- and intramolecular chemistry of 3 will be also presented. Insights into the structure and reactivity of this unusual carbene, obtained computationally using hybrid density functional theory (B3LYP/6-31G*), are discussed.

CHED 734

Intramolecular chemistry of a b-acetoxycarbene

Martin J. Schnermann, and Dasan M. Thamattoor, Department of Chemistry, Colby College, 5750 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, mjschner@colby.edu

A photochemical precursor (1), to the b-acetoxycarbene (2), was prepared in three steps from phenanthrene. Photolysis of 1 in benzene-d6 led to the formation of vinylacetate (3). Under these conditions, there appeared to be no evidence for the formation of the lactone (4) that could have been formed, hypothetically, via an intramolecular C-H insertion reaction. Three different mechanisms for the formation of 3 are discussed. The feasibility of these different pathways examined by computational studies, using methods of hybrid density functional theory (B3LYP/6-31G*), will be presented.

CHED 754

New photochemical source of dichlorocarbene

Jonah A. Barasz, and Dasan M. Thamattoor, Department of Chemistry, Colby College, 5750 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, jabarasz@colby.edu

The photolysis of 8,8-dichloro-2,3,5,6-dibenzobicyclo[5.1.0]octa-2,5-dien-4-one (A), readily available in a single step from dibenzosuberone, was carried out in the presence of cyclohexene. The formation of the adduct B during photolysis indicates that A is a source of dichlorocarbene. A comparison of this new photochemical source of dichlorocarbene with the well known cyclopropaphenanthrene-based system will be presented. In addition, the effect of Lewis acids in influencing the photochemical behavior of A will be discussed.

CHED 763

Photochemical generation of an oxiranylcarbene

Joshua E Weitz, and Dasan M. Thamattoor, Department of Chemistry, Colby College, 5750 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, jeweitz@colby.edu

The cyclopropyloxirane derivative (1), a photochemical precursor to oxiranylcarbene (2), was prepared in three steps starting from phenanthrene. Photolysis of 1 was performed at room temperature in various solvents such as cyclohexane-d12, benzene-d6, and dioxane-d8. In all cases, acetone was observed as the predominant product. The product mixture did not show the presence of methyl vinyl ketone or 1-oxaspiro[2.2]pentane. These results suggest that the putative carbene intermediate (2) prefers to fragment rather than undergo ring-expansion or intramolecular C-H inserion reactions. The other fragmentation partner, acetylene, could not be detected under the photolysis conditions. Results of ab initio molecular orbital calculations employed to model the behavior of 2 will be also discussed.

CHED 826

Synthesis of Montiporynes A and B

Traci J Speed, and Dasan M. Thamattoor, Department of Chemistry, Colby College, 5750 Mayflower Hill, Colby College, Waterville, ME 04901, Fax: 207-872-3804, tjspeed@colby.edu

Montiporynes A and B, that were among the recently isolated diacetylenic ketones from the stony coral Montipora sp., and reported to possess in vitro cytotoxic activity against several human solid tumor cells, have been synthesized in three easy steps. Thus 1-iodononyne, quantitatively prepared from 1-nonyne using NIS and silver nitrate, underwent a copper iodide mediated coupling with propargyl alcohol to afford 2,4-dodecadiynyl alcohol. Subsequent Swern oxidation followed by an in situ Wittig reaction gave Montiporyne A. Upon standing at room temperature for a few hours, Montiporyne A partially isomerized to the isomeric Montiporyne B. The two isomers were separated by flash chromatography and characterized by a variety of techniques such as NMR, IR, and GC-MS.

CHED 872

Determination of nizatidine guest-host complexation constants by electrospray ionization mass spectrometry

Grant C Swisher, and Thomas W. Shattuck, Department of Chemistry, Colby College, 5759 Mayflower Hill Drive, Waterville, ME 04901, Fax: 207-872-3804, gcswishe@colby.edu

Nizatidine, N-[2-[[[2-[(dimethylamino) methyl]-4-thiazolyl]methyl]thio]ethyl]-N'-methyl-2-nitro-1,1, ethenediamine, has been examined for its potential to act as a host to small molecules. This complexation is the result of a number of molecular interactions that may include, to varying degrees, hydrogen bonding and pi-pi interactions. With the use of electrospray ionization mass spectrometry, the binding constants of several guests in aqueous solutions will be determined. These results complement binding studies using high performance liquid chromatography. Guests, such as pyridoxine (vitamin B6) and albuterol, have been proven to have large guest-host binding constants by HPLC. These two guests, as well as benzene-1,2-dimethanol, will be studied in order to explore molecular recognition with the nizatidine host.

CHED 873

Determination of nizatidine-guest complexation constants and computer-aided molecular design

Tamas Juhasz, and Thomas W. Shattuck, Department of Chemistry, Colby College, 5759 Mayflower Hill, Waterville, ME 04901, Fax: 207-872-3804, t_juhasz@colby.edu

Nizatidine, N-[2-[[[2-[(dimethylamino)methyl]-4-thiazolyl]methyl]thio]ethyl]-N'-methyl-2-nitro-1,1, ethenediamine, has been investigated for its potential to act as a host to small molecules. Pyridoxine (vitamin B6) and albuterol have large guest-host binding constants with nizatidine. The binding constants of two other guests, benzene-1,2-dimethanol and sorbitol will be studied by HPLC, using electrospray mass spectrometry as a detector. These studies will complement direct binding studies using electrospray ionization mass spectrometry. Aqueous molecular dynamic calculations will be carried out to study the specific guest-host interactions.