Our research program is based on a combination of organic chemistry and biochemistry directed towards understanding the various roles nitric oxide (NO) performs in biological systems.  Nitric oxide directly participates in the control of blood flow and pressure, neurotransmission, and the immune response and the regulation of NO levels represents a therapeutic strategy for disease states characterized by abnormal NO production.  Our laboratory examines three major areas of NO chemistry:  1) the reaction of nitric oxide and hydroxyurea with hemoglobin to understand the chemistry responsible for the use of these agents as sickle cell disease treatments, 2) the interaction of small molecules with nitric oxide synthase (NOS), the enzyme responsible for biochemical NO production from L-arginine and 3) the synthesis and evaluation of new organic NO delivery agents.  In addition to these projects, our laboratory works to develop new synthetic organic methodology for the preparation of biologically active compounds.

Chemistry of N-Hydroxyureas and Reactions of Nitric Oxide and Hydroxyurea with Heme Proteins

Much of our basic research involves understanding the oxidative chemistry of N-hydroxyureas and the ability of these compounds to liberate nitric oxide or the one electron reduced form of nitric oxide, nitroxyl (HNO).  Nitroxyl displays many of the same pharmacological properties as nitric oxide and can be oxidized to NO by many biological oxidants.  Our results indicate that N-hydroxyureas can be chemically oxidized to the corresponding nitroxide radicals and nitroso compounds, which react with water to produce the corresponding amine, carbon dioxide, and nitroxyl.  The presence of nitroxyl is implied by the gas chromatographic identification of nitrous oxide, the dimerization and dehydration product of HNO.  Current investigations are aimed at more clearly understanding the chemical mechanisms of NOand HNO formation from N-hydroxyureas.

Identification of proteins and enzymes capable of oxidizing hydroxyureas with nitric oxide release represents a major research project in the group.  Our recent efforts have focused on nitric oxide release during the reactions of hydroxyureas with heme-containing proteins including hemoglobin, horseradish peroxidase and catalase.  Hydroxyurea, the simplest hydroxyurea, is an approved treatment of sickle cell anemia and understanding the chemical reactions of this drug with hemoglobins could be important in explaining the action of this drug.  Much of this work is done in collaboration with Dr. Dany Kim-Shapiro, Wake Forest University, Department of Physics.  Each of the above proteins react with hydroxyurea to produce either NO or HNO as determined using a combination of various spectroscopic methods and analytical techniques including electron paramagnetic resonance and ultraviolet spectroscopy as well as chemiluminescence NO detection. Current efforts are focused on determining the site and mechanism of in vivo metabolism of hydroxyurea to NO or HNO.  In addition to the direct oxidation of hydroxyurea to NO, we are also investigating an alternative mechanism of NO production from hydroxyurea which entails the initial hydrolysis of hydroxyurea to hydroxylamine with the subsequent oxidation of hydroxylamine to NO or HNO.  In addition to these studies, our group also participates in collaborative work with laboratories at the National Institutes of Health and the UCLA medical school to determine how hydroxyurea-derived NO benefits sickle cell patients.  These studies examine the ability of hydroxyurea to stimulate soluble guanylate cyclase, which ultimately controls fetal hemoglobin biosynthesis.

In collaboration with Dr. Kim-Shaprio, a series of biophysical experiments designed to evaluate the effects of hydroxyurea on the physical properties of sickle cell hemoglobin and sickle red blood cells has been initiated.  Recently, we have shown that iron nitrosylation has little effect on the solubility of sickle cell deoxyhemoglobin, an important result when one considers the recent suggestion to use inhaled NO gas as a treatment for sickle cell disease.  Other collaborative work with Dr. Kim-Shapiro examines the chemistry and biochemistry of the reactions of nitric oxide with hemoglobin.  An important result from this work is that binding of nitric oxide to partially oxygenated hemoglobin does not follow cooperative binding behavior.

Hydroxyurea also inhibits the enzyme ribonucleotide reductase, the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides.  Inhibition of ribonucleotide reductase blocks DNA synthesis and stops cell division and hydroxyurea has been used as a treatment for a number of cancers.  Hydroxyurea inhibits ribonucleotide reductase by quenching the catalytically essential tyrosyl radical of the enzyme.  This reaction also generates the same hydroxyurea radical formed in the heme protein catalyzed oxidation of hydroxyurea that ultimately forms nitric oxide.  Currently, we are evaluating the reaction of hydroxyurea with ribonucleotide reductase for NO formation.  In addition, we have prepared carbohydrate and peptide-derived hydroxyureas as ribonucleotide reductase inhibitors and are testing these compounds both as inhibitors of ribonucleotide reductase and cytotoxic agents.

Representative Publications in this Area

Huang, J.; Zou, Z.; Kim-Shapiro, D. B.; Ballas, S. K.; King, S. B., “Hydroxyurea Analogs as Kinetic and Mechanistic Probes of the Nitric Oxide Producing Reactions of Hydroxyurea and Oxyhemoglobin,” J. Med. Chem., in press.

Lockamy, V. L.; Huang, J.; Shields, H.; Ballas, S. K.; King, S. B.; Kim-Shapiro, “Urease Enhances the Formation of Iron Nitrosyl Hemoglobin in the Presence of Hydroxyurea,” Biochimica et Biophysica Acta, in press.

King, S. B. “The Nitric Oxide Producing Reactions of Hydroxyurea, Current Medicinal Chemistry, 2003, 10, 437-452.

King, S. B. “A Role for Nitric Oxide in Hydroxyurea-Mediated Fetal Hemoglobin Induction,” J. Clin. Invest. 2003, 111, 171-172.

Xu, X.; Lockamy, V. L.; Chen, K.; Huang, Z.; Shields, H.; King, S. B.; Ballas, S. K.; Nichols, J. S.; Gladwin, M. T.; Noguchi, C. T.; Schechter, A. N.; Kim-Shapiro, D. B. “Effects of Iron Nitrosylation on Sickle Cell Hemoglobin Solubility,” J. Biol. Chem., 2002, 277, 36787-36792

Huang, Z.; Ucer, K.B.; Murphy, T.; Williams, R.T.; King, S.B.; Kim-Shapiro, D. B. “Kinetics of Nitric Oxide Binding to R-State Hemoglobin,” Biochemical and Biophysical Research Communications, 2002, 292, 812-818.

Huang, J.; Sommers, E.; Kim-Shapiro, D. B.; King, S. B. “Horseradish Peroxidase Catalyzed Nitric Oxide Formation from Hydroxyurea,” J. Am. Chem. Soc., 2002, 124, 3473-3480.

Huang, J.; Hadimani, S. B.; Rupon, J. W.; Ballas, S. K.; Kim-Shapiro, D. B.; King, S. B. “Iron Nitrosyl Hemoglobin Formation from the Reactions of Hemoglobin and Hydroxyurea,” Biochemistry, 2002, 41, 2466-2474.

Huang, Z.; Louderback, J.G.; Goyal, M.; Azizi, F.; King, S. B.; Kim-Shapiro, D. B. “Nitric Oxide Binding to Oxygenated Hemoglobin Under Physiological Conditions,” Biochimica et Biophysica Acta, 2001, 1568, 252-260.

Huang, Z.; Louderback, J. G.; King, S. B.; Ballas, S. K.; Kim-Shapiro, D. B. "In Vitro Exposure to Hydroxyurea Reduces Sickle Red Blood Cell Deformability" Am. J. Hematol., 2001, 67, 151-156.

Rupon, J.W.; Domingo, S.R.; Smith, S.V.; Gummadi, B.K.; Shields, H.; Ballas, S.K.; King, S.B.; Kim-Shapiro, D.B. “The Reactions of Myoglobin, Normal Adult Hemoglobin, Sickle Cell Hemoglobin and Hemin with Hydroxyurea,” Biophysical Chem. 2000, 84, 1-11.

Design and Synthesis of Small Molecules that Interact with Nitric Oxide Synthase

Another major research interest of our group focuses on the synthesis and evaluation of new inhibitors and alternative substrates of the nitric oxide synthases.  Based upon the recent X-ray crystallographic structures of the nitric oxide synthases and other mechanistic work, we are examining a series of L-canavanine derivatives, including N-hydroxy-L-canavanine, as alternative NO producing substrates in an effort to define the structural requirements of NO biosynthesis. In addition, we are also examining L-arginine derivatives that contain N-hydroxyphosphonamide or N-hydroxysulfonamide groups (1) as transition state analogs for the conversion of N-hydroxyarginine to L-citrulline and NO.  Small non-amino acid thioureas and S-alkyl isothioureas (2-3) are also being examined as potential isoform selective NOS inhibitors.  These projects rely heavily upon synthetic organic chemistry to prepare potential inhibitors and substrates.  These compounds are evaluated against the enzyme using standard assays for NO or L-citrulline production.

Representative Publications in this Area

Li, X.; Atkinson, R. N.; King, S. B. "Synthesis and Evaluations of New L-Canavanine Derivatives as Inhibitors of Nitric Oxide Synthase, Tetrahedron, 2001, 57, 6557-6565.

Ware, R. W., Jr.; King, S. B. "Evaluation of New L-Thiocitrulline Derivatives as Inhibitors of Nitric Oxide Synthase," Bioorganic and Medicinal Chemistry Letters, 2000, 10, 2779-2781.

 

Synthesis of New Nitric Oxide and Nitroxyl Delivery Agents

Anothermajor research area in our group involves the design, synthesis and evaluation of new organic compounds capable of nitric oxide or nitroxyl release.  A unique approach to nitroxyl release utilizes 9,10-dimethylanthracene-acyl nitroso compound cycloadducts as the nitroxyl delivery agents.  These compounds undergo retro-Diels Alder dissociation to form 9,10 dimethylanthracene and a nitroso compound that can decompose to liberate nitroxyl.  Initial experiments indicate that the cycloadduct of the nitroso compound of hydroxyurea and 9,10-dimethylanthracene decomposes in a first order fashion at 40 °C with nitroxyl release and a half-life of 2.6 hours.  Current efforts to develop water-soluble anthracene-acyl nitroso and polymer derived nitroxyl delivery agents are underway.  Our group also collaborates with the laboratory of Dr. John Toscano, Johns Hopkins to examine the decomposition of these cycloadducts with the formation of acyl nitroso species by time-resolved infrared spectroscopy.  Similar studies of the photolysis of 3, 5-diphenyl-1, 2, 4-oxadiazole-4-oxide reveals the first direct observation of an acyl nitroso species in solution and also provides evidence for the formation of HNO in the presence of nucleophiles.

Representative Publications in the Area

Cohen, A. D.; Zeng, B.; King, S. B.; Toscano, J. P. “Direct Observation of an Acyl Nitroso Species in Solution by Time-Resolved IR Spectroscopy,” J. Am. Chem. Soc. 2003, 1444-1445.

Zeng, B.; King, S. B. “Palladium Catalyzed Synthesis of Water-Soluble Symmetric 9,10-Disubstituted Anthracenes,” Synthesis, 2002, 2335-2337.

Xu, Y.; Alavanja, M.M.; Johnson, V.L.; Yasaki, G.; King, S.B. "Production of Nitroxyl (HNO) at Biologically Relevant Temperatures from the retro-Diels Alder Reaction of N-Hydroxyurea Derived Acyl Nitroso-9, 10-Dimethylantrhacene Cycloadducts," Tetrahedron Lett. 2000, 41, 4265-4269.

Synthetic Organic Methodology

P-Nitrosophosphine oxides (where R = alkyl, aryl) liberate nitrous oxide and act as N-O hetero-dieneophiles in Diels Alder reactions.  These reactive species are generated by the oxidation of the corresponding N-hydroxyphosphonamide.  Asymmetric P-nitrosophosphine oxides have been prepared that diastereoselectively react with simple dienes.  In addition, intramolecular hetero Diels-Alder reactions of these species are being examined as a new method to prepare asymmetric cyclic organophosphorus compounds, like 4.  A number of these compounds are also being studied as anti-cancer agents in collaboration with the National Cancer Institute of the National Institutes of Health.  In addition, P-nitrosophosphates, where R = OR, have also recently been described as new nitric oxide donors and N-O heterodienophiles.

We recently have also disclosed a new method for the preparation of cyclic heterocycles that relies upon the ring opening cross metathesis of 1, 3-cyclopentadiene and heterodienophile cycloadducts.  Current work focuses on elaborating these highly functionalized substrates into natural products, synthetic intermediates or novel polymeric materials.

Representative Publications in this Area

Ware, R. W., Jr.; Day, C. S.; King, S. B. “Diastereoselective and Intramolecular Cycloadditions of Asymmetric P-Nitroso Phosphine Oxides,” J. Org. Chem., 2002, 67, 6174-6180.

Ellis, J.M.; King, S.B. “Ring-opening cross metathesis of 1,3-cyclopentadiene- heterodienophile cycloadducts to produce cyclic hydrazines and hydroxylamines,” Tetrahedron Lett., 2002, 43, 5833-5835.

Ware, R. W., Jr.; King, S. B. "P-Nitroso Phosphate Compounds:  New N-O Heterodienophiles and Nitroxyl Delivery Agents," J. Org. Chem. 2000, 65, 8725-8729.