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King Research Group

Synthesis and Evaluation of New Nitroxyl (HNO) Donors
An increasing amount of evidence highlights the biological importance of nitroxyl (HNO), the one-electron reduced form of nitric oxide (NO).  While nitroxyl-releasing compounds mimic the action of NO donors under some conditions, recent studies reveal distinct biological activities for HNO and NO.  For example, HNO donors clearly improve the contractility of both normal and failing hearts through mechanisms that do not involve NO.  Specifically, nitroxyl appears to stimulate calcium cycling in cardiac muscle and increase the sensitivity of cardiac myofilaments to calcium.  These results suggest the potential of nitroxyl donors for the treatment of heart failure and have prompted the commercial investigation of HNO donors as therapeutic agents (Cardioxyl).

nAngeli’s salt (sodium trioxodinitrate, Na2N2O3) is currently the most widely used HNO donor.  However, with the described emerging picture, new nitroxyl donors will be of increasing importance as both biochemical and pharmacological tools and potential therapeutic agents. Our group has initiated the investigation of other C-nitroso compounds, specifically   a-acetoxy nitroso compounds, as HNO donors.  As a model, the  a-acetoxy nitroso compound-derived from cyclohexanone (1) has been prepared as a bright blue oil by the oxidation of cyclohexanone oxime with lead tetra-acetate (Scheme 1).  Hydrolysis of the acetate group results in decomposition to cyclohexanone and HNO, which has been confirmed by the gas chromatographic identification of nitrous oxide.  In addition, decomposition of 1 in the presence of a ferric porphyrin yields the ferrous porphyrin-NO complex.  The lead tetra-acetate oxidation of cyclohexanone oxime in the presence of an excess of another carboxylic acid results in the formation of a new  a-carboxy nitroso compound, depending on the carboxylic acid.  Oxidation of cyclohexanone oxime in the presence of 10 equivalents of trifluoroacetic acid and p-nitrobenzoic acid yields 2 and 3, respectively (Scheme 1).  Such reactivity allows the preparation of compounds with different profiles of HNO release or physical properties.  Compound 2 hydrolyzes in pH 7.4 buffer while compound 3 is a solid, which has been characterized by X-ray crystallography.  These initial studies provide the foundation for current synthetic work that includes 1) preparing water soluble derivatives, 2) generating long-lasting HNO donors and 3) synthesizing structurally unique HNO donors based on known bio-molecules or drugs. 


Another major group of experiments is aimed at determining the reactivity of these new HNO donors towards simple thiols and thiol containing proteins.  HNO reacts as an electrophile with thiols to yield a condensation product that forms disulfides or sulfinamides depending on the conditions (Scheme 2).   This reactivity has been proposed to be responsible for much of HNO’s biological activity by altering numerous thiol-containing proteins.   Currently, the reactions HNO and  a-acetoxy nitroso compounds with small molecule thiols (Cys and GSH) as well as aldehyde dehydgonease (AlDH) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are being examined by classical analytical and biochemical methods as well as MALDI-TOF and elecdtrospray mass spectrometry.  Other new studies have begun to look at the reactions of these new HNO donors with metal-containing proteins.

Examination of the reactions of HNO with thiol-containing proteins merges with a long-running project designed to detect protein sulfenic acids (RSOH) as important and reversible post-translational modifications that control numerous activities.   In collaboration with Leslie Poole, (WFU biochemistry) our group has generated numerous dimedone-based traps that label protein sulfenic acids both in purified proteins and cells (Scheme 3).  Current work focuses on synthesizing more specific and isotopically labeled labels as well as biological investigations to better define the roles of sulfenic acids in cells.   Along these lines, we also maintain interest in the detection of thiol proteins modified with an S-nitrosothiol group (RSNO) and have recently reported unique reactivity and labeling of a protein-SNO with a water-soluble phosphine (Scheme 3).



Other projects related to HNO include developing new HNO detection methods.   The high reactivity of HNO with itself and other biomolecules prevents HNO’s direct detection and limits our understanding of how this nitrogen oxide elicits its effects, its endogenous production and its development as a therapy.   We have found that HNO reacts with phosphines to generate an aza-ylide species and that properly substituted phosphines undergo Staudinger ligations to give amides as stable end-products (Scheme 4).   Current work is focused on further exploring this method as a biological method of HNO detection.


The Redox Chemistry of Nitrite and Nitrate in Biology
Nitrite has emerged as an important biological mediator over the last decade.  Under hypoxic conditions, nitrite acts as an important NO source and thus represents a hypoxia-mediated non-NOS dependent source of NO.  Nitrite has increasingly become considered as a therapy for numerous vascular conditions.  More recently, nitrate (primarily derived from diet) has been revealed as a potential source of bioactive nitrite as nitrate can be reduced to nitrite (and ultimately to NO) through the action of bacterial enzymes.   The Scheme below (taken from Nature Reviews/Drug Discovery) depicts the generation of nitrite and NO from dietary nitrate and strongly suggests dietary interventions for various vascular conditions including hypertension, stroke and perhaps diabetes.  Projects in this area are in collaboration with Dany Kim-Shapiro (WFU Physics) and the newly formed Translational Science Center (TSC).  Current studies attempt to define optimal nitrate-containing diets, the effects of such diets on brain blood flow and cognition, mechanisms of nitrate conversion to nitrite and NO and the development of organic molecules as sources of nitrate or nitrite.  One particularly interesting area of investigation is the formation of nitrite from common and widely-used nitro-aromatic containing antibiotics.   The fundamental reactions of these simple compounds with biological compounds (especially proteins) also constitute a major research thrust.  These projects are collaborative by nature and often provide students the opportunity to work with biological samples and human subjects. 

Synthesis of Anti-Cancer Agents/Synthetic Reactions
Using a combination of computational modeling and cell biology, Dr. Fred Salsbury and Dr. Karin Scarpinato, (Departments of Physics and Cancer Biology, respectively) have identified a “death conformation” of the MutS homologous proteins that is distinguishable from their “repair conformation” and their repair function.  Reserpine, a naturally occurring alkaloid previously used to treat hypertension, induces this conformation leading to DNA-independent cell death.  These studies have important implications for the identification of compounds that require functional mis-match repair (MMR) proteins to exhibit their full cytotoxicity, which will avoid resistance in cells deficient in MMR proteins.  Compounds capable of specifically stabilizing the death conformation can ultimately be viewed as anti-cancer therapies acting by a unique DNA-independent action.  We have prepared and purified reserpine analogs to investigate the parameters for a small reserpine-like molecule that are required or sufficient to interact with MSH2/MSH6 in a manner that induces MSH2/MSH6-dependent cytotoxic response.  These results also identify rescinnamine as an improved lead compound for the development of this class of agents and current work strives to further modify rescinnamine based on computer modeling as well as generating fluorescently labeled derivatives.

Dr. Suzy Torti, Department of Biochemistry, has been developing multi-walled carbon nanotubes (MWCNT) for the diagnosis and monitoring of advanced breast cancer. Carbon nanotubes offer many advantages for targeted molecular imaging techniques, such as PET, because of their ability to deliver large numbers of imaging agents per each targeted molecular recognition.  These agents can also deliver several different types of imaging agents and can be used for therapeutic applications including chemotherapeutic drug or gene delivery.  MWCNT may be superior to other types of fullerenes (single-walled carbon nanotubes (SWCNT)) for such applications due to their greater surface area and internal volume per particle.  MWCNT are far more efficient transducers of near infrared radiation into heat and are ideal for use in thermal ablation or optical imaging, increasing the possibilities for future development of combined diagnostic and therapeutic applications.  We are currently preparing in collaboration with the Torti group modified MWCNT’s that contain either targeted carbohydrates or peptides/proteins for cancer imaging or therapy.

All of the projects described above are supported by synthetic organic chemistry and synthetic methodology projects are pursued when appropriate.  We recently reported the ring expansion of cyclic ketones to cyclic hydroxamic acids through a unique rearrangement that includes a C-nitroso intermediate (Scheme 5).  These reactions provide a direct route to cyclic hydroxamic acids, which are important components of many iron-binding natural products.  Current work examines both inter and intramolecular acyl nitroso-based ene reactions for generating similar structures.

Recent (since 2008) Publications

El-Armouche, A.; Wahab, A.; Wittkopper, K.;  Schulze, T.; Bottcher, F.; Pohlmann, L.; King, S. B.; Dumond, J. F.; Feroff, C.; Boger, R. H.; Eschenhange, T.; Carrier, L.; Donzelli, S. “ The New HNO Donor, 1-Nitrosocyclohexyl Acetate, Increases Contractile Force in Normal and ß-Adrenergically Desensitized Myocytes,” Biochem. Biophys. Res. Commun. 2010, 402, 340-344.

Vasilyeva, A.; Clodfelter, J. E.; Gorczynski, M. J.; Gerardi, A. R.; King, S. B.; Salsbury, F.; Scarpinato, K. D. “Parameters of Reserpine Analogs that Induce MSH2/MSH6-Dependent Cytotoxic Response,”  J. Nucleic Acids  2010.

Klomsiri, C.; Nelson, K. J.; Bechtold, E.; Soito, L.; Johnson, L. C.; Lowther, W. T.; Ryu, S-E.; King, S. B.; Furdui, C. M.; Poole, L. B. “Use of Dimedone-Based Chemical Probes for Sulfenic Acid Detection:  Evaluation of Conditions Affecting Probe Incorporation into Redox-Sensitive Proteins,” Methods in Enzymology 2010, 473, 77-94.

Huang, Z.; Velazquez, C.; Abdellatif, K.; Chowdhury, M.; Jain, S. Reisz, J.; DuMond, J.; King, S. B.; Knaus, E. “Acyclic Triaryl Olefins Possessing a Sulfohydroxamic Acid Pharmacophore: Synthesis, Nitric Oxide/Nitroxyl Release, Cyclooxygenase Inhibition, and Anti-Inflammatory Studies,” Org. Biomol. Chem. 2010, 8, 4124-4130.

Reisz, J. A.; Bechtold, E.; King, S. B. “Oxidative Heme Protein-Mediated Nitroxyl (HNO) Generation," Dalton Trans., 2010, 39, 5203-5212.

Bechtold, E.; Reisz, J. A.; Klomsiri, C.; Allen W. Tsang, A. W.; Marcus W. Wright, M. W.; Poole, L. B.; Furdui, C. M.; King, S. B. “Water-Soluble Triarylphosphines as Biomarkers for Protein S-Nitrosation," ACS Chemical Biology, 2010, 5, 405-414.

Goetz, B. I.; Shields, H. W.; Basu, S.; Wang, P.; King, S. B.; Hogg, N.; Gladwin, M. T.; Kim-Shapiro, D. B. “An Electron Paramagnetic Study of the Affinity of Nitrite for Methemoglobin,” Nitric Oxide 2010, 22, 149-154.

Choe, C.; Lewerenz, J.; Fischer, G.; Uliasz, T. F.; Espey, M. G.; Hummel, F. C.; King, S. B.; Schwdhelm, E.; Bӧger, R. H.; Gerloff, C.; Hewett, S. J.; Magnus, T.; Donzelli, S. “Nitroxyl Exacerbates Ischemic Cerebral Injury and Oxidative Neurotoxicity,” J. of Neurochemistry, 2009, 110, 1776-1773.

Banerjee, R.; King, S. B. “Synthesis of Cyclic Hydroxamic Acids through –NOH Insertion of Ketones,” Organic Letters, 2009, 11, 4580-4583.

Gorczynski, M. J.; Smitherman, P. K.; Akiyama, T. E; Wood, H. B.; Berger, J. P.; King, S. B.; Morrow, C. S. “Activation of Peroxisome Proliferator-Activated Receptor g (PPARg) by Nitroalkene Fatty Acids: Importance of Nitration Position and Degree of Unsaturation,” J. Med. Chem. 2009, 52, 4631-4639.

Miller, T. W.; Cherney, M. M.; Lee, A. J.; Francoleon, N. E.; Farmer, P. J.; King, S. B.; Hobbs, A. J.; Miranda, K. M.; Burstyn, J. N.; Fukuto, J. M. “The Effects of Nitroxyl (HNO) on Soluble Guanylate Cyclase Activity: Interactions at Ferrous Heme and Cysteine Thiols,”  J. Biol. Chem. 2009, 284, 21788-21796.

Goetz, B.; Wang, P.; Shields, H. W.; Basu, S.; Grubina, R.; Huang, J.; Conradie, J.; Huang, Z.; Jeffers, A.; Jiang, A.; He, X.; Azarov, I.; Seibert, R.; Mehta, A.; Patel, R.; King, S. B.; Hogg, N.; Ghosh, A.; Gladwin, M. T.; Kim-Shapiro, D. B. “Reply to Nitrite-methemoglobin inadequate for hypoxic vasodilation,” Nat. Chem. Biol., 2009, 5, 367.

Reisz, J. A.; Klorig, E. B.; Wright, M. W.; King, S. B. “Reductive Phosphine-Mediated Ligation of Nitroxyl (HNO),” Organic Letters, 2009, 11, 2719-2721.

Bates, D. J. P.; Lively, M. O.; Gorczynski, M. J.; King, S. B.; Townsend, A. J.; Morrow, C. S. “Noncataltyic Interactions between Glutathione S-Transferases and Nitroalkene Fatty Acids Modulate Nitroalkene-Mediated Activation of Peroxisomal Proliferator-Activated Receptor g,” Biochemistry, 2009, 48, 4159-4169.

Alexander, R. L.; Wright, M. W,; Gorczynski, M. J.; Smitherman, P. K.; Akiyama, T. E.; Wood, H. B; Berger, J. P; King, S. B; Morrow, C. S. “Differential potencies of naturally occurring regioisomers of nitrolinoleic acid in PPARg activation,” Biochemistry, 2009, 48, 492-8

Basu, S.; Azarov, N. A.; Font, M. D.; King, S. B.; Hogg, N.; Gladwin, M. T.; Shiva, S.; Kim-Shapiro, D. B. “Nitrite Reductase Activity of Cytochrome C,” J. Biol. Chem. 2008, 283, 32590-32597.

Donzelli, S.; Espey, M. G.; Flores-Santana, W.; Switzer, C. H.; Yeh, G. C.; Huang, J.; Stuehr, D. J.; King, S. B.; Miranda, K. M.; Wink, D. A. “Generation of Nitroxyl by Heme Protein-Mediated Peroxidation of Hydroxylamine but not Hydroxy-L-arginine,” Free Rad. Biol. Med., 2008, 45, 578-584.

He, X.; Azarov, I.; Jeffers, A.; Presley, T.; Richardson, J.; King, S. B.; Gladwin, M. T.; Kim-Shaprio, D. B. “The Potential of Angeli's Salt to Decrease Nitric Oxide Scavenging by Plasma Hemoglobin,” Free Rad. Biol. Med., 2008, 44, 1420-1432.

Nelson, K. J.; Day, A. E.; Zeng, B. B.; King, S. B.; Poole, L. B. “Isotope-coded, Iodoacetamide-Based Reagent to Determine Individual Cysteine pKa Values by MALDI-TOF Mass Spectrometry,” Analytical Biochemistry, 2008, 375, 187-195.