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 and NO show differences in their ability to promote oxidative DNA damage.   Another group of studies highlight the discrete effects mediated by nitroxyl and nitric oxide in both normal and failing heart models.  Specifically, nitroxyl stimulates calcitonin gene-related peptide release while nitric oxide increases cyclic guanylate monophosphate (cGMP) and these results suggest the potential of nitroxyl donors for the treatment of heart failure. These studies have prompted both the theoretical and experimental examination of the thermodynamic properties and chemical reactivity of HNO.

Angeli’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, furhter independent evidence for initial HNO production.  Interestingly, 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.  For example, 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 much current work in this area that includes 1) examining their biological activity, especially in cardiac tissue, 2) generating structurally unique a-acetoxy nitroso compounds based on known biomolecules or drugs, 3) understanding the basic chemistry and reactivity of these molecules (especially with thiols), and 4) their reactions with other thiol containing proteins.  In addition, to these compounds, other work involves new derivatives of Angeli’s salt and Piloty’s acid (PhSO2NHOH) as new HNO donors and developing new organic chemistry based methods for the trapping and detection of HNO.

Reactions of Nitric Oxide and Nitrite with Hemoglobin
 
The reactions of nitric oxide with hemoglobin have drawn considerable interest in an effort to explain how the bioactivity of NO is maintained.  In a collaborative program with Dr. Dany Kim-Shapiro of the Wake Forest University Department of Physics, we have studied the reactions of NO with purified human hemoglobin, red blood cells, and whole blood in an effort to provide a clearer understanding of how NO functions as a vasodilator.  Present work focuses on the reaction chemistry of hemoglobin and nitrite, which recently has been shown to cause vasodilation in vivo.  Deoxyhemoglobin (Fe+2) reacts with nitrite to yield methemoglobin (Fe+3) and NO (Scheme 2).  The NO rapidly binds to unreacted deoxyhemoglobin to form iron nitrosyl hemoglobin (Fe+2-NO) (Scheme 2).  The bioavailability of the NO in iron nitrosyl hemoglobin remains an area of intense research as well as the possible identification of other reaction intermediates capable of nitric oxide release and/or vasodilation.  Further studies show the ability of nitrite to also bind and react with methemoglobin to form nitrogen dioxide and deoxyhemoglobin (Scheme 2).  The nitrogen dioxide can further react with nitric oxide to give N2O3, a powerful nitrosating reagent capable of reacting with many biomolecules.  Thus nitrite and hemoglobin form a nitric oxide redox cycle that may have profound physiological implications in terms of normal blood pressure and flow control. 
 

Another major collaborative project with the Kim-Shapiro lab has been to use nitric oxide, nitroxyl and nitrite donors to alleviate the negative effects of hemolysis.   Much recent work shows that hemolysis (free hemoglobin in the plasma that escapes the red blood cell) greatly interferes with normal nitric oxide physiology and thus these NO donors may restore normal function and could be used as therapies for many conditions (sickle cell disease, transfusion, malaria, various surgeries and even insect/snake bite).     
   
                                                                                  
Synthesis and Evaluation of Fluorescent Probes for Protein Sulfenic Acids as Proteomic Tools for Redox Profiling

Sulfenic acid formation in enzymes and transcriptional regulators as assessed by in vitro experiments is now well documented and is clearly an important component of redox catalysis and regulation   The highly reactive and reversible nature of the Cys-SOH modification gives it a unique suitability to H2O2 and other redox signaling pathways; in addition to the facile reversibility that Cys-SOH shares with disulfide bonds, it also (unlike disulfide bonds) acts as a redox sensor responsive to “oxidative stress” (Figure).  Cys-SOH is a metastable redox form of cysteine which can be stabilized in certain protein environments or can react with proximal thiol groups of other cysteines or external agents (including glutathione) to form disulfide bonds.  Both sulfenic acids and disulfide bonds are readily reducible by cellular thiols (primarily via glutathione or thioredoxin) back to the thiol state and, as such, are appropriately suited for roles in both redox regulation and catalysis.  On the other hand, Cys-SOH lies on the pathway toward oxidative inactivation through irreversible formation of cysteine sulfinic (Cys-SO2H) and sulfonic (Cys-SO3H) acid forms.  Even these irreversible modifications may play a functional role in redox sensing, although this suggestion remains only speculative at this point.

Dimedone has found use as a sulfenic acid probe as it reacts with the sulfenic acid group as a nucleophile to give an adduct.  However, this method is only applicable with purified proteins used in in vitro studies as dimedone bears no useful label for detection or isolation (the radiolabeled reagent is no longer commercially available, and its synthesis is prohibitively expensive).  The addition of dimedone can be detected by the extra mass that it imparts upon adduct formation with a cysteine sulfenic acid, but identification then requires mass spectrometric analysis.  This analysis is practical for only one or a few isolated proteins at a time, although improved mass spectrometric methods and instruments continue to extend our ability to analyze chemical modifications in proteins in more and more complex mixtures of tryptic digests.  As such, we have initiated a program to prepare dimedone analogs with a fluorophore or biotin derivative attached to allow detection through through fluorescence spectroscopy or immunoassay (Scheme 3).  Such compounds would allow facile analyses of the intermediacy of protein sulfenic acids in a large-scale proteomic format

We have developed new Cys-SOH-reactive, fluorophore and biotin-linked reagents (4-5) and have shown them to be at least as reactive as dimedone as a Cys-SOH trapping and labeling agents.  Compound 4 has proven especially useful given its high extinction coefficient (ε = 25,000 M-1 cm-1 at 360 nm) and fluorescence, even in protein adducts resolved on polyacrylamide gels. Compound 5 incorporates a biotin tag into the label was tested for its dimedone-like chemical properties using the Cys-SOH-containing C165S mutant of AhpC and measuring its incorporation by fluorescence and absorbance spectroscopy as well as mass spectrometry.  In one sample, both compound 5 and dimedone were mixed in equal portions to assess their relative reactivity.  Based on these studies, compound 5 reacts with the Cys-SOH of this protein somewhat more efficiently than does dimedone (addition of dimedone decreased the yield of the protein-compound 5 adduct by 20%), indicating that the addition of the hydrocarbon chain and biotin group, and the lack of the dimethyl group present in dimedone, do not interfere with reactivity towards sulfenic acids.  Current work now is directed at using these unique agents to better understand the role of sulfenic acids in normal cellular processes.  Also, these skills are also being applied to the detection of other oxidatively modified thiols (sulfenic chlorides, S-nitrosothiols, sulfinamides and sulfinic acids) in proteins and selective agents are anticipated for these modifications.

Cyclic Hydroxamic Acid Synthesis
 
Our work with various HNO donors led us to the discovery of new reactivity of Piloty’s acids, namely the insertion of the –NOH group into cyclic ketones to give cyclic hydroxamic acids (Scheme 4).  We currently are exploring the scope of this unique reaction as well as the mechanism in an effort to apply this novel reactivity to the preparation of various siderophores, such as mycobactin, that contain cyclic hydroxamic acids as part of their iron chelating system

Recent (since 2005) Publications

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.

55) 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.

Michalek, R. D.; Nelson, K. J.; Holbrook, B. C.; Yi, J. S.; Stridiron, D.; Daniel, L. W.; Fetrow, J. S.; King, S. B.; Poole, L. B.; Grayson, J. S. “The Requirement of Reversible Cysteine Sulfenic Acid Formation for T Cell Activation and Function,” J. Immunol. 2007, 179, 6456-6467.

Poole, L. B.; Klomsiri, C.; Knaggs, S. A.; Furdui, C. M.; Nelson, K. J.; Thomas, M. J.; Fetrow, J. S.; Daniel, L. W.; King, S. B. “Fluorescent and affinity-based tools to detect cysteine sulfenic acid formation in proteins,” Bioconj. Chem. 2007, 18, 2004-2007.

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. “Catalytic Generation of N2O3 by the Concerted Nitrite Reductase and Anhydrase Activity of Hemoglobin,” Nat. Chem. Biol., 2007, 3, 785-794.

Gorczynski, M. J.; Huang, J.; Lee, H.; King, S. B. “Evaluation of Nitroalkenes as NO Donors,” Bioorganic and Medicinal Chemistry Letters, 2007, 17, 2013-2017.

Chakrapani, H.; Gorczynski, M. J.; King, S. B. “Allylic Nitro Compounds as Nitrite Donors,” J. Am. Chem. Soc. 2006, 128, 16332-16337.

Chen, W.; Day, C. S.; King, S. B. “Grignard Reagent-Mediated Conversion of an Acyl Nitroso-Anthracene Cycloadduct to a Nitrone,” J. Org. Chem. 2006, 71, 9221-9224.

Donzelli, S.; Switzer, C. H.; Thomas, D. D.; Ridnour, L. A.; Espey, M. G.; Isenberg, J. S.; Tocchetti, C. G.; King, S. B.; Lazzarino, G.; Miranda, K. M.; Roberts, D. D.; Feelisch, M.; Wink, D. A. “The Activation of Metabolites of Nitric Oxide Synthase by Metals is Both Redox and Oxygen Dependent:  A New Feature of Nitrogen Oxide Signaling,” Antioxidants and Redox Signaling 2006, 8, 1363-1371.

Sha, X.; Isbell, S.; Patel, R. P. P.; Day, C. S.; King, S. B. “Hydrolysis of Acyl Nitroso Compounds Yields Nitroxyl (HNO),” J. Am. Chem. Soc. 2006, 128, 9687-9692.

Alexander, R. L.; Bates, D. J. P.; Wright, M. W.; King, S. B.; Morrow, C. S. “Modulation of Nitrated Lipid Signaling by Multidrug Resistance Protein 1 (MRP1):  Glutathione Conjugation and MRP1-Mediated Efflux Inhibits Nitrolinoleic Acid-Induced PPARg-Dependent Transcription Activation,” Biochemistry, 2006, 45, 7889-7896.

Gorczynski, M. J.; Huang, J.; King, S. B. Regio- and Stereospecific Syntheses and Nitric Oxide Donor Properties of (E)-9 and (E)-10-Nitrooctadec-9-enoic Acids, Org. Lett., 2006, 8, 2305-2308.

Huang, J.; Yakubu, M.; Kim-Shapiro, D. B.; King, S. B. “Rat Liver-Mediated Metabolism of Hydroxyurea to Nitric Oxide,” Free Rad. Biol. Med., 2006, 40, 1675-1681.

Basu, S.; Hill, J. D.; Shields, H.; Huang, J.; King, S. B.; Kim-Shapiro, D. B. “Hemoglobin effects in the Saville Assay,” Nitric Oxide:  Biology and Chemistry, 2006, 15, 1-4.

Donzelli, S.; Espey, M. G.; Thomas, D. D.; Mancardi, D.; Tocchetti, C. G.; Ridnour, L. A.; Paolocci, N.; King, S. B.; Miranda, K. M.; Lazzarino, G.; Fukuto, J.; Wink, D. A. “Discriminating HNO Formation from Other Reactive Nitrogen Oxide Species,” Free Rad. Biol. Med., 2006, 40, 1056-1066.

Parrish, D. A.; Allen, C. L.; Day, C. S.; Zhou, Z.; King, S. B. “A Convenient Method for the Synthesis of N-Hydroxyureas,” Tetrahedron Lett. 2005, 46, 8841-8843.

Poole, L. B.; Zeng, B.; Knaggs, S. A.; Yakubu, M.; King, S. B. “Synthesis of Chemical Probes to Map Sulfenic Acid Modifications in Proteins,” Bioconj. Chem. 2005, 16, 1624-1628.