Our research program is based on a combination of organic chemistry, biochemistry, and biophysics 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 a number of areas of NO chemistry: 1) the nitric oxide producing reactions of hydroxyurea and the interaction of hydroxyurea with biological targets to understand the chemistry responsible for the use of this compound as a sickle cell disease treatment, 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 and nitroxyl (HNO) delivery agents. In addition to these projects, our laboratory works on two other projects: 1) synthesizing compounds to measure the redox status of sulfur containing proteins and 2) the development of new synthetic organic methodology for the preparation of biologically active compounds. Students in our group are exposed to organic synthesis, biochemistry, molecular biology, biophysics (especially spectroscopy) and bio-analytical chemistry.

1. Nitric Oxide (NO)-producing reactions of Hydroxyurea

Hydroxyurea ( 1) is a new approved treatment for sickle cell disease, a condition that affects about 1 in 600 Americans of African descent. Hydroxyurea has been shown to act as a nitric oxide (NO) donor in vivo and the beneficial effects of hydroxyurea therapy in sickle cell disease may be mediated by NO, which is also being considered as a sickle cell treatment. Liberated NO has been proposed to benefit sickle cell patients by two mechanisms: a) stimulation of fetal hemoglobin synthesis through a soluble guanylate cyclase (sGC) mediated pathway and b) by reacting with cell-free hemoglobin, which is abnormally elevated in sickle cell patients, to restore the function of endogenously produced NO. While NO release from hydroxyurea requires oxidation, the chemical and biochemical mechanism of NO release from hydroxyurea and its reactions with these proposed targets (sGC and cell-free hemoglobin) remain poorly understood. Our studies regarding hydroxyurea as a nitric oxide donor have been supported by the National Institutes of Health (HL62198).

The recently published results regarding NO formation from the reaction of hydroxyurea and catalase provides focus for 1) better understanding the mechanism of NO formation from hydroxyurea, 2) defining the site, mechanism and oxidant of in vivo metabolism of hydroxyurea to NO and 3) better understanding the interaction of hydroxyurea with sGC. Scheme 1 depicts our proposed mechanism of NO formation from the reaction of hydroxyurea and catalase. Catalase activation by hydrogen peroxide yields Compound I that oxidizes hydroxyurea ( 1) to the correpsonding nitroso compound ( 2), which rapidly hydrolyzes to nitroxyl (HNO), the one-electron reduced form of NO and a carbamic acid that decomposes to carbon dioxide ammonia. Nitroxyl rapidly reductively nitrosylates the ferric catalase to form a ferrous-NO catalase complex. These results are significant in a number of ways including a) the identification of nitroso compound ( 2) as an intermediate in the conversion of hydroxyurea to NO, b) the proposal of HNO as an intermediate to NO formation from hydroxyurea, c) the finding that catalase, found in high levels in red blood cells and liver, catalyzes NO formation from hydroxyurea and thus targets potential sites of in vivo NO formation and d) the previous identification that ferrous-NO catalase complexes were competent activators of sGC.

Hydroxyurea has also been used to treat various cancers, especially leukemia, since the 1960’s. The anti-cancer activity of hydroxyurea results from its ability to inhibit ribonucleotide reductase, the enzyme responsible for the reduction of ribonucleotides to deoxyribonucleotides and the first committed step in DNA synthesis. Blocking ribonucleotide reductase inhibits DNA synthesis and cell division. Hydroxyurea inhbits ribonucleotide reductase by quenching a catalytically esssential tyrosyl radical in the protein to leave inactivated protein and a hydroxyurea radical (Scheme 2). We propose that the hydroxyurea radical decomposes to NO and that the quenching of ribonucleotide reductatse by hydroxyurea will also produce NO, which has its own cyctoxic properties (blocks cellular respiration, blocks DNA synthesis, direct DNA damage).

This is the first demonstration that the quenching of the ribonucleotide reductase radical by hydroxyurea results in NO formation and suggests that hydroxyureas may act as dual mechanism anti-cancer agents (inhibit ribonucleotide reductase/donate NO). Further experiments are targeted to show the direct formation of NO in this reaction and the identification of the final iron-NO protein complex.

To further extend this work, we have prepared a series of carbohydrate derived hydroxyureas and hydroxamic acids as potential ribonucleotide reductase inhibitors/NO donors/anti-cancer agents. The basic idea is that the carbohydrate portion of the molecules will provide selectivity toward cancer cells and that the hydroxyurea portion of the molecule will act as both an inhibitor of ribonucleotide reductase and a cytotoxic NO donor. While the cyclic carbohydrate-derived hydroxyurea ( 3) acts as an NO donor, this compound only weakly inhibits ribonucleotide reductase and does not show activity in the National Cancer Institute’s screen. However, the acyclic carbohydrate-derived hydroxamic acid ( 4), prepared by the simple condensation of hydroxylamine and d -gluconolactone displays strong activity (nM for 50% growth inhibition) against a select number of tumor cell lines, inhibits ribonucleotide reductase and acts as a nitric oxide donor (Scheme 3). Interestingly this compound shows a different activity profile from hydroxyurea an may be a lead for a new series of cytotoxic, anti-cancer agents. These studies have been also been supported by the National Institutes of Health (HL62198) and the Department of the Army (DAMD17-01-1-0664).

2. 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 ( 5) as transition state analogs for the conversion of N-hydroxyarginine to L-citrulline and NO. Small non-amino acid thioureas and S-alkyl isothioureas ( 6-7) 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.

3 . 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 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, Na 2N 2O 3) 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. Hydrolysis of acyl nitroso species ( 2, Scheme 1) produces HNO and represents a reliable chemical approach for HNO formation. Earlier work shows that cycloadducts ( 8, Scheme 4) of the acyl nitroso species derived from N-hydroxyureas and 9, 10-dimethylanthracene (9, 10-DMA) undergo a retro-Diels-Alder reaction to form acyl nitroso species that hydrolyze to HNO, carbon dioxide and the corresponding amine. While the cycloadducts of N-hydroxyureas derived from simple alkyl and aryl amines release HNO under mild conditions at useful rates, these compounds demonstrate poor water solubility.

Compound 9, a symmetric cycloadduct of the C-nitrosoformamide ( 2) and the water-soluble disodium salt of 9, 10-anthracenedipropionic acid ( 10), represents the proposed parent synthetic water-soluble nitroxyl delivery agent (Scheme 5). Retro-Diels Alder reaction of this compound would directly produce C-nitrosoformamide ( 2), which would hydrolyze to form nitroxyl and the water-soluble anthracene ( 10, Scheme 5). The periodate oxidation of hydroxyurea to 2 in the presence of 10 , which has been prepared in our laboratory, should provide the target molecule 6 through a hetero-Diels Alder reaction. Based upon our previous results, 9 is expected to decompose by a first-order process at a useful rate at 40 °C by a retro-Diels Alder reaction to form 2 and 10. Nitroxyl release from 9 will be determined by gas chromatographic identification of N 2O, the dimerization and dehydration product of HNO. In addition, both UV-vis and EPR spectroscopy will demonstrate the conversion of metHb to HbNO during the decomposition of 9 in the presence of metHb.

Compound 9 represents the parent for a small family of proposed HNO donors that will demonstrate a range of HNO release rates. Cycloadducts of 9, 10-DMA and acyl nitroso species derived from the hydroxyureas of either primary or secondary amines decompose much faster than the cycloadduct ( 9) derived from hydroxyurea. Our preliminary results already confirm that the addition an alkyl group to the –NH 2 portion of cycloadduct 9 increases the rate of decomposition. The periodate oxidation of the proper hydroxyureas in the presence of the water-soluble anthracene ( 10) should produce the cycloadducts 11 and 12, respectively. Based upon the trend observed with the 9, 10-DMA cycloadducts. these compounds should decompose with HNO release faster than 9. The kinetics of decomposition and the ability to release HNO will be determined similarly as for 9. Alternatively, cycloadducts of 9, 10-DMA and acyl nitroso compounds derived from hydroxamic acids undergo retro-Diels Alder dissociation at slower rates than cycloadducts derived from hydroxyureas. Thus, periodate oxidation of commercially available aceto and benzohydroxamic acid in the presence of 10 should produce the cycloadducts 13 and 14, respectively. These compounds should decompose with HNO release at a slower rate than the parent cycloadduct 9 and will be kinetically evaluated for HNO release as previously described. This small family of compounds would be the first group of nitroxyl donors that demonstrates a wide range of HNO-release profiles and would directly complement the diazeniumdiolate family of NO donors. These compounds would be very useful in distinguishing the biological effects of HNO compared to NO. This work has also been supported by the National Institutes of Health (HL62198) and the American Heart Association (0140020N).

Synthetic Organic Methodology

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.

4. Collaborative Projects

Reactions of Nitric Oxide and Nitrite with Hemoglobin-Dr. Dany Kim-Shapiro, Wake Forest Unieversity Department of Physics

Nitric oxide modified hemoglobins, nitrosylated at the iron or S-nitrosated at the b -93 cysteine residue (S-nitrosohemoglobin, -S-N=O functional group), perform important roles in normal blood pressure control and physiological NO transport. 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. Specifically, we have studied the reactions of nitric oxide in whole blood and hemosylates as a function of oxygen saturation. EPR studies show that under these conditions the amount of HbNO that forms is related to the number of free heme groups present. In addition, we have also examined the preference for NO to bind to the heme iron or bond to the b -93 cysteine to form S-nitrosohemoglobin and whether NO can readily transfer between these atoms. These studies have primarily been funded the National Institutes of Health (R01HL58091-06, Kim-Shapiro, PI, King, Collaborator).

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 9). The NO rapidly binds to unreacted deoxyhemoglobin to form iron nitrosyl hemoglobin (Fe +2-NO) (Scheme 9). 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.

Synthesis and Evaluation of Fluorescent Probes for Protein Sulfenic Acids as Proteomic Tools for Redox Profiling-Dr. Leslie Poole, Wake Forest University, Department of Biochemistry

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 H 2O 2 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 to left). 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-SO 2H) and sulfonic (Cys-SO 3H ) 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 10). Such compounds would allow facile analyses of the intermediacy of protein sulfenic acids in a large-scale proteomic format.

Recent Publications  

King, S. B. “N-Hydroxyurea and Acyl Nitroso Compounds as Nitroxyl (HNO) and
Nitric Oxide (NO) Donors,” Curr. Top. Med. Chem. 2005, 5, 665-673.  

Azizi, F.; Kielbasa, J. E.; Adeyiga, A. M.; Maree, R. D.; Yakubu, M.; Frazier, M.; Shields, H.; King, S. B.; Kim-Shapiro, D. B. “Rates of Nitric Oxide Dissociation from Hemoglobin,” Free Rad. Biol. Med., 2005, 39, 145-151.  

Pennington, R. L.; Sha, X.; King, S. B. “N-Hydroxy Sulfonimidamides as New Nitroxyl (HNO) Donors,” Bioorganic and Medicinal Chemistry Letters, 2005, 15, 2331-2334.  

Zeng, B.; Huang, J.; Wright, M. W.; King, S. B. “Nitroxyl (HNO) Release from New Functionalized N-Hydroxyurea Derived Acyl Nitroso-9, 10-Dimethylanthracene Cycloadducts,” Bioorganic and Medicinal Chemistry Letters, 2004, 14, 5565-5568.  

Lockamy, V. L.; Shields, H.; Kim-Shapiro, D. B.; King, S. B. “Iron Nitrosyl Hemoglobin Formation from the Reaction of Hydroxylamine and Hemoglobin under Physiological Conditions,” Biochimica et Biophysica Acta , 2004, 1674, 260-267.  

King, S. B. “Nitric Oxide Production from Hydroxyurea,” Free. Rad. Biol. Med., 2004, 37, 737-744.  

King, S. B. “Mechanisms and Novel Directions in the Biological Applications of Nitric Oxide Donors,” Free Rad. Biol. Med., 2004, 37, 735-736.

Huang, J.; Kim-Shapiro, D. B.; King, S. B. “ Catalase-Mediated Nitric Oxide Formation From Hydroxyurea,” J. Med. Chem., 2004, 47, 3495-3501.

Xu, X.; Cho, M.; Spencer, N. Y.; Patel, N.; Huang, Z.; Shields, H.; Kings, S. B.; Gladwin, M. T.; Hogg, N.; Kim-Shapiro, D. B. “Measurements of Nitric Oxide on the Heme Iron and b -93 Thiol of Human Hemoglobin During Cycles of Oxygenation and Deoxygenation,” Proc. Natl. Acad. Sci. 2003, 100, 11303-11308.

Huang, Z.; Hearne, L.; Irby, C. E.; King, S. B.; Ballas, S. K.; Kim-Shapiro, D. B. “Kinetics of Increased Deformability of Deoxygenated Sickle Cells Upon Oxygenation,” Biophysical Journal, 2003, 85, 2374-2383.

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., 2003, 46, 3748-3753.

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 2003 , 1622, 109-116 . .

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, 125, 1444-1445.

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.

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

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.

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.

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.