Retracted: Nitroxyl (HNO): A Reduced Form of Nitric Oxide ...downloads.hindawi.com/journals/omcl/2016/4867124.pdf · OxidativeMedicineandCellularLongevity O O − − O − O −

RetractionRetracted Nitroxyl (HNO) A Reduced Form of Nitric Oxide withDistinct Chemical Pharmacological and Therapeutic Properties

Oxidative Medicine and Cellular Longevity

Received 27 October 2016 Accepted 27 October 2016

Copyright copy 2016 Oxidative Medicine and Cellular Longevity This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Oxidative Medicine and Cellular Longevity has retractedthe article titled ldquoNitroxyl (HNO) A Reduced Form ofNitric Oxide with Distinct Chemical Pharmacological andTherapeutic Propertiesrdquo [1] The article was found to containa substantial amount ofmaterial from the following publishedarticles

(i) Nazareno Paolocci Matthew I Jackson Brenda ELopez KatrinaMiranda CarloG Tocchetta DavidAWink Adrian J Hobbs Jon M Fukuto The pharma-cology of nitroxyl (HNO) and its therapeutic poten-tial Not just the janus face of NO1 Pharmacologyamp Therapeutics Volume 113 Issue 2 February 2007Pages 442ndash458

(ii) Jon M Fukuto Michael D Bartberger Andrew SDutton Nazareno Paolocci David A Wink and andK N HoukThe Physiological Chemistry and Biolog-ical Activity of Nitroxyl (HNO) The Neglected Mis-understood and Enigmatic Nitrogen Oxide Chemi-cal Research in Toxicology 2005 18 (5) 790ndash801 DOI101021tx0496800

(iii) Christopher H Switzer Wilmarie Flores-SantanaDaniele Mancardi Sonia Donzelli Debashree Basud-har Lisa A Ridnour Katrina M Miranda Jon MFukuto Nazareno Paolocci David A Wink Theemergence of nitroxyl (HNO) as a pharmacologicalagent Biochimica et Biophysica Acta (BBA) - Bioen-ergetics Volume 1787 Issue 7 July 2009 Pages 835ndash840

(iv) Irvine Jennifer C et al Nitroxyl (HNO) the Cin-derella of the nitric oxide story Trends in Pharmaco-logical Sciences Volume 29 Issue 12 601ndash608

(v) Katrina M Miranda The chemistry of nitroxyl(HNO) and implications in biology CoordinationChemistry Reviews Volume 249 Issues 3ndash4 February2005 Pages 433ndash455

References

[1] M E Shoman and O M Aly ldquoNitroxyl (HNO) a reducedform of nitric oxide with distinct chemical pharmacologicaland therapeutic propertiesrdquo Oxidative Medicine and CellularLongevity vol 2016 Article ID 4867124 15 pages 2016

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 4018417 1 pagehttpdxdoiorg10115520164018417

Review ArticleNitroxyl (HNO) A Reduced Form of Nitric Oxide with DistinctChemical Pharmacological and Therapeutic Properties

Mai E Shoman and Omar M Aly

Department of Medicinal Chemistry Faculty of Pharmacy Minia University Minia 61519 Egypt

Correspondence should be addressed to Omar M Aly omarsokkaryahoocom

Received 5 May 2015 Revised 14 August 2015 Accepted 1 September 2015

Academic Editor Lezanne Ooi

Copyright copy 2016 M E Shoman and O M AlyThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Nitroxyl (HNO) the one-electron reduced form of nitric oxide (NO) shows a distinct chemical and biological profile from thatof NO HNO is currently being viewed as a vasodilator and positive inotropic agent that can be used as a potential treatment forheart failure The ability of HNO to react with thiols and thiol containing proteins is largely used to explain the possible biologicalactions of HNO Herein we summarize different aspects related to HNO including HNO donors chemistry biology and methodsused for its detection

1 Nitric Oxide (NO)

Biological activities associatedwith nitrogen oxide species arethe subject of intense and current research interest Muchof this interest stems from the discovery of endogenousNO generation [1] a significant event that represents afundamentally new paradigm in mammalian cell signalingPrior to this finding the idea that a small freely diffusiblereactive molecule (known more for its toxicity) could bebiosynthesized in a highly regulated fashion and elicit specificbiological functions was unheard of and for some at thetime heretical [2] Most attention has been focused on NOsince it is generated directly in mammalian cells by a familyof enzymes referred to as the NO synthases (NOS) whichconverts the terminal guanidino nitrogen of l-arginine intoNO (Scheme 1) [3 4]

NO was described more than 30 years ago as a ligandof the NO-sensitive soluble guanylate cyclase (NO-sensitivesGC) the enzyme that catalyzes the conversion of guanosine51015840-triphosphate to guanosine 3101584051015840-cyclic monophosphate(cGMP) [5] NO activation of sGC involves coordinationto a regulatory ferrous heme on the protein resulting in anincrease in catalysis and a subsequent increase in intracellularcGMP [2] Increased levels of cGMP within vascular smoothmuscle result in vasodilatation [5] thus continual releaseof NO regulates vascular tone and assists in the control ofblood pressure [6] In addition NO increases the level of

cGMP in platelets and this is thought to be the mechanismby which it inhibits platelet function [7] In the vasculatureNO also prevents neutrophilplatelet adhesion to endothelialcells inhibits smoothmuscle cell proliferation andmigrationregulates programmed cell death (apoptosis) and maintainsendothelial cell barrier function [8]

Along with NO significant interest also exists for itsoxidized congeners nitrogen dioxide (NO

2) peroxynitrite

(ONOOminus) and nitrite (NO2

minus) among others This attentionmay be a function of the facility by which NO is oxidizedin aerobic systems and the toxicity of these resulting strongoxidants [9] On the other hand reduced nitrogen oxides(relative to NO) such as hydroxylamine (NH

2OH) nitroxyl

(HNO) and ammonia (NH3) have received much less atten-

tion [10] although there are several reports that suggestthey can be generated endogenously [11] These compoundswere studied in the past with regard to their biologicalactivitytoxicity [12] Among the reduced forms nitroxyl(HNO) is the least understood but is rapidly emerging asa novel entity with distinct pharmacology and therapeuticadvantages over the common nitrogen oxide species [13]

2 Nitroxyl (HNO)

In 1896 the Italian scientist Angeli published the synthesisof the inorganic salt Na

2N2O3[14] and several years later

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 4867124 15 pageshttpdxdoiorg10115520164867124

2 Oxidative Medicine and Cellular Longevity

HO HO

O O O

NH2 NH2

NH2 NH2

Arginine Citrulline

NH

NH

NH2

+

NO synthase2NADPH

2O2

+ NO + 2NADP + 2H2O

Scheme 1 Enzymatic production of NO from l-arginine

proposed its aqueous degradation produced nitrite andNOH[15] The decomposition mechanism of Angelirsquos salt (AS)was revised more than a century later and the productsare now well established to be nitrite and the more sta-ble HNO isomer [16] Nitroxyl (variously called nitrosylhydride hydrogen oxonitrate (IUPAC) nitroso hydrogenor monomeric hyponitrous acid) is a compound related toNO by 1-electron reduction and protonation [17] Nitroxylhas a very unique biological profile which is distinct fromthat of its redox cousin NO [18] Nitroxyl is growing asa potential therapeutic agent for congestive heart failure(CHF) as will be discussed [19 20] Nitroxyl appears tobe a simple triatomic species but it possesses novel andatypical chemistry Prior to 2002 the biologically relevantform of HNO was thought to be the deprotonated anionNOminus since the generally accepted p119870

119886for HNO was 47

[21] However recent studies revised the p119870119886significantly

upward to approximately 114 making HNO the exclusivespecies present at biological pH [22ndash24] In addition thedifferent spin states of the two species HNONOminus complicatethe acid-base relationship between them [17] NOminus which isisoelectric to molecular oxygen O

2 has a triplet ground state

and a singlet lowest excited state while theHNOground stateis a singlet Thus the HNO acid-base equilibrium speciespossess different electronic spin states (ie protonation-deprotonation is spin forbidden) and it is expected that therates of protonation and deprotonation will be extremelyslow compared to normal acid-base reactions [24] This spinforbidden transformation supports the idea that the observedbiological activity is attributed to the protonated form HNO[17 24]

Another complex feature of HNO that complicates thestudy of the production detection and its chemistry isthe high reactivity Chemically HNO is a highly reactiveelectrophile that spontaneously dimerizes to give hyponitrousacid which dehydrates to give nitrous oxide (N

2O) and water

(see (1)) [17 25 26] Detection of N2O as an end product may

serve as a marker for the involvement or at least the presenceof HNO in biological systems [27] This reaction has beenstudied both theoretically and experimentally and severalrate constant values have been offered with the acceptedvalue determined by flash photolysis techniques at roomtemperature being 119896 = 8 times 106Mminus1 sminus1 [24]

HNO +HNO 997888rarr HON=NOH 997888rarr N2O +H

2O (1)

Thus unlikemost commonly used nitrogen oxides HNOcannot be stored or concentrated and is typically studiedusing donor species that release HNO as a decompositionproduct

3 Nitroxyl Donors

31 Angelirsquos Salt As mentioned Angelirsquos salt (AS) or sodiumtrioxodinitrate (Na

2N2O3) is the most common HNO donor

currently used Angelirsquos salt releases HNO with a half-life ofapproximately 2-3min at physiological pH and temperatureNitroxyl release from this compound is observed between pHvalues 4 and 8 [28] through a first-order process and thegenerally accepted mechanism involves protonation of thedianion followed by tautomerization and heterolytic cleavageof the NndashN bond to produce HNO and nitrite (Scheme 2)[16] 15N-labelling confirms that HNO originates solely fromthe nitroso group while the nitritersquos origin is from the nitrogroup [29]

Interestingly when the pH drops below 4 the rate of ASdecomposition significantly accelerates andNObecomes theonly nitrogen-containing end product At higher pH (pH gt8) the decomposition rate decreases Such a decay profile isextremely useful for practical purposes as stock solutions ofAS are relatively stable at high pH while the release profileis pH insensitive near physiological conditions Howeverthe fact that 1 equivalent of nitrite is coproduced during ASdecomposition may disturb the experimental interpretationconsidering the fact that nitrite has its own physiologicalprofile [16]

32 Pilotyrsquos Acid N-Hydroxysulfenamides (Pilotyrsquos acid andits derivatives) are another class of HNO donors that havebeen examined in detail Pilotyrsquos acid shares some similaritieswith AS including pH-dependent first-order decompositionPilotyrsquos acid is stable at low pH and its decomposition rateincreases at higher pH (Scheme 3) Significant HNO releaseoccurs at pH values higher than biological conditions whileat physiological pHmany of Pilotyrsquos acid analogs are oxidizedand subsequently release NO not HNO This pH restrictionhas limited the use of these compounds as HNO donors inbiological studies [30]

33 Cyanamide Cyanamide an antialcoholic drug used inCanada Europe and Japan was found to be bioactivatedvia oxidation by the enzyme catalase leading to an N-hydroxycyanamide intermediate This intermediate speciesspontaneously decomposes to release cyanide and HNO(Scheme 4) which targets a thiol containing enzyme inthe metabolic pathway of ethanol (aldehyde dehydrogenaseALDH) [31 32]

The release of cyanide during this reaction has restrictedthe use of cyanamide and its derivatives in biological settingsHowever this drug demonstrates the feasibility of clinical use

Oxidative Medicine and Cellular Longevity 3

Ominus

OminusOminus

Ominus

Ominus

Ominus

Ominus

Slow

Angelirsquos salt

N+N

+N+

OH+ H

N N NHO

HNO + NO2minus

Scheme 2 Nitroxyl release from AS

OminusOH

minus

Pilotyrsquos acidO

SO

O

S S

O OOH OHNH + HNO+ H2ON

minus

Scheme 3 Nitroxyl release from Pilotyrsquos acid

H

H HN NN NC C

CatalaseH2O2

Cyanamide N-Hydroxycyanamide

HOHNO + CNminus + H+

Scheme 4 Nitroxyl release from cyanamide

Ominus

Ominus

Ominus

N+

Ominus

Ominus

N+

N+

Nminus

NNNHN(1)

(3)NN O

minusN

HIPANO Nitrosamine

OH

+ HNO

Scheme 5 Nitroxyl release from IPANO

of HNO donors and the ability to selectively target proteinthiols as will be seen later [9]

34 Diazeniumdiolates as HNO Donors Diazeniumdiolates(NONOates) which are complexes of secondary aminesand NO share the same chemical class as AS but theyfragment back to NO under the same conditions in whichAS decomposes to HNO [33ndash35] In contrast to these NOdonors decomposition in neutral solution of the amineNONOate produced with primary amines (isopropylamineIPANO) results in HNO release while NO forms only atlower pH values similar to AS [36] Dutton et al [37] showedthat the mechanism of HNO formation is similar to thatof AS starting from protonation of N(1) followed by slowtautomerization forming a higher-energy isomer protonatedat N(3) This tautomer will be a very minor component ofthe equilibrium but once formed it will spontaneously andrapidly dissociate to produce HNO and the deprotonatednitrosamine because of the very small barrier to NndashN bondcleavage (Scheme 5)The resulting nitrosamine is expected tobe unstable and rapidly decompose to the primary alcoholand N

2[38]

35 Acyl Nitroso Compounds Acyl nitroso compounds wereobserved as an oxidation product of hydroxyurea (a drug thatis used for sickle cell disease) and they rapidly hydrolyze toyield N

2O as evidence for HNO release Cycloadducts with

conjugated 13-dienes and NndashO heterodienophiles stabilize

the acyl nitroso compounds and then they can undergoretro-Diels Alder reactions to yield the parent acyl nitrosocompound that rapidly hydrolyses to yield HNO (Scheme 6)[39]

36 Acyloxy Nitroso Compounds A new class of HNOdonors acyloxy nitroso derivatives of cyclohexane has beendeveloped by Kingrsquos group [40 41] They are easily synthe-sized from oxidation of cyclohexanone oxime or dihydro-2H-pyran-4(3H)-one oxime by either lead tetraacetate (LTA)[40] or (diacyloxyiodo)benzene [42] They release HNOupon cleavage of the ester bond (Scheme 7) Modifying theelectronic andor steric properties of the acyl group positionchanges the rate of ester cleavage and thus HNO releaseOne of the major benefits of these HNO donors besidescontrolling the HNO release rate is that nitrite is not aproduct as is the case with AS Another advantage of thesecompounds that makes them very useful in biochemicalstudies is the blue color that is a result of the 119899 rarr 120587lowastelectronic transition of the NndashO bond allowing biochemicalreaction kinetics to be easily monitored [40]

Changing the substitution in such group of compoundsgreatly determines their properties 1-Nitrosocyclohexyltrifluoroacetate decomposes immediately releasing HNOupon addition of water or buffer solution in a man-ner that resembles Angelirsquos salt ability to release HNOwhile 1-nitrosocyclohexyl pivalate is considerably stable with11990512

of 2260min in a 1 1 mixture of MeOH Tris buffer


NH2

H2O

H2NH2N

Hydroxyurea Cycloadduct

O O

OO

O

O

OH

OH

NH NN

HNO + H2N

IO4minus

Scheme 6 Nitroxyl release from acyl nitroso compounds

HydrolysisHNO +

XX

R

+ R-COOH

Acyloxy nitroso compoundR = CH3 C(CH3)3 CF3

X = CH2 O

O

O

O

O

N

Scheme 7 Nitroxyl release from acyloxy nitroso compounds

(50mM pH 76) and hence it is not considered as a commonHNO donor [43]

4 Endogenous Production of HNO

Since the discovery of endogenous NO generation carbonmonoxide (CO) [44] and hydrogen sulfide (H

2S) [45] have

also joined the ranks of gaseoussmall molecule signalingspecies The potency by which HNO elicits many of itspharmacological actions and the apparent selectivity of HNOtowards several of its established biological targets suggeststhat the actions of pharmacologically administered HNO arethe result of preexisting signaling pathways which respondsto HNO Some reports even assumed that HNO can serveas the endothelium-derived relaxing and hyperpolarizingfactor that might contribute to NO claimed vasorelaxingactions [46]However no confirmed evidence exists forHNOendogenous generation and this might be due to the lack ofa specific and sensitive trap for HNO that can be appliedfor biological systems Numerous in vitro chemical studiesprovide possible chemical pathways for that to occur

Several reports showed that HNO can be produced byNOS under certain conditions [47ndash50] especially in theabsence of its vital prosthetic group tetrahydrobiopterin [4751] and via the metabolism of the NOS product N-hydroxy-l-arginine (NOHA) under oxidative stress [52] Nitroxyl canbe also formed under nitrosative stress (eg reaction of NOwith hydroxylamine yields HNO) [53] and by thiolysis of S-nitrosothiols (RSNO a well characterized species formed inbiological systems as a result of NO generation) [2] following

(IV) (III)FeFe

O+ H2O + HNO+ NH2OH

Scheme 8 Oxidation of hydroxylamine to HNO by high-valentiron-oxo complexes

thiol nucleophilic attack on the SNO sulfur (see (2)) [11 26]Hence

RSNO + RSH 997888rarr RSSR +HNO (2)

The direct reduction reactions of NO by mitochondrialcytochrome c [54] xanthine oxidase [55] Cu- and Mn-containing superoxide dismutase (CuMnSOD) [56] andubiquinol [57] are among other reactions reported to generateHNO

Oxidation of NH2OH can serve as an alternative source

for HNO production The generation of high-valent iron-oxo complexes from the reaction of ferric hemes (usuallyperoxidases or catalases) with hydrogen peroxide can oxidizeNH2OH to HNO (Scheme 8) [27 58]

5 Biological Chemistry of HNO

Nitroxyl is a highly reactive electrophile and the literatureon the chemistry of HNO includes multiple examples ofreactivity with nucleophiles oxidants and metalloproteins[59] Bartberger et al [22] used quantum mechanical calcu-lations to predict that HNO reacts somewhat selectively withnucleophiles Theoretical examination predicts that HNOwill not significantly hydrate or react with alcohols howeverit appears to be very reactive towards thiols with the reactionwith amines intermediate between wateralcohol and thiolsThese reactions are different from reactions of NOwith thosenucleophiles Table 1 summarizes the main possible reactionsof NO and HNO with biological reactants

51 Reaction with Thiols To date thiols appear to be amajor site of HNO biochemical reactivity Doyle et al [59]produced one of the earliest reports of thiol reactivity withHNO showing a 98 yield of disulfide and NH

2OH as the

final products of the reaction of HNO with thiophenol in40 aqueous acetonitrile (see (3)) This yield suggested that


Table 1 Comparison of NO and HNO reactivity with biological reactants [28]

Biological reactant NO HNONO No reaction Forms N

2O2

minusHN2O2unknown chemistry

HNO Forms N2O2

minusHN2O2unknown chemistry Dimerization and decomposition to N

2O

O2

Autoxidation leading to nitrosative species that is NO2and

N2O3

Forms a potent 2eminus oxidant that is not ONOOminus

RSHRSminus No direct reaction Forms sulfinamide or disulfide + hydroxylamineFe(II) heme Very stable Fe(II)-NO Forms coordination complex

Fe(III) heme Forms unstable electrophilic nitrosyl first step in reductivenitrosylation Very stable Fe(II)-NO except sGC

Cu2+ No reaction Reduces Cu2+ to Cu+ yielding NOLipid radical Yields lipid-NO Yields lipid-H + NO

N-Hydroxysulfenamide

R

RR

S

SN N

O

OH

H

H+OH

RSH RSSR + NH2OH

NH2

Sulfinamide

Sminus

Scheme 9 Reaction of HNO with thiols

the rate of this reaction significantly exceeds the rate of HNOdimerization (see (1)) [17]

2C6H5SH +HN

2O3

minus997888rarr C

6H5SSC6H5+NH

2OH

+ NO2

minus

(3)

Further thiol reactivity was reported with other bio-logically relevant thiols such as reduced glutathione (GSH)[26] N-acetyl-l-cysteine (NAC) [60] and dithiothreitol(DTT) [61] The mechanism for this reaction was sug-gested to include an attack of the nucleophilic sulfur onthe electrophilic nitrogen atom of HNO leading to theformation of an N-hydroxysulfenamide intermediate TheN-hydroxysulfenamide intermediate can further react withexcess thiol (or a vicinal protein thiol) to give the corre-sponding disulfide and hydroxylamine (Scheme 9) [59] orrearrange to sulfinamide in a competing reaction (Scheme 9)[26]

Significantly disulfide formation is considered to be bio-logically reversible because disulfides are easily regeneratedwhile the generation of the sulfinamide apparently representsan irreversible thiol modification [62] The rate of some ofthese reactions was also investigated and for GSH the rateconstant was found to be 119896 = 2 times 106Mminus1 sminus1 [25] The HNO-thiol reaction was also reported to be condition dependent(under high thiols concentration the disulfide is the mainproduct while under low reduced thiol concentration theN-hydroxysulfenamide intermediate rearranged to the sulfi-namide) [17 26 60]

This reactivitywith thiols indicates that protein sulfhydrylgroups may be a major target for HNO Shen and Englishcharacterized a similar type of chemistry using electron sprayionization mass spectrometry (ESI-MS) upon the reaction ofAS with thiol proteins such as bovine serum albumin (BSA)glyceraldehydes-3-phosphate dehydrogenase (GAPDH) andhuman brain calbindin (HCalB) They used MS characters ofwhole proteins and their digests to demonstrate the abilityof HNO to induce disulfide bond formation when thesereactions were done in the presence of excess cysteinewhile with no excess thiols the sulfhydryl group presentin these proteins was converted mainly to sulfonamide [63]Other reports suggest the ability of these sulfinamides to behydrolyzed to sulfinic acids (SO

2H) [64 65] The rate of the

reaction betweenHNO and thiol proteins appears to be fasterthan that with small thiols as the cysteine in BSA reacts witha rate constant of 119896 = 6times106Mminus1 sminus1 It can be estimated thatGAPDH with a low p119870

119886thiol reacts at a rate gt109Mminus1 sminus1

Thus thiol p119870119886and hydrophobicity of the thiol environment

influence the reactivity [66]Hoffman and coworkers used a tandem mass spectro-

metric analysis including MSMS with collision induceddissociation (CID) and electron capture dissociation to iden-tify the sulfinamide modification introduced by AS to thiolproteinsThese studies revealed a characteristic neutral loss ofHS(O)NH

2fragment (65Da) that is liberated from the mod-

ified cysteine upon CID monitored by mass spectrometryUpon storage partial conversion of the sulfinamide to sulfinicacid was observed leading to neutral losses of 65 and 66Da(HS(O)OH) [64]

These experimental data are supported by theoreticalcalculations for the free energies involved in the reaction ofHNO with 5 different thiols The reaction was found to bedependent on the loss of the SndashH proton a requirement forSndashN bond formation These findings were consistent withproteins whose environment favors the existence of thiolateanions being selectively inhibited by HNO This work alsoshowed that protonation of the HNO oxygen atom mayalso be required before or during the rate limiting stepThe authors strongly suggest that the competition betweenthe two possible pathways would be kinetically controlledAdditionally the calculated values of Δ119866 indicate that thepreferred reaction pathway depends upon the hydrophobicity


of the environment the availability of a local base and theidentity of the thiol substituent In a hydrophobic environ-ment the calculated activation barriers strongly indicate thatformation of a disulfide is favored consistent with the abilityof GSH to prevent the irreversible inhibition of proteins invitro The formation of the sulfinamide would be expectedto become more favorable if a base was present in thelocal environment and with stronger electron-withdrawingsubstituents on the thiol In solution a greater competitionbetween the two pathways is predicted but formation of thedisulfide is expected to be favored in most conditions [67]

52 Reaction with MetalsMetalloproteins Another aspectof HNO chemistry important for its biological activity isits ability to react andor form coordination complexeswith metalloproteins particularly iron heme proteins [68]Nitroxyl reacts with oxidized metals to form reductivenitrosylation products in a single step reaction this reactionwas originally observed upon exposure of metmyoglobin(metMb) ormethemoglobin (metHb) to AS (see (4)) [59 69]

Fe (III) +HNO 997888rarr Fe (II)NO +H+ (4)

Other ferric proteins including cytochromes peroxidases[69] and catalases [70] also undergo reductive nitrosylationby HNO indicating lack of specificity toward nitrogen (histi-dine) sulfur (cysteine or methionine) and oxygen (tyrosine)as the protein part that binds to iron next to theHNObindingsite The protein environment around this site may havesignificant impact on the kinetics of reductive nitrosylation[17]

The suggested mechanism for this reaction is either aninner sphere mechanism whereby direct coordination ofHNO to the metal center occurs followed by electron trans-fer and deprotonation or via an outer sphere mechanisminvolving initial electron transfer to themetal center followedby coordination of NO to the reduced metal center Theexistence of an outer sphere process is evident in the reactionof HNO with ferricytochrome c which only produces theferrous species and NO [59] The rate constant of thesereactions was estimated for the reaction of synthetic ferricporphyrin with AS to be 119896 = 3 times 105Mminus1 sminus1 (comparable tothat with metMb 119896 = 8 times 105Mminus1 sminus1) [71]

Nitroxyl also reacts with oxyhemes (eg oxymyoglobinMbO2 and Fe(II)O

2) converting two MbO

2to two metMb

(Fe(III)) with a rate constant of 107 A two-step reactionwas proposed with the intermediacy of NO and formationof peroxide (see (5)-(6)) the formed peroxide can react togenerate a ferryl-p-cation radical As this reaction has notbeen confirmed there is a possibility that HNO is reducedto H2NO rather than being oxidized to NO with rapid

dissociation of the resulting Fe(III)O2complex (see (7)-(8))

[17 66]

Fe (II)O2+HNO 997888rarr Fe (III) +NO +HO

2

minus (5)

Fe (II)O2+NO 997888rarr Fe (III) +NO

3

minus (6)

N +

+

O

O

O

HN

OH

Phosphine oxide Aza-ylide

HNminus HN

PPh3

+

TPP∙∙PPh3

∙∙PPh3

PPh3 PPh3

Ph3P

Scheme 10 Reaction of HNO with triphenylphosphine

Fe (II)O2+HNO 997888rarr Fe (III) +H

2NO +O

2(7)

Fe (II)O2+H2NO 997888rarr Fe (III) +NH

2OH +O

2(8)

Other metalloproteins also have been reported to reactwith HNO CuZnSOD reacts with HNO to yield NO [69]while reaction with MnSOD results in a MnndashNO complex[72]

53 Reaction with Phosphines Previous reports showedreactions of organic phosphines with various nitroso com-pounds C-Nitroso and S-nitroso compounds react withtriphenylphosphine (TPP) and similar triarylphosphines toform phosphine oxides and aza-ylides [73ndash75] Reisz et alreported a similar type of phosphine reactivity toward HNO(viewed as a simplified nitroso compound) (Scheme 10) [76]

The proposedmechanism for this reactionwas P additionto N or O of the nitroso group forming a three-memberedring intermediate which upon reaction with additionalequivalent of phosphine would give the corresponding aza-ylide and phosphine oxide

This work also showed that in the presence of anelectrophilic ester properly positioned on the phosphine theaza-ylide undergoes Staudinger ligation to yield an amidewith the nitrogen atom derived from HNO (Scheme 11) [76]

54 Other HNO Reactions Nitroxyl reacts with O2to form

an RNS (reactive nitrogen species) which does not appear tobe peroxynitrite (ONOOminus) but has some similar chemistry[77 78] This species is a strong 2eminus oxidant and hydroxy-lating agent and is cytotoxic at low millimolar concentrationand causes DNA strand breaks [53 77] The rate of thisautoxidation of HNO is relatively slow (119896 = 1000Mminus1sminus1)compared to the rate of HNO reactivity with other biologicalreactants likeGSH and the lower concentration ofO

2relative

to the other biomolecules severely limits the formation of thisspecies in vitro and consequently limits the oxidative damagedue to the exposure to HNO in vivo [17]

Other RNS could be formed indirectly from HNO afterits oxidation to NO with superoxide dismutase (SOD) [66]N2O2

minus also can be formed from the reaction of HNO andNO at a rate constant of 5 times 106Mminus1sminus1 [66] Nitroxylcan react with other nucleophilic nitrogen oxides such ashydroxylamine (see (9)) and nitrite (reverse of Scheme 2)

NH2OH +HNO 997888rarr N

2+ 2H2O (9)


COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh

Phosphine oxide amide

Scheme 11 Staudinger ligation of HNO with triarylphosphines

Nitroxyl reduction to NH2OH can easily occur under

physiological conditions This was confirmed by calculationof the reduction potential for the HNO 2H+NH

2OH couple

at pH 7 to be 03 V [24] It was proposed that NH2OHmight

be the product ofHNOoxidation ofNADPHby two electronsto NADP+ though this has not been confirmed [79]

6 Detection of HNO

Several methods implicate the presence or fleeting existenceof HNO As mentioned the detection of N

2O has been

used as evidence for HNO generation since HNO sponta-neously dimerizes to hyponitrous acid (H

2N2O2) that then

decomposes to N2O and water (see (1)) N

2O is usually

detected by gas chromatography-mass spectrometry (GC-MS) However this cannot be applied to biological samplesas an absolute proof of HNO intermediacy since N

2O can be

generated in ways not involving free HNO [80] MoreoverN2O formation is second order in HNO and requires fairly

high concentrations for this reaction to be significantAs discussed earlier HNO reacts readily with thiols In

fact Pino and Feelisch [81] used thiol reactivity as a meansof trapping HNO and distinguishing its actions from thoseof NO Furthermore the identification of the HNO basedthiol modification could serve as a useful method for HNOdetection Donzelli et al [27] used HPLC techniques toidentify both the sulfinamide [GS(O)NH

2] and the oxidized

glutathione (GSSG) as evidence of GSH exposure to ASTransition metals can also be used to trap HNO with the

highest applicability of metMb or synthetic analogs and trap-ping to produce EPR active species [82 83] Other methodsinvolved the oxidation of HNO to NO using ferricyanide andthen detecting the formed NO with chemiluminescence orelectrochemical analysis techniques [84]

Shoeman and Nagasawa [85] reported that nitrosoben-zene could trapHNO to give cupferron (Scheme 12) a speciesthat complexes copper forming a colored complex Howeverthis has not yet been exploited for the detection of HNO ina biological system and the efficiency of this trap has not yetbeen determined

Theuse ofwater soluble phosphines represents a powerfulway to trap HNO in biological samples by tracking the aza-ylide (Scheme 10) that is formed upon the reaction of HNOwith phosphines using simple 31P NMR techniques [76]

HPLC and colorimetricmethodswere developed byReiszet al to quantify HNO release from donor compounds usingphosphine derivatives Ligation of HNO using carbamatecontaining phosphine derivatives yielded HNO derived urea(Scheme 13) and the corresponding phenol Starting with

a nitro derivatized phosphine trap yielded p-nitrophenol thatcan be monitored calorimetrically [86]

HNO reaction with GSH was also used for HNOdetection HNO was trapped by GSH followed by label-ing of GSH with the fluorogenic agent naphthalene-23-dicarboxaldehyde (NDA) and subsequent quantitation byfluorescence difference [87]

A prefluorescent probe 4-((9-acridinecarbonyl)amino)-2266-tetramethylpiperidin-1-oxyl (TEMPO-9-AC) hasbeen used to detect HNO in aqueous solution and todifferentiate it from nitric oxide (NO) TEMPO-9-AC reactswith HNO via net hydrogen abstraction to produce thehighly fluorescent TEMPO-9-AC-H and NO The utility ofTEMPO-9-AC as a probe for HNO has been shown usingthe common HNO donors Angelirsquos salt and Pilotyrsquos acid (PA)along with a recently reported HNO donor 2-bromo-PA[88]

Recently a near-infrared fluorescent turn-on sensor forthe detection of nitroxyl (HNO) using a copper-based probeCuDHX1 was recently developed for selective quantitationof HNO in biological samples The probe contains a dihy-droxanthene (DHX) fluorophore and a cyclam derivative asa Cu(II) binding site Upon reaction with HNO CuDHX1displays fivefold fluorescence turn-on in cuvettes and isselective for HNO over thiols and reactive nitrogen andoxygen species [89]

Clearly the development of sensitive specific and bio-logically compatible trapsdetectors for HNO is essentialin determining whether HNO is physiologically relevantMoreover such methodology will be valuable for futureinvestigations distinguishing the specific actions of HNOfrom those of other nitrogen oxides

7 Biological Activity of HNO

Endogenous HNO generation remains uncertain in mam-malian cells Therefore the question of whether HNO is anatural signalingeffect or species remains open Howevernumerous studies indicate that exogenous HNO administra-tion results in interesting novel and potentially importantpharmacology and toxicology [62]

71 HNO and Antialcoholism The first report of HNOserving as a drug was from the Nagasawa research groupwho were examining the action of the antialcoholic drugcyanamide [31 32 90]The utility of this drug lies in its abilityto inhibit ALDH an enzyme involved in the metabolism ofethanol to acetate Cyanamide has no inherent activity andmust be oxidatively bioactivated to elicit enzyme inhibition


Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron

Scheme 12 Reaction of AS with nitrosobenzene

Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH

Scheme 13 Ligation of AS with arylphosphines producing phenols that can be monitored calorimetrically

Oxidation of cyanamide by catalaseH2O2generates an N-

hydroxy intermediate that decomposes to HNO and cyanideas shown in Scheme 4 Inhibition of ALDH was proposedto occur via reaction of HNO with the active site cysteinethiolate of the enzyme This finding was among the firstindications that HNO was thiophilic and could alter theactions of thiol proteins

72 HNO andVascular Function Similar toNOHNO acts asa potent vasorelaxant [91] Angelirsquos salt elicits vasorelaxationin isolated large conduit [92 93] small resistance arteries[94] and intact coronary [95] and pulmonary [96] vascularbeds in both rabbits [97] and dogs [98 99] Acyloxy nitrosocompounds also caused dilatation in thoracic aorta isolatedfrom Sprague-Dawley rats in a dose-dependent manner [43]Paolocci et al showed that HNO shows selectivity being avenodilator in contrast with NO donors that equally dilateboth arteries and veins In addition they showed HNOrsquos abil-ity to decrease mean arterial blood pressure [99] RecentlyHNO is introduced as a vasodilator for humans that is notsusceptible to tolerance [100]

Because NO elicits vasodilation via activation of sGC[101] it is possible that HNO was being converted to NO inthese experiments Such conversion seemed especially likelybecause HNO itself has been reported to be incapable ofactivating sGC [102] although these studies were performedunder high thiol conditions that probably scavenged HNO

Like NO vasorelaxant responses to HNO are accompaniedby an increase in cGMP [103] and are impaired by thesGC inhibitor 1H-[124]oxadiazolo[43-a]quinoxaline-1-one(ODQ) [92 95 103] indicating that HNO targets sGCWhether HNO itself directly activates sGC or first requiresoxidation to NO or if the preference of HNO for Fe(III) ver-sus Fe(II) targets its actions to the oxidized NO-insensitivesGC isoform which predominates in disease remains tobe determined [104] Zeller et al [105] support the idea ofHNO being oxidized first to NO by the enzyme SOD beforeactivating sGC but further investigations are required

Nitroxyl but not NO increases the plasma level of thevasodilatory neuropeptide calcitonin gene related peptide(CGRP) [98 99] Calcitonin gene related peptide stimulatesendothelial NO release increases vascular smooth musclecAMP and activates K+ channels to elicit vasodilatation [25106] However the use of antagonist CGRP does not preventin vivo vasodilatation ofHNO suggesting that CGRPmay notcontribute to HNOrsquos actions [99] Although CGRP seems tocontribute to ASmediated coronary vasodilation ex vivo [95]it remains unclear howHNO stimulates in vivoCGRP releaseandwhether CGRPplays a similar role in other vascular bedsRecent report suggested that HNO endogenously producedvia the reaction of NO with H

2S can form a disulfide bond

in the sensory chemoreceptor channel TRPA1 resulting insustained calcium influx that causes release of CGRP [107]

HNO also targets K+ channels tomodulate vascular func-tion Multiple studies show that both Kv and KATP channels


were affected in HNO-mediated vascular and nonvascularsmooth muscle relaxation [46 94] By contrast NO onlyactivates KCa channels in this preparation [108] Nitroxylactivation of K+ channels may be direct or cGMP-dependentand HNO-thiol interactions could enable direct modulationof Kv and KATP channels independently of cGMP [94]

The vasodilatory capacity of HNO donors and their abil-ity to target signaling pathways distinct from NOmight offertherapeutic advantages over traditional nitrovasodilatorsSeveral reports demonstrated that unlike organic nitratessuch as glyceryl trinitrate (GTN) AS does not developtolerance after either acute or chronic use [100 103] Giventhat AS inhibits the thiol protein ALDH-2 [9] which hasan important role in GTN biotransformation to NO [109]cross-tolerance between AS and GTN is not likely to happenwhich may be of interest [103] Nitroxyl donors could havenovel utility as vasodilators alone or in conjunction withconventional nitrovasodilators in the treatment of vasculardisorders such as angina and heart failure [13]

73 HNO and Myocardial Function Originally the phar-macological utility of HNO in the cardiovascular systemwas considered limited to vasodilation More recently HNOdonors have been shown to be capable of profoundly affectingmyocardial contractility [18]

Nitroxyl has been shown to have positive inotropic (forceof muscle contraction) and lusitropic (relaxation of cardiacmuscle) properties both properties contribute to increasedcardiac output in both normal and failing canine heartsIn this study Paolocci et al utilized a pressure-volumerelationship approach to examine primary effects of HNOon the heart from changes in vascular loading conditionsand demonstrated for the first time that AS was capable ofincreasing the inotropic status of the heart while fasteningventricular relaxation and unloading of the heart [99]

Nitroxyl-mediated inotropy was not mimicked by anNO donor and is prevented by the HNO scavenger NACindicating a direct HNO effect In dogs with tachypacing-induced congestive heart failure (CHF) HNO improved bothcontractility and relaxation to an extent similar to that foundfor control dogs [98] Nitroxyl inotropy was not dependenton beta-adrenergic pathways and was additive to beta-adrenergic agonists in enforcing myocardial contractilityPositive inotropy unloading cardiac action and peripheralvasodilation are all desirable outcomes in CHF subjectsCurrent CHF cases are usually treated via beta-agonists orphosphodiesterase inhibitors giving inotropic support to theheart and through nitrosovasodilators to unload the heartThat HNO donors provide both effects together is a uniqueadvantage

Tocchetti et al [110] and others [19] have explained theincreased myocardial contractility by showing that HNOelicits the prompt release of Ca2+ from the sarcoplasmicreticulum (SR) in both cardiac and skeletal muscle via activa-tion of ryanodine receptors (RyR2) and skeletal Ca2+ releasechannels (RyR1) These effects appear to occur throughmodifications of certain thiol residues in these receptors Inboth cases the effects were reversible upon the addition of asulfhydryl reducing agent DTT

Froehlich et al [111] suggested that HNO donated byAS can also activate Ca2+ uptake by the cardiac SR Ca2+pump (SERCA2a) by targeting the thiols in phospholamban(PLN a regulatory protein that controls SERCA2a) Theyconcluded that HNOproduces a disulfide bond that alters theconformation of PLN relieving inhibition of the Ca2+ pumpThis increase in uptake keeps the net diastolic Ca2+ low

In addition to these changes in Ca2+ cycling HNO alsoacts as a cardiac Ca2+ sensitizer enhancing myofilamentresponsiveness to Ca2+ and augmenting maximal force ofmyocardial contractility This effect is likely to be due tomodulation of myofilament proteins that have many reactivethiolate groups and are potential targets of HNO Alteringintracellular redox conditions with DTT prevented HNO-induced augmentation in muscle force development consis-tent with the idea of HNO affinity for strategically locatedthiols [112]

These combinedmechanisms suggesting the orchestratedaction of HNOwith cardiac myofilaments and SR to enhanceinotropy and lusitropy at potentially lower energetic costmake HNO an attractive candidate for CHF cases

74 HNO and Ischemia Reperfusion Injury Nitroxyl alsoelicits effects in ischemia reperfusion (IR) injury Ischemiareperfusion injury occurs when tissue is deprived of adequateblood flow for a period of time causing hypoxia whichis the ischemic event and other effects Reperfusion ofoxygenated blood causes the injury and results in necrosis ofthe tissue Ischemia reperfusion injury can be alleviated bypreconditioning the tissue which involves brief occlusionsof the vasculature prior to the actual cessation of bloodflow Administration of AS to ischemic myocardium wasfound to be detrimental as opposed to the beneficial effectsexerted by equieffective doses of NO donors [97] No clear-cut mechanisms for the HNO exacerbation of injury wereidentified though a possible explanation may be the abilityof HNO to induce neutrophil accumulation [9] Pagliaro andcoworkers later showed thatHNO is capable of inducing earlypreconditioning-like effects in isolated rat hearts to an extenteven greater than that induced by NO donor [113] Againthis protective signaling cascade is not clear with Shiva andcoworkers suggesting that mitochondrial thiols are possibleHNO targets [114] Additionally CGRPmaymediate a part ofHNOability to induce protection asCGRPhas been reportedto be protective in this context [115]

Furthermore the distinct role of HNO in cardiovascularsystem made it a very promising strategy in treatmentof cardiomyopathy associated with various diseases suchas diabetes The HNO donor 1-nitrosocyclohexylacetate (1-NCA) was proven to limit cardiomyocyte hypertrophy andLV diastolic dysfunction in a mousemodel of diabetes in vivo[116]

75 HNO and Platelet Aggregation Treatment of plateletswith micromolar concentrations of AS leads to inhibitionof platelet aggregation in both time- and dose-dependentfashion [117]Hoffman et al [64] identified the ability ofHNO


to inducemodifications in 21 proteinswithin humanplateletswith 10 of those proteins showing dose-dependent modifica-tion The function of these proteins ranges from metabolismand cytoskeletal rearrangement to signal transduction

76 HNO and Anticancer Activity In early 2008 Brooksreported the anticancer activity of HNO by demonstratingthe ability of HNO to suppress the proliferation of bothestrogen receptor- (ER-) positive and ER-negative humanbreast cancer cell lines in a dose-dependent manner Thisinhibition was accompanied by a significant decrease in theblood vessel density in HNO-treated tumors Both in vitroand in vivo models of breast cancer were used to evaluateAS One explanation for this activity is the ability of HNO toinhibit the thiol protein glyceraldehydes-3-phosphate dehy-drogenase (GAPDH) blocking the glycolytic cycle insidethe malignant cell and leading to a cascade of signalingmechanisms resulting in the inhibition of angiogenesis [118]Nitroxyl inhibition of GAPDH was previously reportedleading to inhibition of glycolysis in yeast cells [119 120]

In the next year Froehlich also reported the ability ofHNO to suppress the growth of both hormone dependentand independent cancers such as prostate breast pancreaticand lung cancer that overexpress a protein called MAT-8protein (mammary tumor 8 kDa protein) [121 122] MAT-8 ismember of the family of FXYD regulatory proteins it inhibitsthe plasma membrane Na+K+ pump causing collapse ofthe transmembrane Na+ gradient [123] affecting cellularCa2+ levels that protect tumor cells from apoptosis [124]Froehlich demonstrated that HNO inhibited theMAT-8 pro-tein via disulfide bond formation between transmembranecysteine residues relieving the Na+K+ pump inhibition andtriggering endoplasmic reticulum stress rendering the cellsusceptible to apoptosis and destruction [125]

77 HNO and Antioxidant Activity Chemistry predicts thatHNO can serve as an antioxidant with its relatively weak HndashN bond strength of sim50Kcalmol making it a good hydrogenatomdonor with the ability to quench reactive radical species[28] HNO capably inhibits lipid peroxidation in both yeastand in vitromodel systems [126] Nitroxyl inhibits free radicallipid oxidation by interacting with the initiating agent lipidradical or lipid peroxy radical thus terminating the radicalchain chemistry (see (10)ndash(13)) Oxidation of HNO gives NOthat can also quench radical intermediates [9]

LndashH + In∙

997888rarr L∙

+ InndashH (L = lipid In=free radical initiator)

(10)

In∙ +HNO 997888rarr InndashH + ∙NO (11)

L∙ +HNO 997888rarr LndashH + ∙NO (12)

LOO∙ +HNO 997888rarr LOOndashH + ∙NO (13)

78 HNO and Central Nervous System (CNS) Nitroxyl alsointeracts with the thiol residues on theN-methyl-d-aspartate(NMDA) receptor that plays a vital role in neuronal com-munication and synaptic function in the CNS [127] Sinceoverstimulation of the NMDA receptor has been implicatedin the excitotoxicity associated with glutamate the phys-iological outcome of this HNO action affords protectionagainst glutamate-based excitotoxicity In contrast Coltonand coworkers [128] reported that HNO was also capableof blocking glycine-dependent desensitization of the NMDAreceptor resulting in net sensitization of the receptor Thesuggestion was made that the divergent outcomes of thesestudies are at least in part a function of O

2depletion

upon long term use of AS which consumes O2during

decomposition Thus careful attention needs to be paid toexperimental conditions particularly with regard to O

2and

metal content However both studies indicate that HNO canmodify the activity of theNMDA receptor in either a positiveor a negative fashion depending on the oxygen tension of thesystem [9]

79 HNO and Other Thiols In addition to the thiol pro-teins mentioned in the previous sections HNO is a potentinhibitor of some cysteine proteases like papain [129] andcathepsin P [130 131] It also inhibits the metal responsiveyeast transcription factor Ace1 [132] and depletes intracellularGSH levels [53]

710 HNO Toxicity Cyanamide is the only clinicallyapproved drug that functions exclusively through releaseof HNO The therapeutic use of cyanamide induceslittle inherent toxicity at prescribed doses indicating thatadministration of HNO donors is not inherently toxic

However some in vitro experiments showed some toxic-ity associated with the use of AS in very high concentrationscompared to those used for biological studies AS wasfound to significantly reduce cellular viability due to severedepletion of cellular GSH levels Nitroxyl can also inducedouble-stranded DNA breaks The cytotoxicity of Angelirsquossalt was substantially diminished under hypoxic conditionsindicating a role for O

2in the toxicity of HNO These obser-

vations were observed at 1ndash3mM AS which significantlyexceeds the concentrations of AS utilized in pharmacologicalapplicationsThe effects observed with cultured cells may notbe applicable to in vivo systems with intact redox bufferingcapacity [53]

High concentrations of HNO have been proposed tobe responsible for the neurotoxicity observed followingadministration of AS to rats via intranigral infusion Theeffects were both acute and progressive (occurring after thedissipation of HNO) The acute phase was attributed todirect interactions of HNO with cellular components whilethe delayed phase may be a result of excitotoxicity [133]Intrathecal administration of Angelirsquos salt to the lumbarspinal cord of rats resulted in motor neuron injury withouteffect on sensory neurons [134]

Nitroxyl shows some interactions with mitochondria asa redox active species though these interactions were not


reported to be toxic and no pharmacological utility wasreported also [9] Inhibition of GAPDH by HNO inhibitscellular glycolysis [119]

In summary HNO seems to hold very promising thera-peutic potential for cardiovascular diseases (especially CHF)due to its activity profile on the heart and vasculatureEnhancement of intracellular Ca2+ cycling and myofilamentCa2+ sensitization appear to be unique in addition to vasodi-lation and reduction in cardiac load HNO also provides apromising strategy in fighting cancer due to its ability toinhibit GAPDH Moreover the bioactivity of HNO appearsdistinct compared to NO making biological targeting byHNO localized and specific facilitating the use of HNO as anovel therapeutic agent

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] J M Fukuto C L Bianco and T A Chavez ldquoNitroxyl (HNO)signalingrdquo Free Radical Biology amp Medicine vol 47 no 9 pp1318ndash1324 2009

[2] J M Fukuto M D Bartberger A S Dutton N Paolocci DA Wink and K N Houk ldquoThe physiological chemistry andbiological activity of nitroxyl (HNO) the neglected misun-derstood and enigmatic nitrogen oxiderdquo Chemical Research inToxicology vol 18 no 5 pp 790ndash801 2005

[3] O W Griffith and D J Stuehr ldquoNitric oxide synthases proper-ties and catalytic mechanismrdquoAnnual Review of Physiology vol57 pp 707ndash736 1995

[4] S Moncada and E A Higgs ldquoEndogenous nitric oxide phys-iology pathology and clinical relevancerdquo European Journal ofClinical Investigation vol 21 no 4 pp 361ndash374 1991

[5] W P Arnold C K Mittal S Katsuki and F Murad ldquoNitricoxide activates guanylate cyclase and increases guanosine 3101584051015840-cyclic monophosphate levels in various tissue preparationsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 74 no 8 pp 3203ndash3207 1977

[6] G R JThatcher ldquoNOproblem for nitroglycerin organic nitratechemistry and therapyrdquo Chemical Society Reviews vol 27 no 5pp 331ndash337 1998

[7] M AMoro R J R Russell S Cellek et al ldquocGMPmediates thevascular and platelet actions of nitric oxide confirmation usingan inhibitor of the soluble guanylyl cyclaserdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 93 no 4 pp 1480ndash1485 1996

[8] M Rosselli P J Keller and R K Dubey ldquoRole of nitric oxide inthe biology physiology and pathophysiology of reproductionrdquoHuman Reproduction Update vol 4 no 1 pp 3ndash24 1998

[9] N PaolocciM I Jackson B E Lopez et al ldquoThe pharmacologyof nitroxyl (HNO) and its therapeutic potential not just theJanus face of NO1rdquo Pharmacology andTherapeutics vol 113 no2 pp 442ndash458 2007

[10] J M Fukuto A S Dutton and K N Houk ldquoThe chemistry andbiology of nitroxyl (HNO) a chemically unique species withnovel and important biological activityrdquo ChemBioChem vol 6no 4 pp 612ndash619 2005

[11] N Y Spencer N K Patel A Keszler and N Hogg ldquoOxidationandnitrosylation of oxyhemoglobin by S-nitrosoglutathione vianitroxyl anionrdquo Free Radical Biology and Medicine vol 35 no11 pp 1515ndash1526 2003

[12] P Gross and R P Smith ldquoBiologic activity of hydroxylamine areviewrdquo Critical Reviews in Toxicology vol 14 no 1 pp 87ndash991985

[13] J C Irvine R H Ritchie J L Favaloro K L Andrews RE Widdop and B K Kemp-Harper ldquoNitroxyl (HNO) theCinderella of the nitric oxide storyrdquo Trends in PharmacologicalSciences vol 29 no 12 pp 601ndash608 2008

[14] A Angeli ldquoSopra la nitroidrossilamminardquo Gazzetta ChimicaItaliana vol 26 pp 17ndash25 1896

[15] A A A F Angelico ldquoNuove ricerche sopra lacido nitroidrossil-amminicordquo Gazzetta Chimica Italiana vol 33 p 245 1903

[16] A S Dutton J M Fukuto and K N Houk ldquoMechanisms ofHNO and NO production from Angelirsquos salt density functionaland CBS-QB3 theory predictionsrdquo Journal of the AmericanChemical Society vol 126 no 12 pp 3795ndash3800 2004

[17] KMMiranda ldquoThe chemistry of nitroxyl (HNO) and implica-tions in biologyrdquo Coordination Chemistry Reviews vol 249 no3-4 pp 433ndash455 2005

[18] D A Wink K M Miranda T Katori et al ldquoOrthogonalproperties of the redox siblings nitroxyl and nitric oxide in thecardiovascular system a novel redox paradigmrdquo The AmericanJournal of PhysiologymdashHeart and Circulatory Physiology vol285 no 6 pp H2264ndashH2276 2003

[19] E Cheong V Tumbev J Abramson G Salama and D AStoyanovsky ldquoNitroxyl triggers Ca2+ release from skeletal andcardiac sarcoplasmic reticulum by oxidizing ryanodine recep-torsrdquo Cell Calcium vol 37 no 1 pp 87ndash96 2005

[20] A Arcaro G Lembo and C G Tocchetti ldquoNitroxyl (HNO) fortreatment of acute heart failurerdquo Current Heart Failure Reportsvol 11 no 3 pp 227ndash235 2014

[21] M Graetzel S Taniguchi and A Henglein ldquoPulse radiolyticstudy of short-lived by-products of nitric oxide-reduction inaqueous solutionrdquo Berichte der Bunsen-Gesellschaft vol 74 no10 pp 1003ndash1010 1970

[22] M D Bartberger J M Fukuto and K N Houk ldquoOn the acidityand reactivity of HNO in aqueous solution and biologicalsystemsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 5 pp 2194ndash2198 2001

[23] M D BartbergerW Liu E Ford et al ldquoThe reduction potentialof nitric oxide (NO) and its importance to NO biochemistryrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 99 no 17 pp 10958ndash10963 2002

[24] V Shafirovich and S V Lymar ldquoNitroxyl and its anion inaqueous solutions spin states protic equilibria and reactivitiestoward oxygen and nitric oxiderdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 99 no11 pp 7340ndash7345 2002

[25] K M Miranda N Paolocci T Katori et al ldquoA biochemicalrationale for the discrete behavior of nitroxyl and nitric oxide inthe cardiovascular systemrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 16 pp9196ndash9201 2003

[26] P S Wong J Hyun J M Fukuto et al ldquoReaction betweenS-nitrosothiols and thiols generation of nitroxyl (HNO) andsubsequent chemistryrdquo Biochemistry vol 37 no 16 pp 5362ndash5371 1998


[27] S Donzelli M G Espey D D Thomas et al ldquoDiscriminatingformation of HNO from other reactive nitrogen oxide speciesrdquoFree Radical Biology andMedicine vol 40 no 6 pp 1056ndash10662006

[28] C H Switzer W Flores-Santana D Mancardi et al ldquoTheemergence of nitroxyl (HNO) as a pharmacological agentrdquoBiochimica et Biophysica ActamdashBioenergetics vol 1787 no 7 pp835ndash840 2009

[29] F T Bonner and B Ravid ldquoThermal decomposition of oxy-hyponitrite (sodium trioxodinitrate(II)) in aqueous solutionrdquoInorganic Chemistry vol 14 no 3 pp 558ndash563 1975

[30] K M Miranda H T Nagasawa and J P Toscano ldquoDonors ofHNOrdquo Current Topics in Medicinal Chemistry vol 5 no 7 pp649ndash664 2005

[31] E G DeMaster B Redfern and H T Nagasawa ldquoMechanismsof inhibition of aldehyde dehydrogenase by nitroxyl the activemetabolite of the alcohol deterrent agent cyanamiderdquo Biochem-ical Pharmacology vol 55 no 12 pp 2007ndash2015 1998

[32] H T Nagasawa E G DeMaster B Redfern F N Shirota andD J W Goon ldquoEvidence for nitroxyl in the catalase-mediatedbioactivation of the alcohol deterrent agent cyanamiderdquo Journalof Medicinal Chemistry vol 33 no 12 pp 3120ndash3122 1990

[33] C M Maragos D Morley D A Wink et al ldquoComplexes ofsdotNO with nucleophiles as agents for the controlled biologicalrelease of nitric oxide Vasorelaxant effectsrdquo Journal ofMedicinalChemistry vol 34 no 11 pp 3242ndash3247 1991

[34] D Morley C M Maragos X Y Zhang M Boignon D AWink and L K Keefer ldquoMechanism of vascular relaxationinduced by the nitric oxide (NO)nucleophile complexes anew class of NO-based vasodilatorsrdquo Journal of CardiovascularPharmacology vol 21 no 4 pp 670ndash676 1993

[35] J G Giodati A A Quyyumi N Hussain and L K KeeferldquoComplexes of nitric oxide with nucleophiles as agents for thecontrolled biological release of nitric oxide antiplatelet effectrdquoThrombosis and Haemostasis vol 70 no 4 pp 654ndash658 1993

[36] K M Miranda T Katori C L Torres de Holding et alldquoComparison of the NO and HNO donating properties ofdiazeniumdiolates primary amine adducts release HNO invivordquo Journal of Medicinal Chemistry vol 48 no 26 pp 8220ndash8228 2005

[37] A S Dutton C P Suhrada K M Miranda D A Wink JM Fukuto and K N Houk ldquoMechanism of pH-dependentdecomposition of monoalkylamine diazeniumdiolates to formHNO and NO deduced from the model compound methy-lamine diazeniumdiolate density functional theory and CBS-QB3 calculationsrdquo Inorganic Chemistry vol 45 no 6 pp 2448ndash2456 2006

[38] D A Wink K S Kasprzak C M Maragos et al ldquoDNAdeaminating ability and genotoxicity of nitric oxide and itsprogenitorsrdquo Science vol 254 no 5034 pp 1001ndash1003 1991

[39] S B King ldquoN-hydroxyurea and acyl nitroso compounds asnitroxyl (HNO) and nitric oxide (NO) donorsrdquo Current Topicsin Medicinal Chemistry vol 5 no 7 pp 665ndash673 2005

[40] X Sha T S Isbell R P Patel C S Day and S B KingldquoHydrolysis of acyloxy nitroso compounds yields nitroxyl(HNO)rdquo Journal of the American Chemical Society vol 128 no30 pp 9687ndash9692 2006

[41] H A H Mohamed M Abdel-Aziz A A Gamal El-Din and SB King ldquoNew acyloxy nitroso compoundswith improvedwatersolubility and nitroxyl (HNO) release kinetics and inhibitors ofplatelet aggregationrdquo Bioorganic ampMedicinal Chemistry vol 23no 17 pp 6069ndash6077 2015

[42] G Calvet M Dussaussois N Blanchard and C KouklovskyldquoLewis acid-promoted hetero Diels-Alder cycloaddition of 120572-acetoxynitroso dienophilesrdquo Organic Letters vol 6 no 14 pp2449ndash2451 2004

[43] M E Shoman J F Dumond T S Isbell et al ldquoAcyloxy nitrosocompounds as nitroxyl (HNO) donors kinetics reactionswith thiols and vasodilation propertiesrdquo Journal of MedicinalChemistry vol 54 no 4 pp 1059ndash1070 2011

[44] H P Kim S W Ryter and A M Choi ldquoCO as a cellular signal-ing moleculerdquo Annual Review of Pharmacology and Toxicologyvol 46 no 1 pp 411ndash449 2006

[45] R Wang ldquoThe gasotransmitter role of hydrogen sulfiderdquoAntioxidants and Redox Signaling vol 5 no 4 pp 493ndash5012003

[46] K L Andrews J C Irvine M Tare et al ldquoA role for nitroxyl(HNO) as an endothelium-derived relaxing and hyperpolariz-ing factor in resistance arteriesrdquo British Journal of Pharmacol-ogy vol 157 no 4 pp 540ndash550 2009

[47] S Adak Q Wang and D J Stuehr ldquoArginine conversionto nitroxide by tetrahydrobiopterin-free neuronal nitric-oxidesynthase Implications for mechanismrdquo Journal of BiologicalChemistry vol 275 no 43 pp 33554ndash33561 2000

[48] A J Hobbs J M Fukuto and L J Ignarro ldquoFormation offree nitric oxide from L-arginine by nitric oxide synthasedirect enhancement of generation by superoxide dismutaserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 91 no 23 pp 10992ndash10996 1994

[49] H H Schmidt H Hofmann U Schindler Z S Shutenkom DD Cunningham andM Feelisch ldquoNo NO fromNO synthaserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 25 pp 14492ndash14497 1996

[50] R A Pufahl J S Wishnok and M A Marletta ldquoHydrogenperoxide-supported oxidation of 119873G-hydroxy-l-arginine bynitric oxide synthaserdquoBiochemistry vol 34 no 6 pp 1930ndash19411995

[51] K M Rusche M M Spiering and M A Marletta ldquoReactionscatalyzed by tetrahydrobiopterin-free nitric oxide synthaserdquoBiochemistry vol 37 no 44 pp 15503ndash15512 1998

[52] JM FukutoD J Stuehr P L FeldmanM P Bova andPWongldquoPeracid oxidation of an N-hydroxyguanidine compound achemical model for the oxidation of N omega-hydroxyl-L-arginine by nitric oxide synthaserdquo Journal of Medicinal Chem-istry vol 36 no 18 pp 2666ndash2670 1993

[53] D A Wink M Feelisch J Fukuto et al ldquoThe cytotoxicity ofnitroxyl possible implications for the pathophysiological roleof NOrdquo Archives of Biochemistry and Biophysics vol 351 no 1pp 66ndash74 1998

[54] M A Sharpe and C E Cooper ldquoReactions of nitric oxidewith mitochondrial cytochrome c a novel mechanism for theformation of nitroxyl anion and peroxynitriterdquo BiochemicalJournal vol 332 part 1 pp 9ndash19 1998

[55] M Saleem and H Ohshima ldquoXanthine oxidase converts nitricoxide to nitroxyl that inactivates the enzymerdquo Biochemical andBiophysical Research Communications vol 315 no 2 pp 455ndash462 2004

[56] V Niketic S Stojanovic A Nikolic M Spasic and A MMichelson ldquoExposure of Mn and FeSODs but not CuZnSODto NO leads to nitrosonium and nitroxyl ions generation whichcause enzyme modification and inactivation an in vitro studyrdquoFree Radical Biology andMedicine vol 27 no 9-10 pp 992ndash9961999


[57] J J Poderoso M C Carreras F Schopfer et al ldquoThe reactionof nitric oxide with ubiquinol kinetic properties and biologicalsignificancerdquo Free Radical Biology andMedicine vol 26 no 7-8pp 925ndash935 1999

[58] J A Reisz E Bechtold and S B King ldquoOxidative heme protein-mediated nitroxyl (HNO) generationrdquoDalton Transactions vol39 no 22 pp 5203ndash5212 2010

[59] M P Doyle S N Mahapatro R D Broene and J K Guy ldquoOxi-dation and reduction of hemoproteins by trioxodinitrate(II)The role of nitrosyl hydride and nitriterdquo Journal of the AmericanChemical Society vol 110 no 2 pp 593ndash599 1988

[60] D W Shoeman F N Shirota E G DeMaster and H TNagasawa ldquoReaction of nitroxyl an aldehyde dehydrogenaseinhibitor with N-acetyl-L-cysteinerdquo Alcohol vol 20 no 1 pp55ndash59 2000

[61] T Turk and T C Hollocher ldquoOxidation of dithiothreitol duringturnover of nitric oxide reductase evidence for generationof nitroxyl with the enzyme from Paracoccus denitrificansrdquoBiochemical and Biophysical Research Communications vol 183no 3 pp 983ndash988 1992

[62] J M Fukuto C H Switzer K M Miranda and D A WinkldquoNitroxyl (HNO) chemistry biochemistry and pharmacologyrdquoAnnual Review of Pharmacology andToxicology vol 45 pp 335ndash355 2005

[63] B Shen and A M English ldquoMass spectrometric analysis ofnitroxyl-mediated protein modification comparison of prod-ucts formed with free and protein-based cysteinesrdquo Biochem-istry vol 44 no 42 pp 14030ndash14044 2005

[64] M D Hoffman G M Walsh J C Rogalski and J KastldquoIdentification of nitroxyl-induced modifications in humanplatelet proteins using a novel mass spectrometric detectionmethodrdquo Molecular and Cellular Proteomics vol 8 no 5 pp887ndash903 2009

[65] S Mitroka M E Shoman J F DuMond et al ldquoDirect andnitroxyl (HNO)-mediated reactions of acyloxy nitroso com-pounds with the thiol-containing proteins glyceraldehyde 3-phosphate dehydrogenase and alkyl hydroperoxide reductasesubunit Crdquo Journal of Medicinal Chemistry vol 56 no 17 pp6583ndash6592 2013

[66] W Flores-Santana C Switzer L A Ridnour et al ldquoComparingthe chemical biology of NO and HNOrdquo Archives of PharmacalResearch vol 32 no 8 pp 1139ndash1153 2009

[67] M P Sherman W R Grither and R D McCulla ldquoComputa-tional investigation of the reaction mechanisms of nitroxyl andthiolsrdquo Journal of Organic Chemistry vol 75 no 12 pp 4014ndash4024 2010

[68] P J Farmer and F Sulc ldquoCoordination chemistry of the HNOligand with hemes and synthetic coordination complexesrdquoJournal of Inorganic Biochemistry vol 99 no 1 pp 166ndash1842005

[69] K M Miranda R W Nims D D Thomas et al ldquoComparisonof the reactivity of nitric oxide and nitroxyl with heme proteinsa chemical discussion of the differential biological effects ofthese redox related products of NOSrdquo Journal of InorganicBiochemistry vol 93 no 1-2 pp 52ndash60 2003

[70] J HuangD B Kim-Shapiro and S B King ldquoCatalase-mediatednitric oxide formation from hydroxyureardquo Journal of MedicinalChemistry vol 47 no 14 pp 3495ndash3501 2004

[71] S E Bari M A Martı V T Amorebieta D A Estrin andF Doctorovich ldquoFast nitroxyl trapping by ferric porphyrinsrdquoJournal of the American Chemical Society vol 125 no 50 pp15272ndash15273 2003

[72] M R Filipovic D Stanic S Raicevic M Spasic and V NiketicldquoConsequences of MnSOD interactions with nitric oxide nitricoxide dismutation and the generation of peroxynitrite andhydrogen peroxiderdquo Free Radical Research vol 41 no 1 pp 62ndash72 2007

[73] M M Sidky F M Soliman and R Shabana ldquoOrganophos-phorus compounds XXIV The reaction of triphenylphosphineand trialkyl phosphites with 120572-nitroso-120573-naphtholrdquo EgyptianJournal of Chemistry vol 21 pp 29ndash35 1980

[74] E Bechtold J A Reisz C Klomsiri et al ldquoWater-solubletriarylphosphines as biomarkers for protein S-nitrosationrdquoACSChemical Biology vol 5 no 4 pp 405ndash414 2010

[75] M Haake ldquoZur desoxygenierung von tritylthionitritrdquo Tetrahe-dron Letters vol 13 no 33 pp 3405ndash3408 1972

[76] J A Reisz E B Klorlg M W Wright and S B KingldquoReductive phosphine-mediated ligation of nitroxyl (HNO)rdquoOrganic Letters vol 11 no 13 pp 2719ndash2721 2009

[77] K M Miranda Y K-i M G Espey et al ldquoFurther evidence fordistinct reactive intermediates from nitroxyl and peroxynitriteeffects of buffer composition on the chemistry of Angelirsquossalt and synthetic peroxynitriterdquo Archives of Biochemistry andBiophysics vol 401 no 2 pp 134ndash144 2002

[78] K M Miranda A S Dutton L A Ridnour et al ldquoMechanismof aerobic decomposition of Angelirsquos salt (Sodium Trioxodini-trate) at physiological pHrdquo Journal of the American ChemicalSociety vol 127 no 2 pp 722ndash731 2005

[79] A Reif L Zecca P Riederer M Feelisch and H H H WSchmidt ldquoNitroxyl oxidizes NADPH in a superoxide dismutaseinhibitable mannerrdquo Free Radical Biology and Medicine vol 30no 7 pp 803ndash808 2001

[80] J Y Cho A Dutton T Miller K N Houk and J MFukuto ldquoOxidation of N-hydroxyguanidines by copper(II)model systems for elucidating the physiological chemistry of thenitric oxide biosynthetic intermediate N-hydroxyl-L-argininerdquoArchives of Biochemistry and Biophysics vol 417 no 1 pp 65ndash762003

[81] R Z Pino and M Feelisch ldquoBioassay discrimination betweennitric oxide (NO∙) and nitroxyl (NOminus) using L-cysteinerdquo Bio-chemical and Biophysical Research Communications vol 201 no1 pp 54ndash62 1994

[82] A M Komarov D A Wink M Feelisch and H H HW Schmidt ldquoElectron-paramagnetic resonance spectroscopyusingN-methyl-D-glucamine dithiocarbamate iron cannot dis-criminate between nitric oxide and nitroxyl implications forthe detection of reaction products for nitric oxide synthaserdquoFree Radical Biology and Medicine vol 28 no 5 pp 739ndash7422000

[83] Y Xia A J Cardounel A F Vanin and J L Zweier ldquoElec-tron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishesnitric oxide and nitroxyl anion in a redox-dependent mannerapplications in identifying nitrogen monoxide products fromnitric oxide synthaserdquo Free Radical Biology and Medicine vol29 no 8 pp 793ndash797 2000

[84] KMMirandaMG Espey K Yamada et al ldquoUnique oxidativemechanisms for the reactive nitrogen oxide species nitroxylanionrdquo The Journal of Biological Chemistry vol 276 no 3 pp1720ndash1727 2001

[85] D W Shoeman and H T Nagasawa ldquoThe reaction ofnitroxyl (HNO) with nitrosobenzene gives cupferron (N-Nitrosophenylhydroxylamine)rdquo Nitric Oxide vol 2 no 1 pp66ndash72 1998


[86] J A Reisz C N Zink and S B King ldquoRapid and selectivenitroxyl (HNO) trapping by phosphines kinetics and newaqueous ligations for HNO detection and quantitationrdquo Journalof the American Chemical Society vol 133 no 30 pp 11675ndash11685 2011

[87] GM Johnson T J Chozinski D J Salmon A DMoghaddamH C Chen and K M Mirandan ldquoQuantitative detection ofnitroxyl upon trapping with glutathione and labeling with aspecific fluorogenic reagentrdquo Free Radical Biology andMedicinevol 63 pp 476ndash484 2013

[88] M R Cline and J P Toscano ldquoDetection of nitroxyl (HNO) bya prefluorescent proberdquo Journal of Physical Organic Chemistryvol 24 no 10 pp 993ndash998 2011

[89] A T Wrobel T C Johnstone A Deliz Liang S J Lippard andP Rivera-Fuentes ldquoA fast and selective near-infrared fluorescentsensor for multicolor imaging of biological nitroxyl (HNO)rdquoJournal of the American Chemical Society vol 136 no 12 pp4697ndash4705 2014

[90] M J C Lee H T Nagasawa J A Elberling and E G DeMasterldquoProdrugs of nitroxyl as inhibitors of aldehyde dehydrogenaserdquoJournal of Medicinal Chemistry vol 35 no 20 pp 3648ndash36521992

[91] J M Fukuto K Chiang R Hszieh PWong and G ChaudhurildquoThe pharmacological activity of nitroxyl a potent vasodilatorwith activity similar to nitric oxide andor endothelium-derivedrelaxing factorrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 263 no 2 pp 546ndash551 1992

[92] J C Wanstall T K Jeffery A Gambino F Lovren and C RTriggle ldquoVascular smooth muscle relaxation mediated by nitricoxide donors a comparison with acetylcholine nitric oxide andnitroxyl ionrdquo British Journal of Pharmacology vol 134 no 3 pp463ndash472 2001

[93] A Ellis C G Li and M J Rand ldquoDifferential actions of L-cysteine on responses to nitric oxide nitroxyl anions and EDRFin the rat aortardquo British Journal of Pharmacology vol 129 no 2pp 315ndash322 2000

[94] J C Irvine J L Favaloro and B K Kemp-Harper ldquoNO-activates soluble guanylate cyclase andKv channels to vasodilateresistance arteriesrdquo Hypertension vol 41 no 6 pp 1301ndash13072003

[95] J L Favaloro and B K Kemp-Harper ldquoThe nitroxyl anion(HNO) is a potent dilator of rat coronary vasculaturerdquo Cardio-vascular Research vol 73 no 3 pp 587ndash596 2007

[96] B J De Witt J R Marrone A D Kaye L K Keefer and PJ Kadowitz ldquoComparison of responses to novel nitric oxidedonors in the feline pulmonary vascular bedrdquo European Journalof Pharmacology vol 430 no 2-3 pp 311ndash315 2001

[97] X LMa F Gao G-L Liu et al ldquoOpposite effects of nitric oxideand nitroxyl on postischemic myocardial injuryrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 96 no 25 pp 14617ndash14622 1999

[98] N Paolocci T Katori H C Champion et al ldquoPositive inotropicand lusitropic effects of HNONO- in failing hearts inde-pendence from beta-adrenergic signalingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 9 pp 5537ndash5542 2003

[99] N Paolocci W F Saavedra K M Miranda et al ldquoNitroxylanion exerts redox-sensitive positive cardiac inotropy in vivoby calcitonin gene-related peptide signalingrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 98 no 18 pp 10463ndash10468 2001

[100] K L Andrews N G Lumsden J Farry A M Jefferis B KKemp-Harper and J P Chin-Dusting ldquoNitroxyl a vasodilatorof human vessels that is not susceptible to tolerancerdquo ClinicalScience (London) vol 129 no 2 pp 179ndash187 2015

[101] A J Hobbs ldquoSoluble guanylate cyclase the forgotten siblingrdquoTrends in Pharmacological Sciences vol 18 no 12 pp 484ndash4911997

[102] E A Dierks and J N Burstyn ldquoNitric oxide (NO∙) the onlynitrogen monoxide redox form capable of activating solubleguanylyl cyclaserdquo Biochemical Pharmacology vol 51 no 12 pp1593ndash1600 1996

[103] J C Irvine J L Favaloro R EWiddop andBKKemp-HarperldquoNitroxyl anion donor Angelirsquos salt does not develop tolerancein rat isolated aortaerdquoHypertension vol 49 no 4 pp 885ndash8922007

[104] J-P Stasch P M Schmidt P I Nedvetsky et al ldquoTargeting theheme-oxidized nitric oxide receptor for selective vasodilatationof diseased blood vesselsrdquo The Journal of Clinical Investigationvol 116 no 9 pp 2552ndash2561 2006

[105] A Zeller M V Wenzl M Beretta et al ldquoMechanisms underly-ing activation of soluble guanylate cyclase by the nitroxyl donorAngelirsquos saltrdquo Molecular Pharmacology vol 76 no 5 pp 1115ndash1122 2009

[106] S D Brain and A D Grant ldquoVascular actions of calci-tonin gene-related peptide and adrenomedullinrdquo PhysiologicalReviews vol 84 no 3 pp 903ndash934 2004

[107] M Eberhardt M Dux B Namer et al ldquoH2S and NO cooper-

atively regulate vascular tone by activating a neuroendocrineHNO-TRPA1-CGRP signalling pathwayrdquo Nature Communica-tions vol 5 article 4381 17 pages 2014

[108] D K Mistry and C J Garland ldquoNitric oxide (NO)-inducedactivation of large conductance Ca2+-dependent K+ channels(BK(Ca)) in smooth muscle cells isolated from the rat mesen-teric arteryrdquo British Journal of Pharmacology vol 124 no 6 pp1131ndash1140 1998

[109] Z Chen J Zhang and J S Stamler ldquoIdentification of the enzy-matic mechanism of nitroglycerin bioactivationrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 99 no 12 pp 8306ndash8311 2002

[110] C G Tocchetti W Wang J P Froehlich et al ldquoNitroxylimproves cellular heart function by directly enhancing cardiacsarcoplasmic reticulumCa2+ cyclingrdquo Circulation Research vol100 no 1 pp 96ndash104 2007

[111] J P Froehlich J E Mahaney G Keceli et al ldquoPhospholambanthiols play a central role in activation of the cardiac muscle sar-coplasmic reticulum calcium pump by nitroxylrdquo Biochemistryvol 47 no 50 pp 13150ndash13152 2008

[112] T Dai Y Tian C G Tocchetti et al ldquoNitroxyl increases forcedevelopment in rat cardiac musclerdquo Journal of Physiology vol580 no 3 pp 951ndash960 2007

[113] P Pagliaro D Mancardi R Rastaldo et al ldquoNitroxyl affordsthiol-sensitive myocardial protective effects akin to early pre-conditioningrdquo Free Radical Biology and Medicine vol 34 no 1pp 33ndash43 2003

[114] S Shiva J H Crawford A Ramachandran et al ldquoMechanismsof the interaction of nitroxyl with mitochondriardquo BiochemicalJournal vol 379 no 2 pp 359ndash366 2004

[115] Y-J Li and J Peng ldquoThe cardioprotection of calcitonin gene-related peptide-mediated preconditioningrdquoEuropean Journal ofPharmacology vol 442 no 3 pp 173ndash177 2002


[116] N Cao Y G Wong S Rosli et al ldquoChronic administrationof the nitroxyl donor 1-nitrosocyclo hexyl acetate limits leftventricular diastolic dysfunction in a mouse model of diabetesmellitus in vivordquoCirculationHeart Failure vol 8 no 3 pp 572ndash581 2015

[117] E Bermejo D A Saenz F Alberto R E Rosenstein S EBari and M A Lazzari ldquoEffect of nitroxyl on human plateletsfunctionrdquoThrombosis and Haemostasis vol 94 no 3 pp 578ndash584 2005

[118] A J Norris M R Sartippour M Lu et al ldquoNitroxyl inhibitsbreast tumor growth and angiogenesisrdquo International Journal ofCancer vol 122 no 8 pp 1905ndash1910 2008

[119] B E Lopez C E Rodriguez M Pribadi N M Cook MShinyashiki and J M Fukuto ldquoInhibition of yeast glycolysis bynitroxyl (HNO) a mechanism of HNO toxicity and implica-tions to HNO biologyrdquo Archives of Biochemistry and Biophysicsvol 442 no 1 pp 140ndash148 2005

[120] B E Lopez D A Wink and J M Fukuto ldquoThe inhibi-tion of glyceraldehyde-3-phosphate dehydrogenase by nitroxyl(HNO)rdquoArchives of Biochemistry and Biophysics vol 465 no 2pp 430ndash436 2007

[121] H Kayed J Kleeff A Kolb et al ldquoFXYD3 is over expressedin pancreatic ductal adenocarcinoma and influences pancreaticcancer cell growthrdquo International Journal of Cancer vol 118 no1 pp 43ndash54 2006

[122] J P Froehlich and N Paolocci ldquoUp-regulated expression ofthe MAT-8 gene in prostate cancer and its siRNA-mediatedinhibition of expression induces a decrease in proliferationof human prostate carcinoma cellsrdquo International Journal ofOncology vol 24 no 1 pp 97ndash105 2004

[123] K J Sweadner and E Rael ldquoThe FXYD gene family of smallion transport regulators or channels cDNA sequence proteinsignature sequence and expressionrdquo Genomics vol 68 no 1pp 41ndash56 2000

[124] A Ashkenazi and V M Dixit ldquoApoptosis control by death anddecoy receptorsrdquo Current Opinion in Cell Biology vol 11 no 2pp 255ndash260 1999

[125] J P F L Paolocci Use of Nitroxyl (HNO) for the Treatment ofCancers over Expressing MAT-8 USP Office 2009

[126] B E Lopez M Shinyashiki T H Han and J M FukutoldquoAntioxidant actions of nitroxyl (HNO)rdquo Free Radical Biologyand Medicine vol 42 no 4 pp 482ndash491 2007

[127] W-K Kim Y-B Choi P V Rayudu et al ldquoAttenuation ofNMDA receptor activity and neurotoxicity by nitroxyl anionNOrdquo Neuron vol 24 no 2 pp 461ndash469 1999

[128] C A ColtonM Gbadegesin D AWink KMMirandaM GEspey and S Vicini ldquoNitroxyl anion regulation of the NMDAreceptorrdquo Journal of Neurochemistry vol 78 no 5 pp 1126ndash11342001

[129] A J Vaananen E Kankuri and P Rauhala ldquoNitric oxide-related species-induced protein oxidation reversible irre-versible and protective effects on enzyme function of papainrdquoFree Radical Biology and Medicine vol 38 no 8 pp 1102ndash11112005

[130] A J Vaananen P Salmenpera M Hukkanen et al ldquoPersis-tent susceptibility of cathepsin B to irreversible inhibition bynitroxyl (HNO) in the presence of endogenous nitric oxiderdquoFree Radical Biology and Medicine vol 45 no 6 pp 749ndash7552008

[131] A J Vaananen P Salmenpera M Hukkanen P Rauhala andE Kankuri ldquoCathepsin B is a differentiation-resistant target

for nitroxyl (HNO) in THP-1 monocytemacrophagesrdquo FreeRadical Biology and Medicine vol 41 no 1 pp 120ndash131 2006

[132] N M Cook M Shinyashiki M I Jackson F A Leal andJ M Fukuto ldquoNitroxyl-mediated disruption of thiol proteinsinhibition of the yeast transcription factor Ace1rdquo Archives ofBiochemistry and Biophysics vol 410 no 1 pp 89ndash95 2003

[133] A J Vaananen M Moed R K Tuominen et al ldquoAngelirsquos saltinduces neurotoxicity in dopaminergic neurons in vivo and invitrordquo Free Radical Research vol 37 no 4 pp 381ndash389 2003

[134] A J Vaananen R Liebkind E Kankuri P Liesi and P RauhalaldquoAngelirsquos salt and spinal motor neuron injuryrdquo Free RadicalResearch vol 38 no 3 pp 271ndash282 2004

Review ArticleNitroxyl (HNO) A Reduced Form of Nitric Oxide with DistinctChemical Pharmacological and Therapeutic Properties

Mai E Shoman and Omar M Aly

Department of Medicinal Chemistry Faculty of Pharmacy Minia University Minia 61519 Egypt

Correspondence should be addressed to Omar M Aly omarsokkaryahoocom

Received 5 May 2015 Revised 14 August 2015 Accepted 1 September 2015

Academic Editor Lezanne Ooi

Copyright copy 2016 M E Shoman and O M AlyThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Nitroxyl (HNO) the one-electron reduced form of nitric oxide (NO) shows a distinct chemical and biological profile from thatof NO HNO is currently being viewed as a vasodilator and positive inotropic agent that can be used as a potential treatment forheart failure The ability of HNO to react with thiols and thiol containing proteins is largely used to explain the possible biologicalactions of HNO Herein we summarize different aspects related to HNO including HNO donors chemistry biology and methodsused for its detection

1 Nitric Oxide (NO)

Biological activities associatedwith nitrogen oxide species arethe subject of intense and current research interest Muchof this interest stems from the discovery of endogenousNO generation [1] a significant event that represents afundamentally new paradigm in mammalian cell signalingPrior to this finding the idea that a small freely diffusiblereactive molecule (known more for its toxicity) could bebiosynthesized in a highly regulated fashion and elicit specificbiological functions was unheard of and for some at thetime heretical [2] Most attention has been focused on NOsince it is generated directly in mammalian cells by a familyof enzymes referred to as the NO synthases (NOS) whichconverts the terminal guanidino nitrogen of l-arginine intoNO (Scheme 1) [3 4]

NO was described more than 30 years ago as a ligandof the NO-sensitive soluble guanylate cyclase (NO-sensitivesGC) the enzyme that catalyzes the conversion of guanosine51015840-triphosphate to guanosine 3101584051015840-cyclic monophosphate(cGMP) [5] NO activation of sGC involves coordinationto a regulatory ferrous heme on the protein resulting in anincrease in catalysis and a subsequent increase in intracellularcGMP [2] Increased levels of cGMP within vascular smoothmuscle result in vasodilatation [5] thus continual releaseof NO regulates vascular tone and assists in the control ofblood pressure [6] In addition NO increases the level of

cGMP in platelets and this is thought to be the mechanismby which it inhibits platelet function [7] In the vasculatureNO also prevents neutrophilplatelet adhesion to endothelialcells inhibits smoothmuscle cell proliferation andmigrationregulates programmed cell death (apoptosis) and maintainsendothelial cell barrier function [8]

Along with NO significant interest also exists for itsoxidized congeners nitrogen dioxide (NO

2) peroxynitrite

(ONOOminus) and nitrite (NO2

minus) among others This attentionmay be a function of the facility by which NO is oxidizedin aerobic systems and the toxicity of these resulting strongoxidants [9] On the other hand reduced nitrogen oxides(relative to NO) such as hydroxylamine (NH

2OH) nitroxyl

(HNO) and ammonia (NH3) have received much less atten-

tion [10] although there are several reports that suggestthey can be generated endogenously [11] These compoundswere studied in the past with regard to their biologicalactivitytoxicity [12] Among the reduced forms nitroxyl(HNO) is the least understood but is rapidly emerging asa novel entity with distinct pharmacology and therapeuticadvantages over the common nitrogen oxide species [13]

2 Nitroxyl (HNO)

In 1896 the Italian scientist Angeli published the synthesisof the inorganic salt Na

2N2O3[14] and several years later

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 4867124 15 pageshttpdxdoiorg10115520164867124


HO HO

O O O

NH2 NH2

NH2 NH2

Arginine Citrulline

NH

NH

NH2

+

NO synthase2NADPH

2O2

+ NO + 2NADP + 2H2O









2O) and water




2O (1)


3 Nitroxyl Donors









Ominus

OminusOminus

Ominus

Ominus

Ominus

Ominus

Slow

Angelirsquos salt

N+N

+N+

OH+ H

N N NHO

HNO + NO2minus


OminusOH

minus

Pilotyrsquos acidO

SO

O

S S


minus


H

H HN NN NC C

CatalaseH2O2




Ominus

Ominus

Ominus

N+

Ominus

Ominus

N+

N+

Nminus

NNNHN(1)

(3)NN O

minusN

HIPANO Nitrosamine

OH

+ HNO




2[38]









NH2

H2O

H2NH2N


O O

OO

O

O

OH

OH

NH NN

HNO + H2N

IO4minus


HydrolysisHNO +

XX

R

+ R-COOH


X = CH2 O

O

O

O

O

N





2S) [45] have



(IV) (III)FeFe

O+ H2O + HNO+ NH2OH










2OH as the





2O2


HNO Forms N2O2


2O

O2


N2O3






R

RR

S

SN N

O

OH

H

H+OH

RSH RSSR + NH2OH

NH2

Sulfinamide

Sminus



2C6H5SH +HN

2O3

minus997888rarr C

6H5SSC6H5+NH

2OH

+ NO2

minus

(3)





















2to two metMb



[17 66]


2

minus (5)


3

minus (6)

N +

+

O

O

O

HN

OH


HNminus HN

PPh3

+

TPP∙∙PPh3

∙∙PPh3

PPh3 PPh3

Ph3P



2NO +O

2(7)


2OH +O

2(8)







2relative





2+ 2H2O (9)


COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh





2OH couple



6 Detection of HNO


2O has been


2N2O2) that then


2O is usually


2O can be




2] and the oxidized















Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References














































































































































HO HO

O O O

NH2 NH2

NH2 NH2

Arginine Citrulline

NH

NH

NH2

+

NO synthase2NADPH

2O2

+ NO + 2NADP + 2H2O









2O) and water




2O (1)


3 Nitroxyl Donors









Ominus

OminusOminus

Ominus

Ominus

Ominus

Ominus

Slow

Angelirsquos salt

N+N

+N+

OH+ H

N N NHO

HNO + NO2minus


OminusOH

minus

Pilotyrsquos acidO

SO

O

S S


minus


H

H HN NN NC C

CatalaseH2O2




Ominus

Ominus

Ominus

N+

Ominus

Ominus

N+

N+

Nminus

NNNHN(1)

(3)NN O

minusN

HIPANO Nitrosamine

OH

+ HNO




2[38]









NH2

H2O

H2NH2N


O O

OO

O

O

OH

OH

NH NN

HNO + H2N

IO4minus


HydrolysisHNO +

XX

R

+ R-COOH


X = CH2 O

O

O

O

O

N





2S) [45] have



(IV) (III)FeFe

O+ H2O + HNO+ NH2OH










2OH as the





2O2


HNO Forms N2O2


2O

O2


N2O3






R

RR

S

SN N

O

OH

H

H+OH

RSH RSSR + NH2OH

NH2

Sulfinamide

Sminus



2C6H5SH +HN

2O3

minus997888rarr C

6H5SSC6H5+NH

2OH

+ NO2

minus

(3)





















2to two metMb



[17 66]


2

minus (5)


3

minus (6)

N +

+

O

O

O

HN

OH


HNminus HN

PPh3

+

TPP∙∙PPh3

∙∙PPh3

PPh3 PPh3

Ph3P



2NO +O

2(7)


2OH +O

2(8)







2relative





2+ 2H2O (9)


COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh





2OH couple



6 Detection of HNO


2O has been


2N2O2) that then


2O is usually


2O can be




2] and the oxidized















Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References














































































































































Ominus

OminusOminus

Ominus

Ominus

Ominus

Ominus

Slow

Angelirsquos salt

N+N

+N+

OH+ H

N N NHO

HNO + NO2minus


OminusOH

minus

Pilotyrsquos acidO

SO

O

S S


minus


H

H HN NN NC C

CatalaseH2O2




Ominus

Ominus

Ominus

N+

Ominus

Ominus

N+

N+

Nminus

NNNHN(1)

(3)NN O

minusN

HIPANO Nitrosamine

OH

+ HNO




2[38]









NH2

H2O

H2NH2N


O O

OO

O

O

OH

OH

NH NN

HNO + H2N

IO4minus


HydrolysisHNO +

XX

R

+ R-COOH


X = CH2 O

O

O

O

O

N





2S) [45] have



(IV) (III)FeFe

O+ H2O + HNO+ NH2OH










2OH as the





2O2


HNO Forms N2O2


2O

O2


N2O3






R

RR

S

SN N

O

OH

H

H+OH

RSH RSSR + NH2OH

NH2

Sulfinamide

Sminus



2C6H5SH +HN

2O3

minus997888rarr C

6H5SSC6H5+NH

2OH

+ NO2

minus

(3)





















2to two metMb



[17 66]


2

minus (5)


3

minus (6)

N +

+

O

O

O

HN

OH


HNminus HN

PPh3

+

TPP∙∙PPh3

∙∙PPh3

PPh3 PPh3

Ph3P



2NO +O

2(7)


2OH +O

2(8)







2relative





2+ 2H2O (9)


COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh





2OH couple



6 Detection of HNO


2O has been


2N2O2) that then


2O is usually


2O can be




2] and the oxidized















Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References














































































































































NH2

H2O

H2NH2N


O O

OO

O

O

OH

OH

NH NN

HNO + H2N

IO4minus


HydrolysisHNO +

XX

R

+ R-COOH


X = CH2 O

O

O

O

O

N





2S) [45] have



(IV) (III)FeFe

O+ H2O + HNO+ NH2OH










2OH as the





2O2


HNO Forms N2O2


2O

O2


N2O3






R

RR

S

SN N

O

OH

H

H+OH

RSH RSSR + NH2OH

NH2

Sulfinamide

Sminus



2C6H5SH +HN

2O3

minus997888rarr C

6H5SSC6H5+NH

2OH

+ NO2

minus

(3)





















2to two metMb



[17 66]


2

minus (5)


3

minus (6)

N +

+

O

O

O

HN

OH


HNminus HN

PPh3

+

TPP∙∙PPh3

∙∙PPh3

PPh3 PPh3

Ph3P



2NO +O

2(7)


2OH +O

2(8)







2relative





2+ 2H2O (9)


COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh





2OH couple



6 Detection of HNO


2O has been


2N2O2) that then


2O is usually


2O can be




2] and the oxidized















Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References
















































































































































2O2


HNO Forms N2O2


2O

O2


N2O3






R

RR

S

SN N

O

OH

H

H+OH

RSH RSSR + NH2OH

NH2

Sulfinamide

Sminus



2C6H5SH +HN

2O3

minus997888rarr C

6H5SSC6H5+NH

2OH

+ NO2

minus

(3)





















2to two metMb



[17 66]


2

minus (5)


3

minus (6)

N +

+

O

O

O

HN

OH


HNminus HN

PPh3

+

TPP∙∙PPh3

∙∙PPh3

PPh3 PPh3

Ph3P



2NO +O

2(7)


2OH +O

2(8)







2relative





2+ 2H2O (9)


COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh





2OH couple



6 Detection of HNO


2O has been


2N2O2) that then


2O is usually


2O can be




2] and the oxidized















Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References





















































































































































2to two metMb



[17 66]


2

minus (5)


3

minus (6)

N +

+

O

O

O

HN

OH


HNminus HN

PPh3

+

TPP∙∙PPh3

∙∙PPh3

PPh3 PPh3

Ph3P



2NO +O

2(7)


2OH +O

2(8)







2relative





2+ 2H2O (9)


COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh





2OH couple



6 Detection of HNO


2O has been


2N2O2) that then


2O is usually


2O can be




2] and the oxidized















Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References














































































































































COOMeHNO

PPh2

Phosphine ester

CO2Me

PNH

Ph

Aza-ylide

Staudingerligation

CONH2

OPPh

PhPh





2OH couple



6 Detection of HNO


2O has been


2N2O2) that then


2O is usually


2O can be




2] and the oxidized















Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References














































































































































Angelirsquos salt

NN NN+

Ominus

Ominus

Ominus

2Na HNO +

OO

N

Nitrosobenzene

OH

Cupferron


Ph2P

Ph2PPh2P Ph2P

Ph2P

Ph2P

Ph2PNH2

NH2

Cl+

+

O

O

HO

O

O

OO

OO

O

NH

NH

NNH H

NH

NH

NH

NH

NH

O

O

O

O

OO

O

OO

R

R

R

R

R

R

CH2Cl2

AS MeCNH2O

PPh2

a R = minusNO2

b R = minusH





























LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References































































































































































LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References


















































































































































LndashH + In∙

997888rarr L∙


(10)





2depletion



2and














References


















































































































































References




























































































































































































































































































































































































































Documents

Retracted: Nitroxyl (HNO): A Reduced Form of Nitric Oxide ...downloads.hindawi.com/journals/omcl/2016/4867124.pdf · OxidativeMedicineandCellularLongevity O O − − O − O −