Alkyl nitrates, _in vivo_, are metabolized to yield nitric oxide, and thiol groups are considered necessary cofactors. This statement is based on studies that underline how these species potentiate hemodynamic responsiveness to nitrates in patients with ischemic heart disease. However, the role of thiols might be mediated by the formation of corresponding S-nitrosothiols, and a redox process is responsible for the nitrates&#x27; degradation: an enzyme, probably the cytochrome P450, is involved _in vivo_. Here, we report evidence that, in vitro, no reaction between thiols and alkyl nitrates takes place, but that stronger reducing agents, such as iron (II) derivatives, are necessary: alkoxy radicals and the nitrite anion are the reaction intermediates. The latter, in slightly acidic conditions, for instance mimicking ischemic conditions, is shown to nitrosilate thiols to the corresponding S-nitrosothiols: the real NO suppliers. Therefore, the direct release of NO from nitrates is excluded. Finally, the in vivo role of thiols on depletion and tolerance is also accounted for

Loris Grossi

English

Nature Precedings

1The nitrite anion: the key intermediate in alkyl nitrates 
degradative mechanism. 
Loris Grossi 
Dipartimento di Chimica Organica “A. Mangini”, University of Bologna,  
Viale Risorgimento 4, I-40136 Bologna, Italy
loris.grossi@unibo.it
Alky nitrates, in vivo, are metabolized to yield nitric oxide, and thiol groups are 
considered necessary cofactors. This statement is based on studies that underline 
how these species potentiate hemodynamic responsiveness to nitrates in patients 
with ischemic heart disease. However, the role of thiols might be mediated by the 
formation of corresponding S-nitrosothiols, and a redox process is responsible for 
the nitrates’ degradation: an enzyme, probably the cytochrome P450, is involved in 
vivo. Here, we report evidence that, in vitro, no reaction between thiols and alkyl 
nitrates takes place, but that stronger reducing agents, such as iron (II) 
derivatives, are necessary: alkoxy radicals and the nitrite anion are the reaction 
intermediates. The latter, in slightly acidic conditions, for instance mimicking 
ischemic conditions, is shown to nitrosilate thiols to the corresponding S-
nitrosothiols: the real NO suppliers. Therefore, the direct release of NO from 
nitrates is excluded. Finally, the in vivo role of thiols on depletion and tolerance is 
also accounted for. 
Even if a central role of thiols on nitrates degradative reduction has always been 
invoked, no one has never reported a study on exactly what their role is: do they act as 
direct reducing agents or is a different reductant, for example an enzyme, responsible? 
The answer to this query was obtained when thiols such as glutathione (GSH), cysteine 
or benzylthiol, and nitrates such as the 5-phenyl-4-pentene-1-nitrate and the 1-penten-5-
nitrate, in anhydrous solvent, were reacted: thiols and nitrates were recovered 
unchanged. That confirmed the non-involvement of thiols in the initial reductive 
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2process, but indirectly led to hypothesize, in vivo, the predominance of the enzyme path: 
i.e., an Electron Transfer process between a reductant, the enzyme, and an oxidant, the 
nitrate. 
In particular, the cytocrome P450, the enzyme most commonly involved in drugs 
metabolism,1 is characterized by a heme group able to carry out electron transport by 
interconversion between Fe2+ (reduced) and Fe3+ (oxidized) states,2 and therefore able to 
induce the reductive process. In fact, this enzyme, localized in the human heart and 
vessels, seems the most accredited, and its involvement in the biotransformation of 
organic nitrates, via a so-called two-electron mechanism, leading directly to the 
formation of NO, has been suggested.3-6 However, this hypothesis contrasts with other 
reports7-9 that highlight the intermediacy of S-nitrosothiols in NO release. Nevertheless, 
in principle, also via this two-electron degradative nitrate mechanism the formation of 
S-nitrosothiols could be accounted for, and that through the nitrosilation of the 
corresponding thiols. The latter process could be performed by the nitrous anhydride 
(N2O3),10 possibly formed via oxidation of NO, but it has been shown to be irrelevant in 
vivo because negligibly slow.11,12 Scheme 1.  
In contrast, a mono-electron process, leading to alkoxy radicals and the nitrite anion, 
could more readily account for the formation of S-nitrosothiols.8 In fact, the nitrite 
anion, via its acid equilibrium, leads directly to the formation of nitrosilating species. 
Scheme 1.  
To support this hypothesis it was necessary to prove if the reduction of nitrates can 
really be induced by iron (II) derivatives and, to this end, nitrates such as the 5-phenyl-
4-penten-1-nitrate and the 5-nitrate-1-penten should give clear evidence. In fact, the 
hypothesized alkoxy intermediates, besides forming the parent alcohols, can undergo 
rearrangements that lead to compounds whose formation is definitely a proof of a 
radical mechanism. Experiments conducted with these nitrates and anhydrous FeCl2, in
anhydrous solvents, led to the detection of derivatives such as 1-5, Scheme 2. 
The detection of these compounds is an unquestionable result in favor of a radical 
mechanism and of the role of iron (II) as a mono-electron transfer agent. Among the 
reaction products the corresponding di-sulphide and the nitrite anion were also 
evidenced. In particular, as reported in a previous paper,12 the nitrite anion has been 
shown to be able to perform the nitrosilation of GSH, in a buffer solution, starting from 
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3pH 6.86, which is a value consistent with ischemic conditions. Thus, this same 
nitrosilating process, here conducted in vitro, could be hypothesized to occur also in 
vivo, and the S-nitrosothiols, via a homolytic and/or a redox mechanism, act as NO 
releasers.14 Thus, the NO supplementation in vivo, starting from organic nitrates, goes 
through nitrosilated species formed via the nitrite anion.  
One of the most troublesome aspects of organic nitrate ester therapy is the fact that 
patients can become refractory to their effects, i.e., a repeated and prolonged 
administration results in the development of tolerance, characterized by a decrease in 
NO production, together with a decrease in tissue thiols level. As confirmed by the 
renewed responsiveness to nitrate therapy when the thiol groups are restored.15
In particular, it has been hypothesized that the tolerance is affected by enzyme-
activity,3 i.e., it increases with enzyme degradation16 (decrease of enzyme reduced-
form). Indirectly supporting this were the results of experiments in which a cell sample 
treated in advance for 48 hours17 with sodium nitroprusside still showed response to 
glycerol trinitrate treatment, thus leading to the conclusion that only NO coming from 
an enzymatic mechanism induces tolerance. Actually, proof of the role of enzymes on 
tolerance is the time necessary to restore cell bioactivity (bioconversion) in respect to 
nitrates -- most probably the time required to endogenously re-establish the reducing 
capacity of P450. This hypothesis is supported by experiments in which the 
administration of reducing agents (antioxidants) such as vitamin C, vitamin E and 
sulphydryl derivatives,18-22 even if in different percentage, allows the reducing 
properties of P450 to be restored in a shorter period of time. Inside cells, the possibility 
to re-convert P450-iron (III) into P450-iron (II) seems mainly restricted to reductants 
such as thiols, and cysteine and GSH -- the highest in concentration -- being the most 
accredited. To prove this, experiments with nitrates such as the 4-phenyl-1-nitrate and 
the 5-phenyl-4-penten-1-nitrate, and FeCl3, both in the absence and in the presence of 
thiols, were conducted. Experiments carried out in the absence of thiols evidenced no 
interaction, but when repeated in the presence of thiols such as GSH, cysteine, or 
benzylthiol, the nitrate degradative reaction took place, Scheme 3. In particular, 
experiments with the 5-phenyl-4-penten-1-nitrate and FeCl3, in the presence of GSH, 
showed the formation of 1 and 6, Scheme 3. This result was mimicking that obtained by 
reacting the same nitrate directly with iron (II), thus confirming its involvement also in 
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4this process, and its formation via the reduction of iron (III) by GSH. Further evidence 
was obtained running experiments at steady nitrate concentration, and varying the 
thiol/iron (III) molar ratio; the highest yield in nitrate degradation was obtained for a 
2.5:1 molar ratio, i.e., conditions in which the conversion of iron (III) into iron (II) is 
favored. This result supports the role of thiols in delaying tolerance, via restoring the 
reduced form of the enzyme that is responsible in the first nitrate degradative step.
The positive effect of added thiols on nitrates hemodynamic is well known, but 
their action seems confined to the extracellular compartment.23 In fact, through 
experiments with both GSH and N-acetyl cysteine, the possibility of enhancing the 
glycerol trinitrate degradation in whole blood, but not in red blood cells, had been 
reported;24 i.e., showing an accelerating effect apparently mediated in the plasma 
fraction of whole blood. Thus, thiols supplementation is more likely related to a 
pronounced extracellular increase of these species, rather than intracellular. (Plasma) In 
particular, the extracellular hypertensive action seems related mainly to the presence of 
GSH that is synthesized intracellularly (mM concentration), starting from cysteine and, 
in normal conditions, continuously and irreversibly transferred out of the cell. Thus, the 
cysteine, definitely the main active intracellular thiol,25,26 due to its manifold roles, i.e., 
reducing agent (in comparison to the enzyme), supplier of active S-nitroso cysteine and 
GSH producer, will diminish over time: this can accounts for depletion.
To explain the action of added thiols, because no direct interaction between 
nitrates and thiols can take place, in the extracellular compartment it is the presence of 
components such as ascorbate, urate and bilirubin, which can act as antioxidants, 
responsible for the degradative reduction of nitrates to nitrite. Only then will the 
supplemented thiols, under the indirect action of the nitrite anion be transformed into 
the corresponding S-nitrosothiols:7 these will serve as tolerance-reversing mechanism.27
The degradative NO release from alkyl nitrates takes its start from the Electron 
Transfer process between the nitrate, the oxidant, and an iron (II) derivative, most 
probably the reducing enzyme P450, in vivo. This mechanism is supported by 
experiments that lead to the detection of products unquestionably supporting a radical 
mechanism, i.e., which can be accounted for only via an Electron Transfer process. The 
nitrate degradative process, besides alkoxy radicals, leads to the formation of the nitrite 
anion, in principle an inactive NO-derivative, but in slightly acidic conditions is able to 
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5induce the nitrosilation of thiols to the corresponding S-nitrothiols: the real NO 
suppliers. Furthermore, because intracellular thiols, in particular cysteine and GSH, are 
involved in the fundamental processes inside the cell, their concentration will diminish 
over time: this accounts for both depletion and for the less efficiency in the re-
establishment the enzyme reducing capability, tolerance.
Methods 
Reagents. The 5-phenyl-4-penten-1-ol, synthesized as reported,28 was reacted with N-
Bromosuccinimide to lead to the 5-phenyl-4-penten-1-bromo, which under the action of 
silver nitrate guides to the 5-phenyl-4-penten-1-nitrate. The 5-nitrate-1-penten was 
obtained reacting the 5-Bromo-1-pentene, commercial grade, with silver nitrate. 
Gluthathione, cysteine, benzylthiol, ferrous and ferric chloride are commercial products; 
the latter two were carefully kept anhydrous. All solvents, acetonitrile, methanol and 
methylene chloride, were carefully kept anhydrous.
Nitrates degradative reactions. All the reactions were conducted in anhydrous 
solvents and under nitrogen atmosphere, at thermostated temperature (ranging between 
37 and 60 °C). Bromotrichloromethane, or N-Bromosuccinimide, and SO2Cl2 were used 
as spin trap for carbon centered radicals. After the workup, products were identified 
using standard techniques (GC-MS), and compared with data reported in the literature. 
The formation of S-nitrosothiols was shown conducting experiments with two solutions 
containing respectively the nitrate and the iron (II) derivative, which were reacted 
directly in a flat cell inside the spectrometer by a mixing flow system. The characteristic 
S-NO absorption at 540 nm was detectable. 
Acknowledgements.  
I thank L.A. Winter for the helpful discussion, and the Ministero dell’Istruzione, 
dell’Università e della Ricerca (MIUR), Rome, for the financial support (Funds PRIN 
2006).  
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Scheme legends
Scheme 1: Nitrates degradation mechanism. 
Fe++(P450) + R-ONO2
E.T.
NO
NO + O2 2 HNO2N2O3
H2OVery Slow
R-SH+ R-SNOHNO2 N2O3and/or
Two-electron mechanism
Mono-electron mechanism
Fe++(P450) + R-ONO2 NO2 + Fe
+++(P450) + R-O
2 NO2
+ H+
2 HNO2 N2O3
H2O
NitrosilationNa
tu
re
 P
re
ce
di
ng
s 
: h
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9Scheme 2. Electron Transfer process: radical intermediates. 
R + +R
O
+
O
O
O2N
O
NO2
1,5-exo closure
Oxygen
O
O H
O
Hydrogen or 
Halogen donor
O
X
O 1,5-exo closure O
Hydrogen donor
O
R = Phenyl, H
1,2-Hydrogen shift
HO
Halogen donor
HO
X
X = H, Br, Cl
Fe++ Fe+++
H
1
2
4
5
O
+
3
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10
Scheme 3. The role of thiols in nitrates reduction. 
RO NO2 + No Products
RO NO2 +
R-SH
Products
Fe+++
2 R-SH + 2 Fe+++ RS-SR + 2 Fe++
R-SH = GSH, Cysteine, Benzylthiol
Fe+++
The key step :
RO NO2 +  Fe++ + Fe+++ + RONO2
G-SH
+ Fe+++
ONO2 O
O
+
O
H
6 1
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A chemist’s view of the nitric oxide story.

Absence of vascular tolerance in conductance vessels after 48 hours intravenous nitroglycerin in patients with coronary artery disease.

Cytochrome P-450 mediated biotransformation of organic nitrates.

Cytochrome P450s and Other Enzymes in Drug Metabolism and Toxicity.

Dietary supplement with vitamin C prevents nitrate tolerance.

double blind placebo-controlled study of supplemental vitamin E on attenuation of the development of nitrate tolerance.

Escape from Tolerance of Organic Nitrate by Induction of Cytochrome P450.

Formation of nitric oxide from nitrous acid in ischemic tissue and skin. Nitric Oxide 10,

Isoforms of cytochrome P450 on organic nitrate-derived nitric oxide release in human heart vessels.

Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of Snitrosothiols as active intermediates.

Mechanisms for the Pharmacologic Interaction of Organic Nitrates with Thiols. Existence of an Extracellular Pathway for the Reversal of Nitrate Vascular Tolerance by NAcetylcysteine.

Mechanisms of Vasodilatation Induced by Nitrite Instillation in Intestinal Lumen: Possible Role of Hemoglobin.

Methylene blue: a treatment for severe methaemoglobinaemia secondary to misuse of amyl nitrite.

N a t u r e P r e c e d i n g s : h d l :

Nitrate Tolerance in Angina Therapy. How to Avoid It. Drugs 49,

Nitrate Tolerance. A Review or the Evidence.

Nitric oxide: an emerging role in cardioprotection?

NO Release from NO Donors and Nitrovasodilators: Comparisons between Oxyhemoglobin and Potentiometric.

Prevention and reversal of tolerance to nitroglycerin with N-acetylcysteine.

Scheme legends Scheme 1: Nitrates degradation mechanism.

The Biology of Nitric Oxide,

The formation of S-nitrosoglutathione in conditions mimicking hypoxia and acidosis.

Thiol compounds and organic nitrates.

Vitamin E deficiency accelerates nitrate tolerance via a decrease in cardiac P450 expression and increased oxidative stress.

The nitrite anion: the key intermediate in alkyl nitrates degradative mechanism.

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The nitrite anion: the key intermediate in alkyl nitrates degradative mechanism.

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