The reaction of oximes with 4-phenyl-1,2,4-triazoline-3,5-dione to produce nitric oxide – Model compounds for nitric oxide synthase

Article abstract of DOI:10.1016/j.tetlet.2018.04.064

Certain oximes form nitric oxide upon reaction with 4-phenyl-1,2,4-triazole-3,5-dione (PTAD). The oximes appear to undergo an Alder-ene reaction with the PTAD enophile to form a nitroso intermediate capable of dimerization and/or nitric oxide formation. Upon exposure to oxygen, the nitroso compounds eventually form ketones. This reaction may serve as a model for the study of nitric oxide synthase (NOS), the enzyme responsible for physiological production of nitric oxide. NOS is known to produce an oxime intermediate which reacts with oxygen to produce nitric oxide and citrulline.

Full text of DOI:10.1016/j.tetlet.2018.04.064

Accepted Manuscript
The Reaction of Oximes with 4-Phenyl-1,2,4-triazoline-3,5-dione to Produce
Nitric Oxide – Model Compounds for Nitric Oxide Synthase
Anton W. Jensen, Daryl Flotka, Tianzuo Xu, Alexis E. Pullizzi, Wendell L.
Dilling, Joshua C. Doverspike, Mark E. Meyerhoff, Dillip K. Mohanty
PII:
S0040-4039(18)30537-9
https://doi.org/10.1016/j.tetlet.2018.04.064
TETL 49927
DOI:
Reference:
Tetrahedron Letters
To appear in:
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Revised Date:
Accepted Date:
1 December 2017
20 April 2018
23 April 2018
Please cite this article as: Jensen, A.W., Flotka, D., Xu, T., Pullizzi, A.E., Dilling, W.L., Doverspike, J.C., Meyerhoff,
M.E., Mohanty, D.K., The Reaction of Oximes with 4-Phenyl-1,2,4-triazoline-3,5-dione to Produce Nitric Oxide
– Model Compounds for Nitric Oxide Synthase, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.
2018.04.064
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Tetrahedron Letters
The Reaction of Oximes with 4-Phenyl-1,2,4-triazoline-3,5-dione to Produce Nitric Oxide –
Model Compounds for Nitric Oxide Synthase
Anton W. Jensen,† Daryl Flotka,† Tianzuo Xu,† Alexis E. Pullizzi,† Wendell L. Dilling,† Joshua C.
Doverspike,‡ Mark E. Meyerhoff,‡ and Dillip K. Mohanty†
†
Department of Chemistry & Biochemistry, Central Michigan University, Mount Pleasant, Michigan 48859, USA
‡
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Certain oximes form nitric oxide upon reaction with 4-phenyl-1,2,4-triazole-3,5-dione (PTAD).
The oximes appear to undergo an Alder-ene reaction with the PTAD enophile to form a nitroso
intermediate capable of dimerization and/or nitric oxide formation. Upon exposure to oxygen,
the nitroso compounds eventually form ketones. This reaction may serve as a model for the
study of nitric oxide synthase (NOS), the enzyme responsible for physiological production of
nitric oxide. NOS is known to produce an oxime intermediate which reacts with oxygen to
produce nitric oxide and citrulline.
Available online
2017 Elsevier Ltd. All rights reserved.
Oxidation; Oximes; Nitric Oxide; Ene Reaction
 Corresponding author. Tel.: +1-989-774-3125; fax: +1-989-774-3883; e-mail: jense1aw@cmich.edu or mohan1dk@cmich.edu

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Nitric oxide synthase (NOS) is a complex enzyme with
multiple cofactors that is responsible for forming preparing
physiological nitric oxide (NO). The mechanism of formation of
nitric oxide is not entirely understood. What is known is that the
dual nucleophilic and electrophilic character of the nitroso
nitrogen. Its nucleophilic character arises from its lone pair and
its electrophilicity from its similarity to an aldehyde carbon.
enzyme forms an intermediate oxime by hydroxylation of a

terminal nitrogen of arginine substrate. This oxime, N -hydroxy-
L-arginine, reacts enzymatically with oxygen to form nitric oxide
and citrulline (see Scheme 1).1
Scheme 4. (a) Nitroso-Oxime Tautomerization. (b) Nitroso
Dimerization
With these considerations in mind we set out to test the
reactivity of various oximes with one of the most reactive
Scheme 1. Nitric Oxide Synthase Reaction
The NOS oxime intermediate may undergo an enzymatically
facilitated Alder-ene reaction with oxygen to form a nitroso
intermediate. The Alder-ene reaction (also called the ene
reaction), is similar to the Diels-Alder reaction.2 An ene reaction
with an oxime and a typical enophile would theoretically produce
a nitroso compound. Various mechanisms have been proposed
for the ene reaction. These include the concerted one shown
below (Scheme 2), a radical version, and a polar mechanism.3
The same products result regardless of the mechanism. Ene
reactions may involve heteroatoms.4
enophiles known, 4-phenyl-1,2,4-triazole-3,5-dione (PTAD).
Like the Diels-Alder reaction, ene reactions often require high
temperatures, which may not be suitable for observing nitroso
compounds. The reactivity of PTAD allows it to undergo ene
reactions at or below room temperature.12 The double bond
between the two PTAD nitrogens has a reactivity that is similar
to singlet oxygen. Solutions of PTAD are bright red.
Scheme 2. Ene Reaction With Oxime ‘Ene.’
In NOS, the oxime intermediate reacts with an oxygen
molecule that is coordinated to a heme iron cation. This oxygen
molecule could play the role of enophile, in the formation of the
nitroso intermediate, through one of the ene mechanisms
mentioned above. The nitroso intermediate might then be
unstable enough to collapse into citrulline, nitric oxide and
hydroxyl radical (Scheme 3).
PTAD
Acetone oxime reacts with PTAD to produce a somewhat
unstable nitroso compound. The reaction is best done in either
methylene chloride or chloroform solvent due to the
instability/reactivity of PTAD in other solvents. The reaction is
confirmed by loss of the red PTAD color and the appearance of a
blue solution and corresponding UV-visible maximum at of 670
nm, consistent with nitroso compounds. A new singlet also
Scheme 3. Hypothetical NOS Mechanism
To the best of our knowledge, ene reactions with an oxime ene
are not known, nor have they been proposed as a potential
mechanism for NOS. Therefore, we undertook this investigation
to determine 1) whether oxime enes can undergo ene reactions
and 2) if the nitroso products could be observed.
1
1
appears in the H NMR spectrum at 1.79 ppm (similar to the H
NMR signal of 2-methyl-2-nitrosopropane).8b Crude ESI-MS
spectra of the reaction mixture also showed peaks corresponding
An O-alkyl oxime has been reported to undergo an ene
reaction as an enophile.5 Nitroso compounds6 and nitro-like
oxoammonium cations7 also undergo ene reactions as enophiles.
Nitroso compounds have a characteristic n  * absorption
from 630-790 nm, giving them a blue color.8 When nitroso
compounds form with an alpha hydrogen, they rapidly
tautomerize to oximes (Scheme 4a), making it next to impossible
to detect the nitroso tautomer. However, nitroso compounds with
no alpha hydrogens can be observed and even isolated in some
cases. Nitroso compounds are also known to dimerize (Scheme
4b) to form colorless cis and trans dimers,9,10,11 which further
complicate their detection. This dimerization is a result of the

3
to the nitroso compound. This nitroso compound then enters a
complex equilibrium with two competing reactions, 1)
dimerization to cis and trans nitroso dimers, and 2) radical
formation of nitric oxide and an organic radical (Scheme 5).
When even small amounts of oxygen are present, the nitric oxide
reacts with it to form nitrogen dioxide, which then forms an
equilibrium with dinitrogen tetroxide.
stabilizing a radical, and may facilitate loss of nitric oxide from
the nitroso.
In conclusion, acetone oxime, acetophenone oxime,
cyclohexanone oxime, and heptanal oxime react with PTAD at
room temperature. When a benzyl nitroso is formed, it is not
detected by UV. Nitric oxide is produced as the nitroso
compounds decompose. The reaction of oximes with PTAD at
room temperature might be a good model reaction for the
formation of nitric oxide by NOS.
Scheme 5. Reaction of Acetone Oxime with PTAD
The appearance of two colorless nitroso dimers is confirmed
1
by two additional H NMR signals that appear at about 1.8 ppm
1
(similar to the H NMR shift of the 2-methyl-2-nitrosopropane
dimer at 1.6 ppm).8b The formation of nitric oxide is observed by
running the reaction in an NO analyzer, which detects the
presence of NO by reacting it with ozone, forming exited state
NO2, which emits a photon that can be detected. manifested by
the initial formation of a gas (when the reaction is run in a capped
NMR tube the cap pops off as the reaction progresses).
Furthermore, when the reaction is run in a small vial with a
screwed on cap the final solution has a white precipitate and is
colorless (nitric oxide and dinitrogen tetroxide are colorless);
however, when the cap is opened, more oxygen enters the vial
and the equilibrium shifts to yield a yellow-brown gas (nitrogen
dioxide) which evolves from the vial. Also, when the reaction is
run in a closed ESR tube two new radical signals appear
(presumably due to the nitric oxide and organic radical). The
solution also turns pink indicating that some PTAD is reformed.
As the reaction is allowed to run to completion, two additional
products are formed, acetone and a precipitate, 4-phenylurazole
Acknowledgments
The authors wish to thank Central Michigan University for an
FRCE grant used to fund this research. We also thank Dr. Robin
Hood for NMR and MS assistance.
References and notes
1. Santolini, J. J. Inorg. Biochem. 2011, 105, 127-141.
2. Snider, B. B. Acc. Chem. Res 1980, 13, 426-432.
3. Alberti, M. N.; Orfanopoulos, M. Chem. Eur. J. 2010, 16, 9414-
9421.
4. (a) Clarke, M. L.; France, M. B. Tetrahedron 2008, 64, 9003-
9031. (b) Borzilleri, R. M.; Weinreb, S. M. Synthesis 1995, 347-
360. (c) Seymour, C. A.; Greene, F. D. J. Am. Chem. Soc. 1980,
102, 6384-6385.
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1996, 52, 13079-13110.
6. Adam, W.; Krebs, O. Chem. Rev. 2003, 103, 4131-4146.
7. Pradhan, P. P.; Bobbitt, J. M.; Bailey, W. F. Org. Lett. 2006, 8,
5485-5487.
1
(confirmed by their H NMR signals). The products are both
derived from the organic radical formed from the initial nitroso
compound. No other major products are observed in the reaction
of acetone oxime with PTAD. A mechanism consistent with
these observations is shown below.
8. (a) Gowenlock, B. G.; Richter-Addo, G. B. Chem. Rev. 2004,
104, 3315-3340. (b) Homer, S. R.; McKinnon, S. J.; Whittenburg,
S. L. J. Chem. Ed. 1986, 63, 1103-1004.
9. Glaser, R.; Murmann, R. K.; Barnes, C. L. J. Org. Chem. 1996,
61, 1047-1058.
Other oximes also react with PTAD. Acetophenone oxime
reacts slowly with PTAD, possibly due to steric crowding or
conjugation of the ene with aromatic ring. The major product is
acetophenone, but other minor products are also formed,
consistent with a radical reaction. No nitroso peak is observed in
the UV-visible spectrum, possibly because the nitroso rapidly
falls apart to NO and a benzyl radical. The reaction of
cyclohexanone oxime with PTAD is very fast and similar to the
reaction of acetone oxime with PTAD – a nitroso intermediate is
observed by UV and the final product is almost exclusively
cylcohexanone. Heptanal oxime also reacts rapidly with PTAD,
and a fleeting nitroso compound is observed briefly by UV,
which most likely tautomerizes to the oxime.
10. Lee, J. L.; Chen. L.; West, A. H.; Richter-Addo, G. B. Chem. Rev.
2002, 102, 1019-1065.
11. Beckett, A. H.; Jones, G. R.; Coutts, R. T. Tetrahedron 1976, 32,
1267-1276.
12. Vassilikogiannakis, G.; Elemes, Y.; Orfanopoulos, M. J. Am.
Chem. Soc. 2000, 122, 9540-9541.
13. Sha, X.; Isbell, S.; Patel, R. P.; Day, C. S.; King, S. B. J. Am.
Chem. Soc. 2006, 128, 9687-9682.
14. Mohamed, H. A. H.; Abdel-Aziz, M.; Abuo-Rahma, G. E.-D. A.
A.; King, S. B. Bioorg. Med. Chem. 2015, 23, 6069-6077.
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Org. Chem. 1999, 64, 5634-5643.
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Org. Chem. 1987, 52, 3254-3263.
Supplementary Material
The alpha nitrogen present in all of the nitroso compounds
made in this study may have something to do with their
instability and radical decomposition. Indeed, similar compounds
with an alpha acetate, such as 1-nitrosylcyclohexyl acetate, are
much more stable, but form HNO, and cyclohexanone when the
acetate is hydrolyzed.13,14 An examination of the radical
stabilizing ability (radical sigma value)15,16 of an amine ( =
0.69), relative to methoxy ( = 0.24), or methyl group ( =
Supporting Information is available and includes experimental
procedures and spectra (NMR, UV, EPR, NO analysis, and ESI-
MS).
0.11), indicates that the adjacent nitrogen atom is much better at

4
Graphical Abstract
The Reaction of Oximes with 4-Phenyl-1,2,4-
triazoline-3,5-dione to Produce Nitric Oxide –
Model Compounds for Nitric Oxide Synthase
Leave this area blank for abstract info.
Anton W. Jensen,* Daryl Flotka, Tianzuo Xu, Alexis E. Pullizzi, Wendell L. Dilling, Joshua C. Doverspike, Mark
E. Meyerhoff, and Dillip K. Monhanty*

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Highlights
 Oximes react with PTAD to produce nitric
oxide and ketones.
 An intermediate nitroso compound is
detected.
 The mechanism may provide insights into
the mechanism of nitric oxide synthase.