Use of nitroxides in biological applications
Angelika Gurianov, Alex M. Szpilman
Medicinal chemistry department, Arial University Center of Samaria, Ramat Hagolan St 65, Ariel city, Israel.
E-mail: olga_angelika@yahoo.com
Abstract: Nitroxides are kinetically persistent
structures, which can avoid various diseases caused
by oxidative stress. Nitroxides can also be used as
spin labelling molecules, that are detected by EPR
in biological studies. This review gives an overview
of nitroxides utilization in biological applications
as oxidative stress suppressors and as spin labeling
species.
Keywords: nitroxide • EPR • ROS • spin labelling
1. Introduction
Reactive oxygen species (ROS) are vital in very small
amounts for maintaining certain physiological
functions; the equilibrium between ROS and
antioxidant system is required for homeostasis.
However, oxidative stress, a surplus amount of ROS can
result in significant damage to human tissue and cellular
components such as: proteins, DNA and lipids.
Oxidative stress has been associated with a vast number
of significant diseases including cancer, Alzheimer,
Parkinson, rheumatoid arthritis, atherosclerosis,
inflammation, ischemia, diabetes, gastric ulceration,
asthma and more. ROS are originated from both intrinsic
and extrinsic sources. Intrinsic sources of ROS mostly
include mitochondria, inflammatory cells, and several
enzymatic cellular complexes, whereas extrinsic
sources of ROS include radiation, pro-oxidant
environmental toxins, and diverse chemical compounds,
including alcohol, drugs and smoke. Energy is produced
via the oxidation of energy rich biomolecules (e.g. fatty
acids, monosaccharides) using oxygen as an electron
sink. Living cells require energy to survive and the
majority of oxidative chemistry takes place within the
mitochondria, organelle that presents in eukaryotic cell.
Nevertheless, reactive oxygen species such as
superoxide, hydrogen peroxide and hydroxyl radical are
produced by only partial reduction of oxygen (figure 1).
1
On the one hand, there are two enzymes that counter the
high ROS levels by converting them to harmless
compounds. Superoxide dismutase (SOD) detoxifies
superoxide and converts it to oxygen and hydrogen
peroxide. Catalase enzyme converts hydrogen peroxide
to oxygen and water. On the other hand, hydroxyl radical
is not removed by any enzyme. This radical species is
very reactive and reacts with almost any substance, and
has a very short lifetime. In
Figure 1. Reduction of oxygen molecule
addition, hydrogen abstraction is one of the most
common reactions initiated by hydroxyl radicals. As a
result, other radicals are generated, often carbon
centered radical, like peroxyl radicals. Hydroxyl
radicals are found in living systems mainly because of
Fenton reaction. The reaction involves interaction
between peroxide and ferrous iron as will be described
later.
Aside from natural antioxidants such as
glutathione, Vitamin C, SOD and catalase, Nitroxides
are among the most effective artificial antioxidants that
have been utilized in therapies to combat oxidative stress
and its destructive effects. Nitroxides are kinetically
persistent structures, which contain free radical that is
delocalized over nitrogen and oxygen atoms. This
delocalization prevents dimerization of nitroxide
radicals, and contributes to its paramagnetic property.
Due to nitroxides paramagnetic state, it can easily
undergo both oxidation and reduction reactions
(figure 3). Although the stability of nitroxide arises from
electronic delocalization between nitrogen-oxygen
bond, the nature of substituents adjacent to the nitrogen
has a significant role in persistence of nitroxides. For
instance, tertiary α carbon substitution limits the access
to reactive substances and prevents radical-radical
interaction. An example of commonly encountered
stable nitroxides include: Tempo (1), Peroxyl (2), and
isoindoline (3). This stability is due to the absence of
disproportion of phenyl substituted nitroxides and the
absence of disproportion of nitroxides with hydrogen
bounded to the α carbon. Also, the substituent group R
(figure 2) can be directed to specific hydrophobic or
hydrophilic zones in the cellular microenvironment. [1,2]
While nitroxide radicals cannot dimerize, they can react
with carbon centered radicals. The effectiveness of these
reaction depends on steric and polar effects, as well as
solvent effects (i.e. viscosity and polarity), chain length
and configuration effects occurring during
polymerization. [3]
Figure 2. Common structures of stable nitroxides. (1)
Tempo (R=H). (2) Peroxyl (R=H). (3) isoindoline.
Site directed spin labelling (SDSL) study introduces a
persistent radical (i.e. Nitroxides), called 'spin label', into
a biomolecule that is not naturally paramagnetic such as
DNA and protein. The complex that is formed is
characterized by Electron paramagnetic resonance
spectroscopy (EPR). EPR is the only direct technique
that detects unpaired electron and its transition in an
applied magnetic field. EPR spin labeling is used for
tagging macromolecules in specific regions, determine
solvent accessibility, and ascertain biopolymers
intermediate to long distances. In contrast to NMR or X-
Ray, EPR yields information about conformational
changes, dynamics of conjugated biomolecules and use
those dynamics to provide structural insights into ligand
binding.[4]
2
This review will give an overview of Tempol nitroxide
utilization as oxidative stress suppressor. Its activity as
SOD mimetic, as pro apoptotic agent, Inhibitor of
Fenton reaction and lipid peroxidation, will be discussed
in detail. In addition to Nitroxides utilization as
oxidative stress suppressors, this review will introduce
nitroxides as SDSL in DNA and proteins.
2. Tempol synthesis
Nitroxides are prepared by oxidation of the
corresponding secondary amines. It is achieved with
hydrogen peroxide and catalytic sodium tungstate
(Na2WO4) or with m-CAPA. Na2WO4 (4) and H2O2 gives
Na2[W(O2)4] (5). N,N-Dimethylacetamide (DMA) solvent
coordinates to tungsten and stabilizes the catalyst (6) .
Without DMA, (5) is decomposed to oxygen and (4).
DMA is exchanged after introducing the secondary
amine. Next, the alkoxide ligand (7) undergoes
intramolecular exchange and eventually hydroxylamine
is produced (figure 3).[5]
Figure 3. proposed mechanism of hydroxylamine
formation
Acetone (8) is the initial reagent for preparing the
corresponding secondary amine (figure 4). First, acetone
undergoes aldol condensation under acidic condition to
give mesityl oxide (9). Nucleophilic addition of mesityl
oxide with ammonia and dehydration proceed to give
imine intermediate (10). Amine intermediate (11) reacts
with acetone to generate 2,6-dimethylhepta-2,5-dien-4-
imine (12). Afterwards, 12 undergoes Michael addition
and intramolecular cyclization with ammonia to yield
intermediate (14), which is converted to triacetonamine
(15) by nucleophilic substitution with water. Then,
triacetonamine is reduced by sodium borohydride
(NaBH4) and secondary amine with hydroxyl residue (16)
is formed (figure 4).[6] In other words, the secondary
amine (16) is converted to hydroxylamine (17) as shown
in figure 3. Then, hydroxylamine is oxidized to
oxammonium salt (18). Eventually, it is proposed that 18
is disproportionated with 17 to give nitroxide (19).
Another option is the oxidation of hydrogen peroxide to
oxygen by 18 to give the final product.[7]
Figure 4. Proposed tempol synthesis
3. Applications of Antioxidative
Nitroxides in biological systems
The ability of Nitroxides to participate in redox
reactions enables them to applicate their antioxidant
activity in biological systems. The antioxidant activity
of nitroxides is associated with redox cycle (figure 5 ).
Tempol is a widely used antioxidant piperidine
nitroxide, and all the examples shown below are related
to the applications of Tempol in biological systems
(figure 4).
3
Figure 5. Redox cycle of nitroxide
3.1. SOD mimetic activity
Unlike other antioxidants, nitroxides act as catalysts
and they mimic SOD in biological conditions. SOD is
an metalloenzyme that catalyzes superoxide, a cell
damaging radical, and forms harmless or less harmful
molecules : oxygen and hydrogen peroxide
respectively. Nitroxide has dual activity, where it can
be oxidized and reduced, depending on the pH values.
Oxidation in low pH values converts nitroxide radical
into oxammonium cation, and forms hydrogen
peroxide (20). Then, oxammonium cation can be
reduced by another superoxide back to the nitroxide
radical, and converting superoxide to oxygen (21). [8]
RR'NO. + O2.- + 2H+ → [RR'NO+] + H2O2 (20)
[RR'NO+] + O2.- → RR'NO. + O2 (21)
Combining equations 20 and 21 yields the following:
2O2.- + 2H+ → H2O2 + O2 (22)
It was found that superoxide enhances the contractility
of blood vessels and hypertension by many mechanisms
including: bioactivation of nitric oxide, enhancing the
peripheral sympathetic nervous system and enhancing
renal tubular NaCl reabsorption. Wilcox and colleagues
have shown that six-member ring piperidine (i.e.
Tempol) nitroxides act as SOD mimics. Their
hypertensive effect was evaluated by redox midpoint
potential, using EPR, cyclic voltammetry and bulk
electrolysis. It was detected that six-member ring
nitroxides are kinetically more effective than five
member rings in redox reactions. This can be explained
by the conformation transformation between "boat" and
"chair" structures that facilitates access to reactants. As
a result, five-member nitroxides act less efficiently as
SOD mimics than six-member nitroxides. [9]
3.2. Inhibiting Fenton reactions
Fenton reaction generates hydroxyl radical, a strong
oxidant that harms cell's essentiality, by catalyzation of
hydrogen peroxide with iron cation. As a result, the
reduced metal (Fe2+) is oxidized by hydrogen peroxide,
forming the oxidized metal (Fe3+), hydroxyl radical and
hydroxide (23). Nitroxides can prevent the generation of
hydroxyl radicals by accepting the electron from the
reduced metal (24). In other words, a low oxidation state
of iron (Fe2+) is oxidized to higher oxidation state (Fe3+).
As a result, nitroxides act as antioxidants and prevent
from Fe2+ to react with hydrogen peroxide to produce
hydroxyl radical.[8]
Fe2+ +H2O2 → Fe3+ 2.OH + -OH (23)
H+ + RR'NO. + Fe2+ → RR'NOH + Fe3+ (24)
According to Zhao and colleagues, the role of nitroxides
would depend on the availability of reducing agent.
Nitroxides would oxidize the iron back to Fe3+ in the
presence of a reducing agent such as glutathione that
reduces Fe3+ to Fe2+ . Also, nitroxides might act as pro
oxidants when the reducing agent is absent. In this case,
nitroxides reduce Fe3+ to Fe2+ that may react with
hydrogen peroxide and produce the undesired hydroxyl
radicals. However, the reduction potentials of RR'NO. /
RR'NOH and RR'NO+/ RR'NO. are +0.61V and +0.75
respectively. Therefore, it is favorable for nitroxides to
be antioxidant and oxidize than pro oxidants and reduce.
In addition, there are many reducing agents in blood
plasma that keep transition metals and their complexes
in lower oxidation states. This reducing environment
may help nitroxides to act as antioxidants rather than pro
oxidants and prevent from metals to participate in
Fenton reaction.[10]
3.3. Detoxification of hypervalent heme proteins
In addition, stable nitroxides can neutralize the toxicity
of hypervalent heme proteins such as ferrylmyoglobin
(MbFe4+). When the heme iron of myoglobin (Mb) is
4
storing and delivering oxygen, its oxidative state is +2
(oxyMbFe2+). However, oxymyoglobin can undergo
oxidation and yield MbFe3+ and superoxide which
disproportionates to oxygen and hydrogen peroxide (22).
Metmyoglobin, the oxidized form of oxygen carried by
hemeprotein, has catalase-like activity. Reaction
between hydrogen peroxide and metmyoglobin
produces .MbFe4+ oxoferryl complex attached to globin
radical (25). Both the globin radical and the oxoferryl
complex are chemically reactive species. The oxoferryl
complex oxidize cellular components, whereas globin
radical is damaging biological systems by oxidation,
peroxidation and epoxidation of biological substrates.
The ferryl moiety and the globin radical can oxidize
another hydrogen peroxide molecule, forming
ferrylmyoglobin (26). The significance of MbFe4+ stems
from its ability to oxidize important biological targets
such as: membranal lipoproteins, fatty acids and can
utilize ubiquitous cofactors to generate additional
hydrogen peroxide molecules. Although the lifetime of
the globin radical has been estimated to be in a scale of
few milliseconds, the life time of oxoferryl species
ranged in higher scales of minutes and hours. Therefore,
reduction of oxoferryl species is essential for protecting
biological systems. The unique feature of nitroxide is its
catalytic nature, enabling the transformation of 1-
electron between three oxidation states of nitroxide as
shown in figure 2. In addition, stable nitroxides have
been found to react ferryl ion and decompose hydrogen
peroxide without damaging the heme vitality. However,
no detailed studies have been made about the nitroxides
mechanism of catalase-like activity. It was found that
Tempol is enhancing the depletion of hydrogen
peroxide, by measuring oxygen evolution, hydrogen
peroxide depletion and redox transitions between
various oxidation states of hemoglobin. [11]
MbFe3+ +H2O2 → .MbFe4+ + H2O. (25)
.MbFe4+ + H2O2→ MbFe4+ + O2.- + 2H+ (26)
3.4. Inhibition of lipid peroxidation
Cellular injury is mediated by lipid and protein
peroxidation, a process that forms radicals and causing
membrane disintegration. An overproduction of free
radicals in the cell and low levels of natural antioxidants amidinopropane)dihydrochloride (AAPH) as radical
(e.g. GSH, vitamin C & E), may lead to peroxidative initiator in a presence of different nitroxides. The
damage in cellular membrane and other critical targets. samples were introduced into agarose gel
Lipid peroxidation process occurs in three steps (27-29), electrophoresis, and their relative protection was tested.
where lipid cellular (LH) lipids decomposes into lipid Even though TEMPO and Tempol are capable
radicals (L.), peroxyl radicals (LOO.) and alkoxyl scavenging only carbon centered radicals, the
radicals (LO.). First, cellular lipids are damaged by free experimental findings show that nitroxides are not
radical (X.) in initiation step (27). Afterwards, oxidized variable with indolinonic or quinolinic nitroxides in
lipids (L.) react with oxygen in propagation steps (28a-d) protective ability. If so, it appears that protection is due
until oxygen is depleted. Finally, the levels of oxygen or to trapping alkyl radicals, thereby reducing the
other substances are used in the cycle of reactions and possibility of direct attack on DNA and indirect attack
termination step occurs (29a-c). Nitroxide radicals via reaction with oxygen to give peroxyl radicals.[12]
(RR'NO.) and hydroxylamines (RR'NOH) inhibit the The lipophilicity of Nitroxides must be
initiation step by interacting with free radicals (30a-b) . considered for drug designing. This is due to blood brain
In addition, they also inhibit the propagation step by barrier permeability and stability between nitroxides and
interacting with oxidized lipids (31a-c).[8] the lipid derived radicals via van der Waal's forces.
Alkyl-substituted nitroxides at 𝛼 position may enhance
LH + X. → L. + XH (27) the lipophilicity of nitroxides. The correlation between
L. + O2 → LOO. (28a) half maximal inhibitory concentration (IC50) and
lipophilicity (Po/w) of nitroxides was detected. Nitroxides
LOO. + LH → L. + LOOH (28b)
with two bulky positions had low IC50 values in contrast
LOOH → LO. + .OH (28c)
to nitroxides with one bulky position. Bulky alkyl
LO. + LH → L. + LOH (28d) substituents induce resistance against lipid radicals, and
L. + L. → LL (29a) therefore are less potent. Conversely, nitroxide that was
LOO. + LOO. → LOOL + O2 (29b) substituted with a methyl group had a better protecting
LOO. + L. → LOOL (29c) effect than the nitroxide that was substituted with ethyl
group. These results suggest that the protecting effect
X. + RR'NO. → RR'NOX (30a)
against lipid peroxidation depends in two factors: the
X. + RR'NOH → RR'NO. + XH (30b) steric effect and the affinity of nitroxides with lipid
L. + RR'NO. → RR'NOL (31a) radicals. [13]
L. + RR'NOH → RR'NO. + LH (31b)
LO. + RR'NOH → RR'NO. + LOH (31c) 4. Nitroxides as pro-apoptotic agents
Cancer disease remains one of the most complicate
Among the different kinds of free radicals that were
diseases and its complexity is attributed to its
mentioned above, peroxyl radical plays a prominent role
multifaceted characteristics. Apoptosis is the cell's
in DNA damage. It has recently been demonstrated that
natural mechanism for death, but also a promising target
peroxyl radicals are able to cleave the phosphodiester
for anticancer therapy. complex I (NADH-ubiquinone
bond of DNA. The resulting strand may also be cleaved
reductase) and complex III (succinate-ferricyanide
by another peroxyl radical at 5-methyl position of
reductase) are mitochondrial enzymes that participate in
thymidine. The radical that was formed at 5th position
respiratory chain process under high membrane
can reach further reaction, forming oxidized products.
potential and in producing ROS particles. Almost all
Therefore, antioxidants should be used as protectors
forms of DNA damage are associated with ROS, causing
against oxidative DNA damage. Olmo and colleagues
cancer initiation and cancer development. However,
treated DNA with 2,2'-azobis(2-
ROS opens the mitochondrial permeability transition

5
pore, and thus interferes with mitochondrial electron Isoindoline, a rigid nitroxide spin label, is converted into
transfer chain and induce the release of Cytochrome-c. spin label analogue of nucleotides found in DNA. This
The combination of Cytochrome-c, apoptotic peptidase analogue is constructed upon cytidine group, called C-
activating factor 1 (Apaf-1), and procaspase-9 in cytosol spin. As a result, the terminal aromatic ring forms three
forms apoptosomes which activates caspase-9. The hydrogen bonds with guanidine (figure 6), and highly
latter activates effector caspases (e.g. caspase-3), that rigid conformation is shown by X-Ray crystallography
results in the cleavage of cellular proteins and apoptotic analysis.
cell death. In other words, ROS can also induce
apoptotic cell death, which is an important approach in
cancer therapeutics.[2]
Previously, it has been shown that nitroxides
such Tempol have antioxidant effect and pro-oxidation
by producing hydrogen peroxide upon superoxide
dismutation (20). Accordingly, nitroxides have been
reported to induce either anti or proapoptotic effects,
depending upon type of mammalian cells and their
Figure 6. Design of the rigid nitroxide spin label (left),
microenvironment. Complex I is the main entry point
that is paired with guanidine (right) by hydrogen bonds.
into the electron transport chain. Thus, targeted
inhibition of complex I can stop the flow of electrons
Two steps were done by Sigurdsson and co-workers for
through the transport chain, resulting bioenergetic
designing the rigid nitroxide spin label. The first step in
depletion. Tempol is reduced by ubiquinol within
the synthesis of rigid nitroxide spin label is preparing
mitochondria. The resulting hydroxylamine inhibits
Isoindoline derivative (figure 7a). Diazotization and
complex I and therefore inducing mitochondrial
hydrolysis of compound 32 converts it into phenol 33.
dysfunction that leads to apoptosis. In conclusion, ROS
Afterwards, nitration of compound 33 creates compound
and Tempol have dual function that may be used in
34. Finally, reduction of 34 forms 35 in 45% overall yield.
therapeutic manner. [14]
The second step in the synthesis of rigid nitroxide spin
label is combining isoindoline moiety with nucleoside
5. Incorporation of rigid nitroxide spin (figure 7b). A cytidine derivative (36) is activated by
label into DNA PPh3 and CCl4 in a reaction called Appel reaction . Then,
isoindoline was introduced in presence of DBU, a strong
DNA is a universal carrier of genetic information, which base that stabilize the positive charge that is formed
dictates biological functions. In nucleic acid studies, during nucleophilic addition between 35 and 36. The
SDSL has been applied for revealing DNA dynamic resulted product (37) was heated in the presence of KF to
behavior; obtaining distance constrains, monitoring afford the ring closure (38). Then, oxidation of secondary
rotational diffusive motions, and probing local and amine in isoindoline derivative was done in the presence
global dynamics. In future, site specificity in DNA may of sodium tungstate, as mentioned previously (figure 3),
play a key role in gene regulation and even repairing to form nitroxide spin label 39 in 29% overall yield. [4]
proteins by altering DNAs flexability. [15] Rigid nitroxide
spin label has several advantages over other existing
spin labels for nucleic acids. Firstly, flexible conjugation
decreases the accuracy of EPR measurements, so there
is a need for developing rigid spin labels. Secondly, the
nucleoside becomes fluorescent upon mild reducing
agents (e.g. DTT or sodium dithionite), therefore a
complementary spectroscopic technique can be used.

6
6. Incorporation of nitroxide spin label
into protein
product, i.e. imine that is substituted with imidazoline
SDSL technique can also provide a useful information
nitroxide. The benzoxazine ring, is formed through
about proteins structure, structural changes and
further reaction with formaldehyde and the resulted
characterize membrane protein dimerization or
product was investigated.
oligomerization within bilayers as well as association of
According EPR analyzing, the GAPDH/CP12
proteins on membranal surface. Protein-protein
complex did not contribute to spectral shape when
interactions can also be studied with SDSL technique,
comparing the free label spectrum. This result indicates
revealing function and biology of cell. [16] The common
that the tyrosine residue is not directly involved in the
use of this technique is based on introduction of
complexes interaction because C-terminal region
nitroxide at cysteine residue. Although it is rare to find
remains highly flexible. This observation proves the
cysteine residues in proteins, they are frequently
proposed assumption, interaction region involving
involved in crucial structural elements, especially as
central α helix of CP12 rather than C-terminus of the
binding of metal co-factors and disulfide bridges.
However, one of the main regulatory proteins of protein, where the spin label is attached. [17]
photosynthesis, chloroplast protein from green alga
(CP12), and its partner protein glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) are forming a
complex that is widely studied. CP12 contains four
cysteine residues that are involved in two disulfide
bridges, where GAPDH/CP12 complex is formed, and
only one tyrosine residue at the end of C-terminus
region. Therefore, introduction of nitroxide probe to
Figure 8. Spin labelling of CP12 at tyrosine residue
tyrosine residue rather than cysteine is required. The
tyrosine containing protein is substituted with mannich

7
7. Conclusions and future directions
ROS species have a wide scale degenerative
consequence in biological systems that is expressed in
numerous types of diseases. Therefore, the use of low
molecular weight antioxidants, and the search for more
potent species is a goal for many biochemists, polymer
scientists, nutritionists and others. Nitroxides
effectiveness as antioxidants was mentioned in this
review, and therefore have potential performance as
valuable therapeutics.
Aside from its therapeutic use, the efficiency of
nitroxide synthesis is reflected by their restricted
conformational spin labelling, and its applications in
EPR study of biomolecules. However, the strategy to
label proteins and nucleic acids is still challenged by the
balance between flexibility and biostability. Many
future developments in this field are apparent, including
use of multiple labels to enhance the accuracy of data
interpretation. As it was mentioned before, site
specificity in DNA may play a key role in gene
regulation and even repairing proteins by altering DNAs
flexibility. Furthermore, the dual spectroscopic activity
of nitroxide spin label enables the preparation of a redox
sensor that controls biological activities. In addition to
nucleic acid and proteins, which were mentioned in this
review, other biomolecules are about to be studied (e.g.
carbohydrates) in SDSL technique [4,7].
Abbreviations
CP12 chloroplast protein
EPS Electron paramagnetic resonance spectroscopy
GAPDH glyceraldehyde 3- phosphate dehydrogenase
DBU 1,8-Diazabicyclo(5.4.0)undec-7-ener
DMA N,N-Dimethylacetamide
DTT Dithiothreitol
IC50 half maximal inhibitory concentration
ROS Reactive oxygen species
SDSL Site directed spin labelling
SOD Superoxide dismutase
8. Reference
[1] Prescott C. & Bottle S. E. (2016). Biological
Relevance of Free Radicals and Nitroxides. Cell
Biochemistry and Biophysics, 75(2), 227–240.
8
[2] Aggarwal V., Tuli H. S., Varol A., Thakral F.,
Yerer M. B., Sak K., Varol M., Jain A., Khan A. &
Sethi G. (2019). Role of Reactive Oxygen Species in
Cancer Progression: Molecular Mechanisms and
Recent Advancements. Biomolecules, 9(11), 735.
[3] Bagryanskaya E. G. & Marque S. R. A.
(2014). Scavenging of Organic C-Centered Radicals
by Nitroxides. Chemical Reviews, 114(9), 5011–
5056.
[4] Barhate N., Cekan P., Massey A. P., & Sigurdsson
S. T. (2007). A Nucleoside That Contains a Rigid
Nitroxide Spin Label: A Fluorophore in Disguise.
Angewandte Chemie International Edition, 46(15),
2655–2658
[5] Hida T. & Nogusa H. (2009). Practical and versatile
oxidation of alcohol using novel Na2WO4–H2O2
system under neutral conditions. Tetrahedron,
65(1), 270–274.
[6] Tian J., Chen L., Zhang C., Yan X. & Li Y.
(2014). Continuous synthesis of triacetonamine
over sulfonic acid-functionalized mesoporous
silicas. RSC Advances, 4(34), 17860
[7] Haugland M. M., Lovett J. E. & Anderson E. A.
(2018). Advances in the synthesis of nitroxide
radicals for use in biomolecule spin labelling.
Chemical Society Reviews, 47(3), 668–680.
[8] Soule B.P., Hyodo F., Matsumoto K., Simone L.N.,
Cook J.A., Krishna M.C. & Mitchell J.B.
(2007). The chemistry and biology of nitroxide
compounds. Free Radical Biology and Medicine,
42(11), 1632–1650.
[9] Patel K., Chen Y., Dennehy K., Blau J., Connors S.,
Mendonca M., Tarpey M., Krishna M., Mitchell
J.B., Welch W.J., Wilcox C. S. (2006). Acute
antihypertensive action of nitroxides in the
spontaneously hypertensive rat. American Journal
of Physiology-Regulatory, Integrative and
Comparative Physiology, 290(1), R37–R43.
[10] Shi F., Zhang P., Mao Y., Wang C., Zheng M. &
Zhao Z. (2017). The nitroxide Tempo inhibits
hydroxyl radical production from the Fenton-like
reaction of iron(II)-citrate with hydrogen peroxide.
Biochemical and Biophysical Research
Communications, 483(1), 159–164.
[11] Krishna M. C., Samuni A., Taira J., Goldstein S.,
Mitchell J. B., & Russo A. (1996). Stimulation by
Nitroxides of Catalase-like Activity of
Hemeproteins. Journal of Biological Chemistry,
271(42), 26018–26025.
[12] Damiani E., Kalinska B., Canapa A., Canestrari S.,
Wozniak M., Olmo E. & Greci0 L. (2000). The
effects of nitroxide radicals on oxidative DNA
damage. Free Radical Biology and Medicine, 28(8),
1257–1265.
[13] Yamasaki T., Ito Y., Mito F., Kitagawa K.,
Matsuoka Y., Yamato M. & Yamada K.
(2011). Structural Concept of Nitroxide As a Lipid
Peroxidation Inhibitor. The Journal of Organic
Chemistry, 76(10), 4144–4148.
[14] Pathania, D., Millard, M., & Neamati, N.
(2009). Opportunities in discovery and delivery of
anticancer drugs targeting mitochondria and cancer
cell metabolism. Advanced Drug Delivery
Reviews, 61(14), 1250–1275.
[15] Popova A. M., Kálai T., Hideg K. & Qin P. Z.
(2009). Site-Specific DNA Structural and Dynamic
Features Revealed by Nucleotide-Independent
Nitroxide Probes. Biochemistry, 48(36), 8540–8550.
[16] FANUCCI, G., & CAFISO, D. (2006). Recent
advances and applications of site-directed spin
labeling. Current Opinion in Structural Biology,
16(5), 644–653.
[17] Lorenzi M., Puppo C., Lebrun R., Lignon S.,
Roubaud V., Martinho M., Mileo E., Tordo P.,
Marque S.R.A., Gontero B., Guigliarelli B. & Belle
V. (2011). Tyrosine-Targeted Spin Labeling and
EPR Spectroscopy: An Alternative Strategy for
Studying Structural Transitions in Proteins.
Angewandte Chemie International Edition, 50(39),
9108–9111.

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