Exploiting the role of TEMPO as radical scavenger

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1. Introduction 
1.1 Aim of the thesis 
 
Since 1960, TEMPO radical molecule has been attracting more and more attention of 
scientific community, in particular for its antioxidant behaviour which is directly 
correlated to its ability of scavenging reactive radicals, with a consequent effect on 
reducing oxidative stress and reactive oxygen species in biological mediums. 
The aim of this work thesis is to obtain TEMPO grafted polymers, PEI, PEG, hydrogels 
and nanogels, using different methods of functionalization, to reach different degrees 
of grafting, to be tested as antioxidants. 
The purpose is to synthetize and characterize PEG and PEI grafted with TEMPO, and 
to use them as building blocks respectively of hydrogels and nanogels. These materials 
are expected to exploit anti-inflammatory, cytoprotective and antioxidant properties. 
Resulting hydrogels and nanogels find different pharmaceutical applications, in 
particular they are largely used for tissue repairing and drug delivery, due to their 
biocompatibility, flexibility and ability to permit a local drug release. 
On the other hand, TEMPO grafted branched PEI could be also as non-viral vector for 
gene delivery, to form complexes with DNA and convey the genetic material inside 
the target cells. 
This work can be divided into three parts: 
• Synthesis of a selected library of TEMPO-derivatives bearing different functional 
groups for further grafting on polymers; 
• Synthesis of a selected library of TEMPO-tagged PEG and PEI (both linear and 
branched) derivatives with different grafting degree; 
• Synthesis of TEMPO-labelled hydrogels and nanogels. 
All the sections are linked together: the results coming from each unit are essential for 
the following steps of the work.
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1.2 Nitroxides 
 
Nitroxides, also named nitroxyl radicals, constitute a family of free stable radical 
species with different properties and reactivity (Figure 1). The term stable for the 
nitroxides implies that the nitroxide compounds can be safely handled and stored 
under ambient conditions, without precautions of temperature, oxygen or moisture 
exposition. With their unpaired electron spin they display a unique reactivity towards 
various environmental factors, enabling applications ranging across the life and 
physical sciences. The two most exploited properties are paramagnetism and redox 
activity, which are typically found in inorganic materials, especially in metals. Metal-
based materials, which often carry significant health, safety and environmental 
concerns, can be replaced with organic-based materials thanks to the analogous 
properties. [1] 
 
 
Figure 1: Examples of nitroxides 
It’s important to distinguish between persistence, stability and reactivity, which are 
terms frequently used to describe free radicals. Lifetime or persistence of a radical is 
due to kinetic stability: these radicals have an extended lifetime due to steric hindrance 
around the radical centre, which makes the radical physically difficult to react with 
other molecules (radicals, solvent or other substrates). On the other hand, stable 
radicals are those for which delocalization of free-electron density leads to a 
thermodynamic stabilization that significantly decreases radical reactivity. Lastly, 
reactive radicals have only a brief existence. They don’t have sufficient kinetic or 
thermodynamic stability to avoid reactions and prevent them from reacting 
immediately with radicals or substrates present in the reaction mixture.
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Nitroxides stability is the consequence of the stable electronic configuration of the 
nitrogen-oxygen group, rather than to the steric or electronic influences of the groups 
attached to the nitrogen. This property results from the presence of a three electron π 
bond in the aminoxyl group (N–O•). The electronic structure of the N–O• group is 
represented by the contribution of two resonance forms (A) and (B) (Figure 2). 
 
 
Figure 2: Representation of two resonance structures of nitroxides [2] 
The lifetime of nitroxides is diminished by their ability to react with themselves by 
disproportionation: the nitroxides are in fact persistent only if the groups attached on 
nitrogen atom do not allow the radical to react with itself. Typically, nitroxides having 
one or more hydrogen atoms on the α-carbons (Cα) of the nitrogen atom are not stable 
due to a disproportionation reaction that leads to the formation of the corresponding 
nitrones and hydroxylamine’s (Figure 3). 
 
 
Figure 3: Disproportionation reaction of a nitroxide with α-hydrogens 
When there is no α-hydrogen atom to be abstracted, disproportionation is not possible, 
and so the nitroxides are effectively as stable as any diamagnetic organic material. The 
dimerization of nitroxides is not thermodynamically favoured because it does not 
correspond to a gain in bonding electrons. [2] 
Cyclic hindered nitroxides, also known as aminoxyls or nitroxyls, are stable free 
radicals. They have also a long lifetime thanks to the presence of the methyl groups at 
the α position in ring structures. These methyl groups confer persistency to the
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radicals, preventing disproportionation and limiting access to reactive substances, 
which can quench the radical species. One of the most prominent cyclic nitroxides is 
(2,2,6,6-tetramethylpiperidin-1-yl)oxyl, also known as TEMPO. 
Thanks to the unusual radical stability and to the possibility to conserve the radical 
properties during reactions, nitroxides find a wide range of applications related to the 
fields of organic chemistry, physical-chemistry, material science, biology and 
medicine, used as synthetic tools, such as catalysts or building blocks, as imaging 
agents and probes, for medicinal antioxidant applications and in energy storage. [1] 
 
1.3 TEMPO and derivatives 
 
One of the most popular nitroxyl radicals is 2,2,6,6-tetramethylpiperidin-1-oxyl, 
commonly known as TEMPO, a stable radical, which has a lot of applications in 
organic synthesis since it was first synthesised by Lebelev and Kazarnowskii in 1960. 
It is a heterocyclic compound, made by a piperidine ring with four methyl groups. 
Depending on the applications, TEMPO molecule could be modified with different 
functional groups. Some of the most used TEMPO derivatives are shown in Figure 4. 
 
 
 
Figure 4: TEMPO and some derivatives [3]
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The stability of TEMPO and its derivatives is due to the delocalization of one unpaired 
electron over the nitrogen oxygen bond (thermodynamic stabilization), but also to an 
inability to disproportionate to the corresponding nitrone and hydroxylamine because 
of a lack of α hydrogen atoms and an inability to recombine with itself because of steric 
hindrance of four methyl groups, that confer an additional stability (kinetic 
stabilization) [3].  
 
1.4 Applications of TEMPO 
 
TEMPO radical and its derivatives are extensively used as oxidation catalysts in 
organic synthesis, mediators for controlled polymerization, spin probes for 
biochemical research, magnetic resonance imaging (MRI) contrast agents, dynamic 
nuclear polarization (DNP) agents for NMR spectroscopy, building blocks for the 
preparation of organic magnets, and antioxidants in biological systems [3]. 
 
1.4.1  Paramagnetic spin label 
 
Thanks to the presence of an unpaired electron, TEMPO and derivatives can be 
detected via electron paramagnetic resonance (EPR) and magnetic resonance imaging 
(MRI), non-destructive investigative techniques for soft matter research. TEMPO can 
be used as a spin label contrast agent as it influences the time of relaxation of protons 
in physiological water, to produce clearer images of the organ studied. TEMPO has to 
be properly modified to enable its incorporation into a larger framework (a polymer, 
a surface, or a biomolecule such as a protein, nucleic acid, sugar or lipid), and thus to 
be used as a probe. Spin labelling is used to monitor physical, biophysical or 
biochemical properties of these frameworks, being sensitive to the physical 
surrounding. Such spin probes are ideally suited to investigate the dynamic aspects of 
molecular interactions. [4-5] 
In general, spin labels should ideally fulfil several criteria: i) the framework of the label 
must stabilise the radical against redox processes, ii) the radical must possess desirable
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properties for the magnetic resonance experiment (such as chemical stability and 
persistence), and iii) the label must be readily attached without structural distortion of 
the system under study. [4] In addition, spin labels don’t have to provoke health 
damages. The most commonly used compounds for contrast enhancement are 
gadolinium-based systems, but their safety has been questioned due to acute adverse 
effects (neurological and non-neurological, involving also nephrogenic systemic 
fibrosis and gadolinium deposition disease) and the toxicity during the retention 
phase of the dechelated Gd
3+
 in the biological environment. The use of TEMPO as 
magnetic contrast agent, which presents a higher biocompatibility, a prolonged half-
life and degradation properties, could be considered as a leading strategy to avoid the 
use of heavy metals. 
Many examples of TEMPO applications as spin label can be found in literature. In 
particular, Mauri et al. developed a microwave-based method for the fabrication of an 
MRI traceable TEMPO – labelled hydrogel that can be imaged in vivo throughout the 
whole time period of a longitudinal study, without using toxic compounds that could 
be hostile for the target tissue (Figure 5). [6] 
 
 
Figure 5: MR longitudinal and axial images of: (A and C) CTRL gel without TEMPO 
conjugation; (B and D) gel with linked TEMPO for 6 h after injection [6]
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1.4.2  Antioxidant effect 
 
Generation of reactive oxygen-derived species (ROS) occurs continuously in biological 
systems as a result of both endogenous and exogenous factors, in a variety of normal 
metabolic processes. 
 
  
Figure 6: Examples of ROS [7] 
Living cells require energy to survive, produced by oxidation of molecules which are 
present to store and transfer energy when necessary, such as carbohydrates, lipids, 
proteins, enzyme co-factors, etc. The source of oxidative power is oxygen: the 
reduction of oxygen into water occurs via the rapid addition of four electrons by a 
single enzyme and four protons. If, for any reason, the addition of these electrons does 
not occur in the appropriate manner, partially reduced oxygen-based species can be 
produced. These species and their derivatives are generally described as “reactive 
oxygen species” (ROS) (Figure 6). When present in very small amounts, they are 
important to maintain specific physiological functions. They are part of the immune 
defense mechanism against microbial infections, but their action must be very selective 
since ROS can also destroy healthy cells. The problem is present when ROS are 
produced in amounts larger than those expected . Their presence in excess can result 
in significant damage to cellular components, called oxidative stress (OS). [8] 
OS refers to the imbalance due to excess ROS or oxidants over the capability of the cell 
to mount an effective antioxidant response. Oxidative stress results in macromolecular 
damage and is implicated in various disease states such as the deposition of arterial 
plaques in atherosclerosis, diabetes, cancer, neurodegeneration, chronic inflammation,
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renal failure and aging. As a result, all aerobic organisms utilize a series of antioxidant 
defenses for protection against oxidative damage, including antioxidant compounds, 
metal chelators, enzymes and proteins. [9] 
Antioxidants can scavenge ROS or inhibit their production. Cells naturally contain an 
array of antioxidants, including nicotinamide adenine dinucleotide (NADH) and its 
phosphorylated derivative (NADPH), the tripeptide glutathione (L-γ-glutamyl-
Lcysteinylglycine, ‘GSH’), vitamins A (retinol), and especially C (L-ascorbic 
acid/ascorbate) and E (α-tocopherol), β-carotene and related terpenes [8]. However, 
most of them, appear not to be suitable drug candidates, because they show poor 
solubility and inability to cross membrane barriers.  
In recent years, nitroxides have been extensively utilised in biological studies, thanks 
to their anti-inflammatory, cytoprotective and antioxidant properties. They can 
degrade superoxide and peroxide, inhibit Fenton reactions, and undergo radical–
radical recombination, especially for carbon centred radicals. [10] By modifying 
oxidative stress and altering the redox status of tissues, nitroxides have been found to 
interact with and alter many metabolic processes. These interactions can be exploited 
for therapeutic and research use, including protection against ionizing radiation, 
cancer prevention and treatment, control of hypertension, and protection from 
damage resulting from ischemia/reperfusion injury. Many applications have been well 
studied and some are currently being tested in clinical trials. [11] 
 
 
Figure 7: Proposed Mechanism for the Radical-Trapping Antioxidants 
activity of TEMPO [12]