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BMPO

BMPO, BocMPO or 5-BMPO cpd is the short form available for 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide. It is novel spin trap, which is butoxylated. It molecular structure is diagrammatically depicted in Figure 1. The detailed protocol for its synthesis has already been documented in the scientific literature

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Figure 1 The Molecular Structure of 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide. Other short names include BMPO, BocMPO or 5-BMPO cpd.

In normal or we can say low concentrations Reactive Oxygen Species ROS and Reactive Nitrogen Species RNS are produced during the aerobic metabolism. They are not only the key players in the normal metabolism but also maintains the cellular homeostasis as they regulate cellular signaling and functions. In abnormal or we can say high concentrations, they cause oxidative stress and become harmful to the cell structure and function as they negatively impact all the vital biomolecules of the cell, particularly, lipids, DNA and proteins. They cause peroxidation of lipids, mutations in DNA via cleaving it and denaturation of proteins; all of these modifications are irreversible in nature. Moreover, these modifications are so intense that cell cannot repair them. Thus, high levels of ROS and RNS lead the cell to apoptosis, a type of cell death which is programmed. In short, ROS and RNS both are critical in the physiology as well as in the pathology.

Thus, generally, the detection of Reactive Oxygen Species ROS and Reactive Nitrogen Species RNS in the biological systems is very important; it is specifically important for those radicals which act as precursors for other radicals, for example, Superoxide radical anion acts as a precursor for peroxynitrite radical and hydroxyl radical. It is important as their concentration can only be effectively treated/ managed if there is some method by which they can be detected. Interestingly, the rate constant of the reaction between BMPO and superoxide, which is shown in Figure 1, is a big value which suggests that this reaction is very fast.  This is in fact very useful as we know that Superoxide radical anion is highly unstable (Mitchell et al., 2013).

The Continuous Wave (CW) Electron Paramagnetic Resonance (EPR) Spin Trapping is also known as Electron Spin Resonance (ESR) Spectroscopy (Hawkins and Davies, 2014). As is a valid, gold standard and sensitive method; it is used to identify and quantify the radicals which are intrinsically unstable due to their short life span, mostly the Reactive Oxygen Species ROS and Reactive Nitrogen Species RNS in the range of nM to μM concentration present in the biological systems. Various cyclic nitrones may be used as a spin trap in this method to identify and quantify ROS and RNS, BMPO is one these cyclic nitrones which is particularly used to quantify super oxide anion and hydroxyl radicals (Misak et al., 2020). Various unique advantages are associated with the use of BMPO as a spin trap; including all the BMPO adducts formed from thiyl radical, hydroxyl radical, glutathionyl radical and superoxide anion demonstrate a unique Electron Paramagnetic Resonance (EPR) spectrum/ spectral pattern. Moreover, these BMPO adducts are stable. BMPO superoxide adduct does not decompose into some other radical adduct like BMPO hydroxyl adduct. Last but not the least, BMPO adducts also shows a high signal to noise ratio. Owing to these benefits, using BMPO is preferred if detection of radicals is required (Zhao et al., 2001).

Electron Paramagnetic Resonance (EPR) Spin Trapping using BMOP is more useful method to detect superoxide than the cytochrome c spectrophotometric reduction assay (550 nm). This is particularly due to the fact that EPR signal monitoring is the key feature which is available in the former one (Schroeder et al., 2011).

Electron Paramagnetic Resonance (EPR) Spin Trapping follows a general principle which is irrespective of the chemical nature of the used spin trap/ cyclic nitrone and the free radical. This principle is diagrammatically depicted as a chemical equation in Figure 2, although the reaction shown in Figure 2 is a bit specific as it has superoxide anion in the reactants (Mitchell et al., 2013). This mechanism involves an addition reaction between the radical and the spin trap, that results in the formation of a spin adduct, that is stable. This spin adduct is different depending upon either the radical or spin trap, which is then detected by the Electron Paramagnetic Resonance (EPR) spectroscopy. In this method, the radicals are identified by their characteristic and unique spectrum.

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Figure 2 The mechanism of a General Reaction for the formation of Spin Trap Adduct from the spin trap as first reactant and the superoxide radical. As it is a general reaction equation, R is different for each of the spin trap; it is CO2C(CH3)3 for BMPO spin trap. The product of this addition reaction is an adduct (Mitchell et al., 2013).

While selecting an optimal spin trap in the detection of free radicals, 5,5-dimethyl-pyrroline N-oxide (DMPO) is often encountered. But it is not used in the research studies due to its limitations which are adequately compensated by 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide BMPO. These are already mentioned in the advantages of using BMPO.

BMPO is solid at room temperature, which makes it very easy to handle. It is soluble in water, and it exists as a mixture of two optical isomers, i.e., S-BMPO and R-BMPO, in the aqueous solution. It is important to note that the signal recorded as EPR spectrum is the sum of two individual signals by diastereoisomers (Misak et al., 2020). All the products of these reactions which are shown in Figure 3, are physically paramagnetic, while chemically they are nitroxide radicals and very stable. The half life of BMPO-OH is 30 minutes.

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Figure 3 The reaction of either of the optical isomers, i.e., S and R with superoxide anion and hydroxyl radicals. They are different with respect to the orientation of tert-butoxycarbonyl group. The resultant products or adducts are diastereoisomeric in nature, with respect to the orientation of tert-butoxycarbonyl group (Misak et al., 2020)

BMPO is also widely used in the research for the detection of free radicals in the non biological systems as well. In in vitro research study, BMPO is used as spin trapping agent to trap two distinct free radicals, i.e., oxygen superoxide anion and hydroxyl radical. Although, this research was done to indicate the enhancement in the x-ray imaging (Chang et al., 2016).

In another in vitro research study, BMPO is used as spin trapping agent to trap two distinct free radicals, i.e., hydroperoxyl and hydroxyl radicals. This research study was aimed to detect these free radicals as the products of the iodide−peroxide system. The system composition, the detected products and their respect EPR spectrum is diagrammatically depicted in Figure 4. By this study, this system was known to produce free radicals. Later on, this system was recognized as the easiest way to produce free radicals in the laboratory environment.

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Figure 4 The use of BMPO in the detection of free radicals

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Figure 5 The chemical reaction, i.e., addition, between BMPO and Hydroperoxyl Radical. In this addition reaction, hydroperoxyl radical binds to the BMPO at the nitroxide ring, which leads to the formation of adduct shown in C

 



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