TEMPO nitroxides spin labeling

Figure 26 also depicts the distances calculated from fluorescence quenching experiments by enzyme-bound Co2+ on the s-adenosine moiety of s-ATP adenylylated glutamine synthetase (e-AMP-GS). Additional data were gathered by an EPR method that measured the decrease in EPR amplitude of the nitroxide spin-labeled adenylylated enzyme (TEMPO-AMP-GS) owing to enzyme-bound Mn2+ (122). Distances between Mn2+ and the N—O of the spin label are also shown in Fig. 26. 

One technique, Overhauser Dynamic Nuclear Polarization (ODNP), is based on the well-known chemical shift of water in NMR spectra. Ordinarily, the liquid water signal intensity is low however, intensity can be magnified 1000-fold by addition of a nitroxide spin label such as TEMPO. Precession of the unpaired electron in TEMPO at the Larmor frequency results in Nuclear Overhauser-mediated polarization of the protons in water. These get polarized within 15 A of the spin labels and then relax with a relaxation time determined by the local diffusivity, i.e. in bulk water, the diffusivity is high and so relaxation is rapid by contrast, in hydration layers, relaxation takes 10-fold longer than in bulk water. Next, the trick is to covalently tether spin labels to surfaces of interest and measure how relaxation rates in hydration layers change as adhesive proteins approach and locally dehydrate the surfaces. 

Let us consider the application of Equation (S) to the one-electron reduction of p-benzoquinone and to the oxidation of nitroxide spin label Tempo. 

The starting point for every kind of EPR study is the choice of the radical that is best suited to deliver valuable information on the systems that are investigated. In this chapter only three types of radicals are used for this purpose. Nitroxide radicals such as TEMPO (2,2,6,6-tetramethylpiperidine-l-oxyl Pig. 2), its derivatives, and its five-ring analogs (Fig. 2) are the by far most widely used spin probes and spin labels and they are the spin probes of choice for the majority of EPR spectroscopic applications in naturally diamagnetic systems [2, 19]. 

Spin Labels. - Spin labels have traditionally been a tool of ESR spectroscopy, where the ESR resonance of the free radical label (usually a nitroxide-containing species, such as TEMPO) is observed directly. However, spin labels have increasingly become a tool of NMR as T1 relaxation enhancing agents. 

The fluorescence-photochrome technique was first applied to study the molecular dynamics of a stilbene fiuorescence-photochrome molecule, SITC, attached covalently to the terminal amino group of sperm whale myoglobin [18]. The same myoglobin residue was also labeled with a spin label, 4-iodoacetamide-TEMPO. The kinetics of the stilbene trans-cis photoisomerization (k pp) and the rotational diffusion frequency of nitroxide radicals (v ) was monitored by fiuorescence and E S R... 

A spin label is a stable paramagnetic molecule that contains an atom or group of atoms with an unpaired electron spin that can be bonded to another molecule. In this way, one can detect molecules that otherwise would not give an ESR spectrum. Most spin labels are nitroxide compounds. For example, 2,2,6,6-tetramethylpiperidine-A-oxyl (known as TEMPO) is a common spin label with a characteristic three-line ESR spectrum, seen in Figure 3.87. 

Nitroxide radical spin labels can be used to measure inter-spin distances by pulsed electron paramagnetic resonance (EPR) spectroscopy, and to allow study of structure and folding of DNA and RNA ohgonucleotides [27-30]. Spin-labeling of nucleobases by (TuAAC in solution has first been demonstrated using the radical azide 4-azido-2,2,6,6-tetramethylpiperidine 1-oxyl (4-azido-TEMPO) [31], Alkyne modifications at position 7 of 7-deazapurines and C5-modified pyrimidines allowed attachment of the spin label in the major groove of duplex DNA [31]. In the same year, click chemistry on solid support to introduce spin labels into DNA was reported [32]. 

The in vivo distribution of a new, polymer-nitroxide probe, spin-labelled dextran (termed TEMPO-DX) was investigated by Saito et al, who also compared its distribution and pharmacokinetics to that of 3-carbamoyl-proxyl. As expected, it was found that the half-life of the larger molecule was considerably longer than that of 3-carbamoyl-proxyl, and that the TEMPO-DX concentrated in the tumour of a mouse with an Ehrlich s ascites carcinoma. 

Electron spin resonance (ESR), often called electron paramagnetic resonance (EPR), requires the presence of unpaired electrons in the sample [5, 15, 16, 135]. Since most organic molecules do not carry free radieals, the most common method is to simply dissolve stable free radieals as spin probes in the sample. Alternatively, free radical groups can be ehemically bonded to the molecule of interest providing a spin label. Probably the most frequently used spin probes are nitroxide free radicals like 2,2,5,5-tetra-methyl-piperidinoxide (TEMPO). 

Coordination causes electron-spin density redistribution in the N-O fragment the contribution of resonance structure II increase. The redistribution of spin density results in changes in the parallel component of the nitrogen hyperfine tensor. TEMPO and anthraquinone (AQ) have been used in this way to probe the Lewis acidity of alumina and Li and Mg doped alumina matrices.176 The differences in the Lewis acidic strength towards TEMPO and anthraquinone are discussed. An interesting study has appeared aimed to study the guest-host interaction between poly(amidoamine) dendrimers labelled with nitroxides and several porous solids including alumina.177... 

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