Nitroxides, spin-labeled derivatives

The u.v.-visible spectrum of the 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl-methyl-cobinamide is very similar to methyl-cobin-amide itself and as a result this technique cannot be used to rigorously identify the spin labeled derivative. The spin labeled compound does show a spectral change with pH between pH 7.0 and pH 2.0 which methyl-cobinamide does not exhibit. Despite the similarities between methyl-cobinamide and nitroxylmethylcobinamide, the circular dichroism spectrum of the two derivatives are quite different. Fig. 23 shows the marked difference in C. D. spectra of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, methylcobinamide, and a methylcobinamide solution containing an equimolar amount of uncoordinated nitroxide. 

One other aspect of the photolysis of coordinate spin labeled derivatives is of interest. Nitroxides are good free radical scavengers (123). As a result, when methyl-cobalamin is photolyzed in the presence of a nitroxide, the methyl radical generated will react with the free nitroxide and cause disappearance of the ESR spectrum (123). However, once the nitroxide is coordinated it is no longer susceptible to attack by free radicals. Thus the nitroxyl function is quite well protected from approach by other species. 

The distance measured by EPR is the point dipole distance between the two paramagnetic centres. When applied to structures of spin-labelled biomolecules the desired distance is between sites on the biomolecules. A key question is the conformation and conformational flexibility of the spin label. X-ray crystal structures of four spin-labelled derivatives of T4 lysozyme have been reported.54 Preferred rotational conformations of the linkage between the cysteine introduced by site-directed mutagenesis and the spin label were observed. The electron density associated with the nitroxide ring in different mutants was inversely correlated with the mobility of the label observed in fluid solution EPR spectra. 

Since malonate (Structure I) is able to fulfill the role of the anion in transferrin, it seemed reasonable to see whether spin-labeled derivatives of malonate could serve as probes of the active sites. Two such spin-labled derivatives were prepared and tentatively identified as having structures II (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonamide) and III (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonate). Similar results were obtained with each (Figure 3). Upon mixing Fe(III), transferrin, and II at low pH, and then raising the pH to near-neutrality with C02-free ammonia, the characteristic orange-red color of the ternary Fe-transferrin-anion complex is promptly displayed. However, the anticipated EPR signal of the nitroxide spin-label is not observed, presumably because it is broadened beyond detectability by its proximity... 

A review of the synthesis and chemistry of nitroxide spin labels includes a number of steroid derivatives. Novel spin-labelled steroids have been prepared by esterification with the nitroxyl carboxylic acid derivative (17),for use in spin immunoassays (SIA) as an alternative to radioactive labelling. The prednisolone ester (18), for example, exhibits an e.s.r. spectrum with narrow lines when it is in a free state in solution, but when bound to antibody the rate of tumbling is reduced, and linewidths are broad. Signals from bound and unbound derivatives are easily distinguished and measured, so SIA of antibody-bound prednisolone provides a potentially useful serum assay method. 

Platinum - NMR spectroscopy has been used to derive long-range electron-proton distance in a DNA duplex containing a nitroxide spin-labelled platinum... 

Nitroxide lipid spin label derivatives 14-SASL, stearic acid bearing the spin label group on the 14-C atom. 14-PCSL phosphatidylcholine bearing the spin label on the 14-C atom of the sn-2 chain. 14-CLSL cardiolipin (diphos-phatidylglycerol) bearing the spin label on the 14-C atom of a single chain. 

Structural and dynamic information derived from singly-attached nitroxide spin labels... 

Another chemically more interesting spin labeled B12 derivative involves coordinate attachment of the nitroxyl function to the cobalt atom of a cobinamide. Fig. 22 shows a reaction in which an alkyl cobin-amide is mixed with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl. The nitroxide displaces water from the 6th coordination position very slowly and therefore this reaction is usually allowed to proceed for a few days with a large excess of nitroxide. From the properties of the coordinated nitroxide derivative discussed below, it is certain that the cobalt is coordinated by the N—O functional group. An analogous compound to that shown in Fig. 22 can be made with a similar nitroxide in which the 4-hydroxyl-group is missing. In this case the N—O-function is the only basic site on the molecule and therefore must be the position of attachment to the cobalt 119). 

Spin labeled 5 -deoxyadenosylcobinamide has been used as a cofactor for ethanolamine-ammonia-lyase and the ESR spectrum followed during catalysis (123). This spin labeled coenzyme is biologically active in this enzyme. Enzyme kinetics showed this derivative to have the same Vmax as the cofactor 5 -deoxyadenosylcobinamide, but it has a higher Km value of 5.1 X 10-6 M compared to 4.6 X 10-6 for 5 -deoxyadenosylcobinamide (123). When the spin labeled coenzyme was incubated with apoenzyme to give the enzyme-coenzyme complex, the nitroxide ESR spectrum resembled that of free spin label but the lines are broadened significantly. 

Oxidation of 2,2,4,4-tetraalkyloxazolidines gives stable nitroxide radicals (265) which are used as spin labels for probing biomolecular structures (69ACR17, B-76MI41800). The stearic acid derivative 12-doxylstearic acid (266) is commercially available. 

Fluorescein-labelled oestradiol has been used as a probe for anti-oestradiol antibody. The probe material was prepared by condensing the 6-carboxy-methyloximino-derivative of oestradiol with fluorescein amine . It had a fluorescence emission spectrum similar to that of fluorescein, and permitted a sensitive immunoassay of oestradiol. A system suitable for spin-immunoassay of steroids, using nitroxides for spin-labelling, is described on p. 264. 

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