Spin labeled steroid

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. 

Distances between the nitroxide of bound steroid and several assigned protons in or near the active site could be determined on the basis of the paramagnetic effects of the nitroxide on the relaxation rates of the active site protons. These distances could then be used to dock the structure of the spin-labeled steroid into a partially refined 2.5-A resolution X-ray structure of the isomerase 128). However, in order to rationalize the chemical evidence that Asp-38 is involved in the catalytic mechanism, it was necessary to assume that steroid substrates and the spin-labeled steroid (16) are bound not only with reversal of the C-3 and C-17 positions, but also with reversal of the planes of the steroid ring systems ( up-sidedown binding), positioning Asp-38 above the C-4j8 and C-6j3 protons. 

It is clear from a variety of spectroscopic techniques that biomembranes are dynamic not static structures. It is also known that certain membrane functions depend critically on the fluidity of the membrane lipids. Spin-labelled and fluorescent-labelled lipid probes are found to perform rotational motions in the nanosecond timescale in fluid lipid bilayers and membranes. For diffusive rotation the characteristic correlation times are given by the Debye equation (t = n V/kT) and correspond to effective viscosities in the range q 0.1-1 poise. A spin-labelled steroid analogue of cholesterol for instance rotates rapidly about its long... 

Another example comes from the work of Johnson, et a/.18 These workers studied spin labels dissolved in lipid bilayer dispersions of dipalmitoylphos-phatidylcholine and cholesterol (9 1 by weight) in the hope that anisotropic rotational diffusion of the spin label would mimic the motion of the bilayer components. In addition to 5-DS, which is sensitive to rotational motion about the NO bond, they used the steroidal nitroxide 8, which tends to rotate about an axis perpendicular to the N-O bond. ESR measurements were carried out at both 9 and 35 GHz and at temperatures ranging from 30 to 30 °C. Rather different results were obtained with the two spin labels, largely as a result of the different axes of rotation. Because the rotation rates were very slow, ESR spectra appeared as powder patterns rather than isotropic spectra and special methods were needed to extract the motional data. 

In another variant on the use of paramagnetic species to probe protein structure, Zhao et al.2m determined the secondary structural features of A8-3-ketosteroid isomerase using 3D NMR techniques on the l5N,l3C-labelled protein. This enzyme catalyses the conversion of A5- to A4-3-ketosteroids and is a homodimer of 125 amino acids per subunit. The NMR studies were undertaken on the steroid-bound protein. The amino acids near to the steroid were confirmed by binding a steroid incorporating a spin label and monitoring the disappearance from the 15N- H HSQC spectrum of the cross-peaks associated with these residues. 

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. 

The nature of the catalytic base involved in the intramolecular proton transfer between the C-4j8 and C-6/8 positions of substrate has not been unequivocally identified. However, the carboxyl group of Asp-38 is a reasonable candidate. Photodecarboxylation of this residue, in the presence of 19-nortestosterone, to give Ala-38 results in complete loss of enzyme activity (123-125). Moreover, mutagenesis of Asp-38 to Asn-38 leads to a 10 -fold decrease in ifccat (126). Kuliopulos et al. have formulated a tentative model of the productive enzyme-substrate complex in which Asp-38 is proposed to be the catalytic base above the ]8 face of bound steroid (127) (Fig. 5). Their model building studies are based on the finding that the spin-labeled substrate analog, spiro[doxyl-2,3 -5 a-androstan]-17 /3-ol (16), binds at the active site of the isomerase. 

The nitroxide (467), a free-radical species with potential value as a spin label, has been synthesized in three steps (Me2S04. CH2=CH—CH2MgBr, and m-chloroperbenzoic acid) from the lactam (468) The formation of some novel steroids with fused heterocyclic rings is described on pp. 326, 346,351,355, and 359 and a novel method for the introduction of thio- and nitrogen-containing substituents at C-5 on p. 357. 

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