The present invention relates to the synthesis of conjugated polyene sterol derivatives, the compounds obtained and to their use as fluorescent probes for cellular membranes.
A membrane probe is essentially a research tool used to probe (investigate) the environment of a membrane. Membrane probes find application, for example, in determining the effect of environmental pollutants and dietary constituents on cell membranes. Cell membranes are very selective and will permit only selected molecules to permeate the membrane. As well, the interactions between the membrane and its environment (i.e. the molecules in the environment) are very selective. Membrane probes are useful in the investigation of the structure and function of membrane constituents such as proteins and enzymes. Cholesterol is a major membrane constituent. The molecular structure of the probes of the present invention is very similar to cholesterol and thus the probes behave in a manner similar to cholesterol.
The probes of the present invention are fluorescent, so their behaviour in the membrane environment and their effect on the membrane can be easily determined by simply shining light on the membrane and observing the light emitted by the probe. Because of the very great sensitivity of fluorescence techniques, only very small amounts of extrinsic probe material need be incorporated into the sample of interest. This contrasts with other spectroscopic techniques such as nuclear magnetic resonance, absorption and electron spin resonance spectroscopy, which require significantly greater amounts of probe substance, with much greater risk of altering the system being investigated. Techniques using radio-isotopes have a high degree of sensitivity but by their nature are more hazardous and the radioisotopic probe materials have limited shelf life.
By studying the behiavour of the probe of the invention more can be learned about the behaviour of cholesterol. The probes of the present invention have potential for use in determining cholesterol levels and cholesterol properties in membranes and cell membrane properties and can be applied to clinical assays and diagnoses involving cholesterol.
Aromatic olefins, particularly diphenylhexatriene (DPH), have been used extensively as fluorescent probes of membrane fluidity, (see for example, L. A. Chen, R. E. Dale, S. Roth and L. Brand, J. Biol. Chem., 1977, 252, 2163). Such molecules, however, are not natural membrane constituents and can provide only indirect insight into protein-lipid and lipid-lipid interactions.
Cholesterol is an important lipid component, but its role in influencing membrane structure and dynamics is poorly understood. Some cholesterol-type molecules in which the C-3 alcohol function has been modified have been synthesized. (See for example, R. R. Rando, F. W. Bangerter and M. R. Alecio, Biochim. Biophys. Acta, 1981, 684, 12.) For example, the compounds ##STR1##
Apart from a loss of the amphipathic nature of the cholesterol moiety in some of these derivatives (e.g. 1(a)), in all cases the C-3 hydroxy function has been drastically altered. Other membrane probes have been synthesized in which the C-3 hydroxy group is intact. (See for example, R. Bergeron and J. Scott, Anal. Biochem., 1982, 119, 12H; F. Schroeder, FEBS Lett., 1981, 1335, 127.) For example, the compounds ##STR2## However, the unsaturation in the ring systems 2(a) and 2(b) drastically changes the molecular geometry. Such probes would be expected to pack differently from cholesterol in membranes and the lipid-probe interactions would be different from those of lipid-cholesterol.
Fluorescent chromophores in the C-17 side-chain of cholesterol have been reported. (See for example, Y. J. Kao, A. K. Soutar, K. -Y. Hong, H. J. Pownall and L. C. Smith, Biochemistry, 1978, 17, 2689.) For example, the compounds ##STR3##
In compounds of structures 3(a), 3(b) and 3(c) the hydrophobic character of the C-17 side-chain is much different from that of cholesterol. The membrane probes of the present invention, which have an olefinic fluorescent chromophore in the C-17 side-chain, resemble cholesterol more closely both in geometry and in amphipathic nature.