Photochemistry and photophysics of cholesta-5,7,9(11)-trien-3β-ol: a fluorescent analogue of cholesterol

Abstract

7,9(11)-trien-3β-ol (9,11-dehydroprovitamin D 3 , CTL) is used as a fluorescent probe to track the presence and migration of cholesterol in vivo. CTL is known to be photochemically active, but little consideration has been given to the formation efficiency and possible toxicity of its photoproducts. In degassed tetrahydrofuran (THF) solution, we isolated the photoproduct of CTL and of its 25-hydroxy derivative (HOCTL), and X-ray crystal structures were obtained for HOCTL and the photorearrangement product. The X-ray crystal structure and its 1 H NMR spectrum confirm the product structure as a pentacyclic HOCTL isomer. In the presence of air in THF, endoperoxide formation via [2+4] addition of 1 O 2 * across the B ring of CTL or HOCTL becomes the dominant photoreaction. The UV spectrum and decay kinetics of the triplet state of HOCTL, the precursor of 1 O 2 *, are determined by transient absorption spectroscopy. We confirm the proposed structure of the endoperoxide by X-ray crystallography. Kinetics analysis of quantum yields provides rate constants for photophysical and photochemical events.

Photochemistry and Photophysics of Cholesta-5,7,9(11)-trien-3β-ol in Ethanol

“…HOCTLP, a major product from the irradiation of HOCTL in ethanol in the presence of air [ 13 ], was also purified by semi-preparative HPLC. During a purification run, we noticed the formation of small crystals in one of the fractions.…”

“…The quantum yield measurement of HORP 1 formation in ethanol was carried out in parallel with the previously described experiment [ 13 ] in THF. HOCTL contaminated with 11% Pro based on 1 H NMR and UV analysis was used.…”

“…In the presence of air, peroxide formation becomes the dominant photoreaction. Furthermore, as in THF [ 13 ], the relative yield of peroxide increases at higher CTL or HOCTL concentrations. Measurements in ethanol were carried out in parallel with those described for THF [ 13 ].…”

“…Because CTL and DHE are photochemically active, their fluorescence intensity decreases on prolonged excitation leading to photobleaching and fluorescence recovery studies [ 12 ]. Our initial studies of the photochemistry and photophysics of CTL and its 25-hydroxy derivative, HOCTL, were carried out in tetrahydrofuran (THF), an aprotic solvent [ 13 ]. The presence of the OH substituent at C 25 of CTL led to facile crystallization of photoproducts and allowed us to confirm results from early pioneering studies by the Windaus and Barton groups [ 14 , 15 , 16 ] using X-ray crystallography ( Scheme 1 ) [ 13 ].…”

“…Followed by UV, the loss of CTL or HOCTL in THF is accompanied by the appearance of a structureless peak at 261 nm due to the formation of the rearranged products RP 1 or HORP 1 [ 13 ]. In contrast, a 1930 study found that upon direct excitation of DHE in ethanol, the λ max of the photoproduct mixture is at 243 nm [ 19 ].…”