[Retinoids].

Retinoid includes vitamins A and synthetic relatives except provitamins A such as carotenes. In this review, recent developments on the chemistry and physiological functions of retinoid (retinol, retinal and retinoic acid etc.) were surveyed, special emphasis being laid on the following subjects: (1) vision (retinal and photo-sensitivities; molecular mechanism of scotopic and photopic vision). (2) systemic actions of vitamins A (multiple physiological functions and their active intermediates). (3) reproduction and morphogenesis (extracellular-transport proteins, intracellular-cytoplasmic transfer proteins, and intracellular-nuclear transacting factors; RAR and RXR; morphogen). (4) chemical prevention of cancer (synthetic retinoid derived from retinoic acid). (5) biological significance of cis-retinoid.

Optical resolution of (+/-)-all-trans-3-hydroxyretinal by use of high-performance liquid chromatography.

Racemic all-trans-3-hydroxyretinal (3-OH-RAL) (1) was converted by a reaction with (-)-camphanic acid chloride (CpCl) into a diastereomixture of camphanates (2a) and (2b) which was separated by preparative high-performance liquid chromatography (HPLC) to give two esters (2a) and (2b) in pure state, respectively. Saponification of (2a) and (2b) independently afforded optically active (3S)- and (3R)-3-OH-RALs (3a) and (3b), respectively, whose absolute structures were determined by circular dichroism (CD) spectra. Racemic 3-OH-RAL was separated to two peaks by HPLC using chiral column (ChiraSpher, Merck). Cochromatography with authentic specimens (3a) and (3b) showed that the peak with a short retention time corresponded to (3R)-isomer and the other to (3S).

Mechanism of isomerization of rhodopsin studied by use of 11-cis-locked rhodopsin analogues excited with a picosecond laser pulse.

Primary photochemical behaviors of cattle rhodopsin analogues (Rh5 and Rh7) having cyclopenta- and cycloheptatrienylidene 11-cis-locked retinals (Ret5 and Ret7, respectively) were studied by excitation with a picosecond laser pulse (wavelength 532 nm; duration 21 ps). Picosecond absorption and fluorescence measurements of Rh5 showed formation of only a long-lived excited singlet state (tau l/e = 85 ps). The excited state of the retinal analogue having a five-membered ring was stabilized in protein (Rh5) more than in solvent (protonated Schiff base of Ret5; PSB5). Excitation of Rh7 produced two ground-state photoproducts, Rh7 (580) and Rh7 (630). According to the analysis of photon density dependency, Rh7 (580) was a single-photon product of Rh7, while Rh7 (630) was the photoproduct of Rh7 (580). Fluorescence emitted from a seven-membered ring system like Rh7 or a protonated Schiff base of Ret7 (PSB7) was weaker than that in a corresponding five-membered ring system, especially in protein (Rh7). The difference in photoreaction between Rh5 and Rh7 may originate from the difference in fixation of the 11-cis form. On the basis of the spectral and kinetic similarities between Rh7 (580) and photorhodopsin, a precursor of bathorhodopsin, it was proposed that both have twisted all-trans chromophores in the way of the isomerization. The protein moiety of rhodopsin which fixes the chromophore at both ends seems to accelerate the rotation of the C11-C12 double bond and to prevent it from going through relaxation processes other than the isomerization. This may be a plausible reason why rhodopsin has a large quantum yield (0.67).

Retinoid composition in the compound eyes of insects.

Retinoids in the compound eyes of insects in ten orders were extracted by the oxime method and analysed by HPLC. Four geometrical isomers (13-cis, 11-cis, 9-cis and all-trans) of syn and anti retinal oximes, and syn and anti 3-hydroxyretinal oximes were separated in a single analysis by a stepwise eluent condition. The amounts of the two isomers, syn 11-cis and syn all-trans, were quantified. 11-Cis 3-hydroxyretinal was detected in six orders: Lepidoptera, Diptera, Coleoptera, Neuroptera, Hemiptera and Odonata, and retinal and 3-hydroxyretinal were found together in the compound eyes of some species of Coleoptera and Odonata. We conclude that early in their phylogeny, insects had the ability to use 3-hydroxyretinal as the chromophore of visual pigment. Peaks corresponding to syn 9-cis and 13-cis 3-hydroxyretinal oximes were observed on the chromatogram of extracts from fly heads and compound eyes of cicadas. Retinol and 3-hydroxyretinol were also analysed and quantified relative to retinal and 3-hydroxyretinal. Larger amounts of the alcohols than the aldehydes were found in the compound eyes of butterflies, hornets, cicadas and grasshoppers, which are diurnal insects. 3-Dehydroretinal has not been detected in insects.

A fly, Drosophila melanogaster, forms 11-cis 3-hydroxyretinal in the dark.

All-trans and 11-cis 3-hydroxyretinals were synthesized and the presence of these substances in the head of Drosophila melanogaster was shown by using high performance liquid chromatography. Even when the head extract was prepared in the dark from the flies reared successively in the dark, both of the 3-hydroxyretinal isomers were detected. In the culture medium, they were not present. D. melanogaster must have an 11-cis 3-hydroxyretinal forming-system that does not need light.

Studies on structure and function of rhodopsin by use of cyclopentatrienylidene 11-cis-locked-rhodopsin.

The photochemical reaction of cyclopentatrienylidene 11-cis-locked-rhodopsin derived from cyclopentatrienylidene 11-cis-locked-retinal and cattle opsin was spectrophotometrically studied. The difference absorption spectrum between the cyclopentatrienylidene 11-cis-locked-rhodopsin and its retinal oxime had its maximum at 495 nm (P-495). Irradiation of P-495 at -196 degrees C with either blue light or orange light caused no spectral change, supporting the cis-trans isomerization hypothesis for formation of bathorhodopsin. Upon irradiation of P-495 at 0 degree C with orange light, however, its absorption spectrum shifted to a shorter wavelength owing to formation of a hypsochromic product. The difference absorption spectrum between this product (P-466) and its retinal oxime showed its maximum at 466 nm. Analysis of retinal isomers by high-performance liquid chromatography showed that this spectral shift was not accompanied by photoisomerization of the chromophore. P-466 could almost completely be photoconverted to the original pigment (P-495) by irradiation at 0 degree C with blue light with little formation of the other isomeric form of its chromophore. The alpha-band of the circular dichroism spectrum of P-495 was very small in comparison with that of rhodopsin, while that of P-466 was comparable to it. These facts suggest that P-495 has a planar conformation in the side chain of the chromophore and that P-466 has a twisted one, probably at the C8-C9 single bond. Cyclic-GMP phosphodiesterase in frog rod outer segment was activated by neither P-495 nor P-466. This result suggests that the isomerization of the retinylidene chromophore of rhodopsin is indispensable in the phototransduction process.

Cytotoxic action of retinoidal butenolides on mouse neuroblastoma and rat glioma cells.

Proliferation and death were measured in cultures of mouse neuroblastoma N18TG -2 and rat glioma C6BU -1 cells when treated with up to 100 micron retinoidal butenolides (RB 1-6). The number of viable cells in each case was measured with various concentrations of the compounds, of which RB-3 (5-hydroxy-4-[2-(2,6,6,-trimethyl-l- cyclohex en-l-yl) ethenyl ]-2(5H)- furanone ) was the most potent in destroying the cells after 2 days' incubation. ED50 of RB-3 was about 5 X 10(-7) M for both types of cell. RB-3 was 80 times more potent than retinoic acid. Ten analogues of RB-3 had a similar inhibitory effect on DNA synthesis in N18TG -2 cells. The degenerative changes caused by RB-3 in C6BU -l cells were irreversible even when the cells were exposed to it for 2 h. Tumor weights of N18TG -2 cells that had been inoculated subcutaneously onto the backs of A/J mice were 30-40% lower than those of untreated controls after 14 days of single daily i.p. injections of RB-3 doses of 100 mg/kg of body weight. The results indicate that RB-3 is cytotoxic in murine tumor cells originating from the nervous system and has an inhibitory effect on neuroblastoma-tumor growth in mice.

Spectral analysis and high-performance liquid chromatography of the cis-trans geometrical isomers of 5,6-Epoxyretinal.

Spectral characterization of 5,6-epoxyretinal isomers (II) prepared by a regioselective epoxidation of the parent retinal isomers have been described. High-performance liquid chromatographic separation of epoxyretinals and a photoisomerization behavior of all-trans-5,6-epoxyretinals (IIa) have been investigated.

Thermal stereoisomerization of all-(E)-beta-carotene: (Z)-beta-carotenes and electrocyclized beta-carotenes.

Thermal isomerization of synthetic all-(E)-beta-carotene yielded a multi-component mixture composed of more than sixteen (Z)-beta-carotenes and two thermo-cyclized isomers. They were separated and purified by high-performance liquid chromatography. Ten of these were characterized by spectroscopic methods as four mono-(Z)-, five di-(Z)-, and one tri-(Z)-beta-carotenes; the 7-(Z)-, 9-(Z)-, 13-(Z)-, 15-(Z)-, 9.13-(Z)-, 9.15-(Z)-, 9.13'-(Z)-, 13.15-(Z)-, 7.13'-(Z)-, and 9.9'.13-(Z)-isomers were included. Stereochemistry of six additional (Z)-isomers obtained is, as yet, unknown, though one of them might have the 9.9'-(Z)-geometry in the molecule. This is the first report on the formation and identification of the 'sterically hindered' 7-(Z)-isomers as well as the (5 leads to 10)-thermo-cyclized products in the field of alicyclic carotenoids.

Activation of phosphodiesterase in frog rod outer segment by rhodopsin analogues.

Activation of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35) in frog rod outer segment membrane by rhodopsin analogues has been investigated. A rhodopsin analogue modified at the Schiff-base linkage (N-retinyl-opsin) or the beta-ionone ring (3-dehydro-rhodopsin) in the retinylidene chromophore of rhodopsin has some ability in activation of the enzyme. In consideration of our previous observation that opsin including a retinal-oxime can activate the enzyme, it seems likely that the Schiff-base linkage is not always necessary for the phosphodiesterase activation. On the other hand, a change in the length of the side chain of retinal (complex of opsin and beta-ionone, beta-ionylideneacetaldehyde or retinylideneacetaldehyde) or dissection of the conjugate double-bond system of the side chain (retro-gamma-rhodopsin) remarkably reduces the activation ability. However, 5,8-epoxy-rhodopsin having a similar dissected conjugate double-bond system induces some enzyme activation because of its rigid conformation around C7-C8-C9 single bonds. Consequently, it is suggested that the necessary portion of rhodopsin chromophore for the activation of the enzyme is the rigid conjugate double-bond system between the beta-ionone ring and the Schiff-base linkage in its all-trans form.

Phosphorylated polyenols of biochemical interest: cis=trans stereoisomeric retinylphosphates.

All-trans biscyclohexylammonium retinylphosphate was synthesized using bis (triethylammonium) phosphate as the phosphate donor. It was shown to be pure from physico-chemical evidence including ultraviolet, infrared, 1H- and 13C-nuclear magnetic resonance and mass spectrometry. In contrast to previous reports, the synthesized product is quite stable toward alkaline hydrolysis. Direct or iodine-catalyzed photoisomerization of the all-trans compound can produce the corresponding 13-cis isomer. Simultaneous quantification of cis-trans stereoisomeric retinylphosphates was demonstrated by ion-pairing reversed-phase liquid chromatography with an isocratic mobile phase consisting of aqueous methanol and with ultraviolet detection at 300 nm. The mobile phase also contains tetrabutylammoniumphosphate as the counter-ion. The feasibility and extent of stereoisomerization of all-trans retinylphosphate have been compared with those of carotenoidal and retinoidal polyenes.

Structural elucidation of the main cis beta-carotenes.

Different isomerization experiments on all trans-beta-carotene (I) yielded multi-component mixtures of cis isomers. Separation and isolation of these isomers were achieved by classic adsorption chromatography on a lime column. Investigation of their 200 MHz 1H- and 50.3 MHz 13C-NMR spectra led to the structural elucidation of the main cis-beta-carotenes, namely the 9-monocis structures (II) for neo beta-carotene U, and the 13-monocis structure (III) for neo beta-carotene B. Formation of other hindered monocis (7-cis and 11-cis) and 15-monocis isomers should be carefully examined by a suitable method such as high-performance liquid chromatography.