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The crystal structures of the unbound carotenoids, synthetic astaxanthin (3S,3'S:3R,3'S:3R,3'R in a 1:2:1 ratio), canthaxanthin and (3R,3'S, meso)-zeaxanthin are compared with each other and the protein bound astaxanthin molecule in the carotenoprotein, beta-crustacyanin. Three new crystal forms of astaxanthin have been obtained, using different crystallization conditions, comprising a chloroform solvate, a pyridine solvate and an unsolvated form. In each structure, the astaxanthin molecules, which are similar to one another, are centrosymmetric and adopt the 6-s-cis conformation; the end rings are bent out of the plane of the polyene chain by angles of -42.6 (5), -48.9 (5) and -50.4 (3) degrees , respectively, and are disordered, showing the presence of both R and S configurations (in a 1:1 ratio). In the crystal packing of the chloroform and pyridine solvates, the astaxanthin molecules show pair-wise end-to-end intermolecular hydrogen bonding of the adjacent 3-hydroxyl and 4-keto oxygens, whereas in the unsolvated crystal form, the hydrogen-bonding interaction is intermolecular. In addition, there are intermolecular C-H hydrogen bonds in all three structures. The canthaxanthin structure, measured at 100 and 293 K, also adopts the 6-s-cis conformation, but with disorder of one end ring only. The rotation of the end rings out of the plane of the polyene chains (ca -50 degrees for each structure) is similar to that of astaxanthin. A number of possible C-H hydrogen bonds to the keto O atoms are also observed. (3R,3'S, meso)-zeaxanthin is centrosymmetric with a C5-C6-C7-C8 torsion angle of -74.9 (3) degrees ; the molecules show pair-wise hydrogen bonding between the hydroxyl O atoms. In addition, for all the crystal structures the polyene chains were arranged one above the other, with intermolecular distances of 3.61-3.79 A, indicating the presence of pi-stacking interactions. Overall, these six crystal structures provide an ensemble of experimentally derived results that allow several key parameters, thought to influence colour tuning of the bathochromic shift of astaxanthin in crustacyanin, to be varied. The fact that the colour of each of the six crystals remains red, rather than turning blue, is therefore especially significant.

The principal (>96% of total) carotenoid in the novel, extremely halophilic eubacterium Salinibacter ruber, here called salinixanthin (1), has been assigned the structure (all-E,2'S)-2'-hydroxy-1'-[6-O-(13-methyltetradecanoyl)-beta-D-glycopyranosyloxy]-3',4'-didehydro-1',2'-dihydro-beta,psi-caroten-4-one by spectrometric (vis, EIMS, (1)HNMR, CD, GCMS) and chemical methods.

In previous work we reconstituted salinixanthin, the C40-carotenoid acyl glycoside that serves as a light-harvesting antenna to light-driven proton pump xanthorhodopsin, into a different protein, gloeobacter rhodopsin expressed in E. coli, and demonstrated that it transfers energy to the retinal chromophore (Imasheva et al. 2009. Biochemistry 48, 10948). The key to binding of salinixanthin was the accommodation of its ring near the retinal β-ionone ring. Here we examine two questions: do any of the native Gloeobacter carotenoids bind to gloeobacter rhodopsin, and does the 4-keto group of the ring play a role in binding. There is no salinixanthin in Gloeobacter violaceous, but a simpler carotenoid, echinenone, also with a 4-keto group that lacks the acyl glycoside, is present in addition to β-carotene and oscillol. We show that β-carotene does not bind to gloeobacter rhodopsin, but its 4-keto derivative, echinenone, does and functions as a light-harvesting antenna. This indicates that the 4-keto group is critical for the carotenoid binding. Further evidence for this is that salinixanthol, an analogue of salinixanthin in which the 4-keto group is reduced to hydroxyl, does not bind and is not engaged in energy transfer. According to the crystal structure of xanthorhodopsin, the ring of salinixanthin in the binding site is turned out of the plane of the polyene conjugated chain. Similar conformation is expected for echinenone in the gloeobacter rhodopsin. We suggest that the 4-keto group in salinixanthin and echinenone allows for the twisted conformation of the ring around C6-C7 bond and probably is engaged in interaction that locks the carotenoid in the binding site.

Aplanospores of Haematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of /B,-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3'S)-astaxanthin diester (34%), (3S,3'S)-astaxanthin monoester (46%), (3S,3'S)-astaxanthin (1%) and (3R,3'R,6'R)-lutein (6%).The astaxanthin esters were examined by TLC and HPLC and VIS, H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Milller, 1979) after anaerobic hydrolysis.The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.

Concentrations of all-E-, 9Z- and 13Z- geometrical and (3R,3'R), (3R, 3'S) and (3S,3'S) optical isomers of astaxanthin were determined in rainbow trout liver, gut tissues, kidney, skin and blood plasma to evaluate their body distribution. Two cold-pelleted diets containing predominantly all-E-astaxanthin (36.9 mg/kg astaxanthin, 97% all-E-, 0.4% 9Z-, 1.5% 13Z-astaxanthin, and 1.1% other isomers, respectively) or a mixture of all-E- and Z-astaxanthins (35.4 mg/kg astaxanthin, 64% all-E-, 18.7% 9Z-, 12.3% 13Z-astaxanthin, and 2.0% other isomers, respectively), were fed to duplicate groups of trout for 69 d. Individual E/Z isomers were identified by VIS- and 1H-NMR-spectrometry, and quantified by high-performance liquid chromatography. Significantly higher total carotenoid concentration was observed in plasma of trout fed diets with all-E-astaxanthin (P < 0.05). The relative E/Z-isomer concentrations of plasma, skin and kidney were not significantly different among groups, whereas all-E-astaxanthin was higher in intestinal tissues and 13Z-astaxanthin was lower in liver of trout fed all-E-astaxanthin (P < 0.05). The relative amount of hepatic 13Z-astaxanthin (39-49% of total astaxanthin) was higher than in all other samples (P < 0.05). Synthetic, optically inactive astaxanthin was used in all experiments, and the determined dietary ratio between the 3R,3'R:3R, 3'S (meso):3S,3'S optical isomers was 25.3:49.6:25.1. The distribution of R/S-astaxanthin isomers in feces, blood, liver and fillet was similar to that in the diets. The ratio between (3S,3'S)- and (3R,3'R)-astaxanthin in the skin and posterior kidney was ca. 2:1 and 3:1, respectively, regardless of dietary E/Z-astaxanthin composition. The results show that geometrical and optical isomers of astaxanthin are distributed selectively in different tissues of rainbow trout.

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