Synthesis of (3S,3′S)- and meso-Stereoisomers of Alloxanthin and Determination of Absolute Configuration of Alloxanthin Isolated from Aquatic Animals

In order to determine the absolute configuration of naturally occurring alloxanthin, a HPLC analytical method for three stereoisomers 1a–c was established by using a chiral column. Two authentic samples, (3S,3′S)- and meso-stereoisomers 1b and 1c, were chemically synthesized according to the method previously developed for (3R,3′R)-alloxanthin (1a). Application of this method to various alloxanthin specimens of aquatic animals demonstrated that those isolated from shellfishes, tunicates, and crucian carp are identical with (3R,3′R)-stereoisomer 1a, and unexpectedly those from lake shrimp, catfish, biwa goby, and biwa trout are mixtures of three stereoisomers of 1a–c.

In 1967, Campbel et al. demonstrated that these two carotenoids were identical with alloxanthin [5]. Therefore, cynthiaxanthin and pectenoxanthin were synonyms of alloxanthin. The absolute configuration of alloxanthin isolated form algae was deduced to be 3R,3′R by X-ray analysis of degradation product of fucoxanthin and in view of biogenetic grounds [6]. Bartlett et al. reported that the ORD spectra of alloxanthin specimens from Cryptomonas algae and tunicate showed an identical shape each other and that both specimens are assumed to have an identical absolute configuration [7]. Since then, alloxanthin was isolated from several aquatic animals, such as shellfishes [8,9], starfishes [10], tunicates [11,12] and freshwater fishes [13,14], etc. These alloxanthin specimens showed similar non-conservative CD with weak negative Cotton effects.
Carotenoids such as astaxanthin, zeaxanthin, lutein, and tunaxanthin in animals are known to exist as a mixture of stereoisomers. Namely, astaxanthin in crustaceans and marine fishes exists as a mixture of three stereoisomers at C3 and C3′-positions [15,16]. Zeaxanthin [17], lutein [18], and tunaxanthin [19] in marine fishes also consist of these stereoisomers. Their absolute configurations were determined by CD spectra and chiral HPLC analyses. Due to its non-conservative CD, absolute configurations of alloxanthin in several origins could not be determined exactly by CD spectra.
In order to determine the absolute configuration of naturally occurring alloxanthin, we synthesized stereoisomers of alloxanthin (1a-c) and established a HPLC analytical method using a chiral column. Applying this method, the absolute configurations of alloxanthin specimens isolated from shellfishes, tunicates and fishes were investigated. Here, we describe these results.

Determination of Absolute Configuration of Alloxanthin Isolated from Aquatic Animals by HPLC
In order to determine the absolute configuration of naturally occurring alloxanthin, a HPLC analytical method for three stereoisomers 1a-c was investigated. As a result, three synthetic stereoisomers of alloxanthin can be separated using a chiral column (CHIRALPAK AD-H; Daicel) as shown in Figure 3.
Next, alloxanthin specimens isolated from scallop Mizuhopecten yessoensis, oyster Crassostrea gigas, pacific pearl oyster Pinctada margaritifera, freshwater bivalve Unio douglasiae, tunicate Halocynthia roretzi, and crucian carp Carassius auratus grandoculis were subjected to the HPLC method to find that these consist of only (3R,3′R)-stereoisomer 1a. On the other hand, alloxanthin specimens isolated from lake shrimp Palaemon paucidens, catfish Silurus asotus, biwa goby Gymnogobius isaza, and biwa trout Oncorhynchus masou rhodurus consisted of three stereoisomers 1a-c (Table 1).  Previously, one of the authors reported that zeaxanthin in plants, shellfishes, and tunicates consisted of only (3R,3′R)-stereoisomer, whereas zeaxanthin in fishes consisted of three stereoisomers [17]. Similar results were obtained in the case of alloxanthin in aquatic animals. Alloxanthin is de novo synthesized in Chryptophyceae and Euglenophyceae micro algae [22]. However, origin of alloxanthin in aquatic animals was remained uncertain. Patrali et al. (1989) [22] and Liaaen-Jensen (1998) [23] reported that alloxanthin in Mytilus edulis might be a terminal metabolite of fucoxanthin through intermediates, halocynthiaxanthin and isomytiloxanthin, based on observation in feeding experiment. However, conversion of isomytiloxanthin into alloxanthin is too complex and there were no direct evidences for the conversion, especially in aquatic animals. In our experience, isomytiloxanthin has not been isolated from these animals [24].
Shellfishes (bivalves) and tunicates are filter-feeders, which accumulate carotenoids from micro algae. Therefore, alloxanthin in these animals is assumed to originate from Chryptophyceae and Euglenophyceae micro algae, etc. Thus, these alloxanthin specimes consist of only (3R,3′R)-stereoisomer. Crucian carp is omnivorous and feeds not only animal planktons belonging to Cladocera but also micro algae. Therefore, alloxanthin in crucian carp is also assumed to originate from micro algae. On the other hand, alloxanthin in lake shrimp, catfish, biwa goby, and biwa trout exist as a mixture of three stereoisomres. These crustacean and fishes are carnivorous. Especially, lake shrimp contains a large amount of (3S,3′S)-and meso-alloxanthin (Table 1). Lake shrimp is a one of the major food of catfish and biwa trout. Therefore, (3S,3′S)-and meso-alloxanthin in these fishes might be originated from lake shrimp. However, origin of (3S,3′S)-and meso-alloxanthin in lake shrimp is uncertain.
Catfish is a top predator in Japanese freshwater ecosystems. Catfish ingests astaxanthin from crustaceans whose astaxanthin exists as a mixture of three stereoisomers. Catfish can convert astaxanthin into zeaxanthin [24]. Therefore, zeaxanthin in catfish exists as a mixture of three stereoisomers. Although the origin of stereoisomers of alloxanthin in catfish is uncertain, it might be naturally formed by epimerization of 7,8,7′,8′-tetradehydroastaxanthin originated from crustacean at C3 and C3′-positions and subsequent reduction at C4 and C4′-positions. Further studies are need to reveal the origin of (3S,3′S)-and meso-alloxanthin in crustaceans and fishes. This is the first report of the occurrence of (3S,3′S) and meso-alloxanthin in nature.

General
IR spectrum was measured on a Perkin-Elmer FT-IR spectrometer (Perkin-Elmer, Yokohama, Japan), spectrum 100. 1 H and 13 C NMR spectra were determined on a Varian Gemini-300 superconducting FT-NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) and the chemical shifts were referenced to tetramethylsilane. Mass spectrum was taken on a Thermo Fisher Scientific Exactive spectrometer (Thermo Fisher Scientific, Bremen, Germany). CD spectra were measured on a Shimadzu-AVIN 62A DS circular dichroism spectrometer (Shimadzu, Kyoto, Japan).
NMR assignments are given using the carotenoid nmbering system.

Animal Materials
Scallop Mizuhopecten yessoensis was provided from Hokkaido Research Organization, Abashiri Fisheries Research Institute, Hokkaido, Japan. Oyster Crassostrea gigas, and sea squirt Halocynthia roretzi were purchased from fisheries market at Kyoto city. Pacific pearl oyster Pinctada margaritifera was provided from a pearl aquaculture industry, Ishigaki city, Okinawa Prefecture. Freshwater bivalve Unio douglasiae, crucian carp Carassius auratus grandoculis, and catfish Silurus asotus were purchased from Katata fisheries cooperative, Shiga Prefecture. Biwa trout Oncorhynchus masou rhodurus was purchased from Nango Fisheries Center, Shiga Prefecture. Biwa goby Gymnogobius isaza and lake shrimp Palaemon paucidens were purchased from fisheries market at Maibara city.

Isolation of Alloxanthin from Aquatic Animals
According to our routine methods, carotenoid was extracted with acetone from animal tissue. The extract was partitioned between Et 2 O-n-hexane (1:1) and water in separating funnel. The organic phase was evaporated and saponified with 5% KOH/MeOH at room temperature for 2 h. Then, unsaponifiable compounds were extracted with Et 2 O-n-hexane (1:1, v/v) from the reaction mixture by addition of water. The organic layer was dried over Na 2 SO 4 and evaporated. The residue was subjected to silica gel column chromatography increasing percentage of Et 2 O in n-hexane. The fraction eluted with Et 2 O was subjected to HPLC on silica gel with acetone-n-hexane (3:7) to afford alloxanthin. Purity of alloxanthin was checked by UV-Vis, 1 H-NMR, and MS spectral data. Then alloxanthin obtained from aquatic animals was subject to configurational analysis using a chiral column described above.

Conclusions
In conclusion, we synthesized stereoisomers of alloxanthin (1a-c) and established a HPLC analytical method using a chiral column to identify them for naturally occurring alloxanthin. Application of this method to various alloxanthin specimens of aquatic animals demonstrated that those isolated from shellfishes, tunicates, and crucian carp are identical with (3R,3′R)-stereoisomer 1a, and unexpectedly those from lake shrimp, catfish, biwa goby, and biwa trout are mixtures of three stereoisomers of 1a-c. This is the first report of the occurrence of (3S,3′S) and meso-alloxanthin in nature. The analytical method can be a powerful tool to identify stereoisomers of alloxanthin in nature in a straightforward manner.