*2.1. Synthesis of (3S,3ȝS)-Alloxanthin (1b) and meso-Alloxanthin (1c)*

We previously reported [20] stereoselective total synthesis of (3 *R*,3<sup>ȝ</sup>*R*)-alloxanthin (**1a**) by use of C15-acetylenic tri*n*-butylphosphonium salt **5a** (Scheme 1) as a versatile synthon for syntheses of acetylenic carotenoids. This time, (3*S*,3<sup>ȝ</sup>*S*)-alloxanthin (**1b**) and its *meso*-stereoisomer **1c** were newly synthesized using (3*S*)-phosphonium salt **5b**, which was prepared from 3-epi-actinol **6** [21] in the same procedure [20] as preparation of (3 *R*)-one **5a**. 

## **Scheme 1.** Synthesis of C15-acetylenic tri*n*-butylphosphonium salts **5a** and **5b**.

Reagents: (**a**) (i) TMSCl, Et3N, DMAP, (ii) TMSCǓCH, *n*BuLi, then aq. KOH, (iii) Ac2O, pyridine, (iv) CuSO4, xylene, reflux (Dean-Stark), (v) LiAlH4; ( **b**) TESCl, Et3N, DMAP, (**c**) (i) vinyl bromide **6**, Pd(PPh3)4, CuI, BHT, *i*Pr2NH, (ii) MsCl, LiCl, ·-collidine, (iii) P*n*Bu3, Et3N. 

Compound **6** was converted into terminal alkyne **3b** via the addition of lithium acetylide in 72% yield over six steps. The high enantiomeric purity of **3b** (99% ee) was confirmed by HPLC analysis [CHIRALPAK AY-H; Daicel, 2-PrOH– *n*-hexane (5:95)]. Compound **3b** was then transformed into the phoshonium salt **5b** via Sonogashira cross-coupling of the triethylsilyl (TES)-protected terminal alkyne **4b** with vinylbromide **6** in 59% over four steps. 

Wittig condensation of C10-dialdehyde **7** with excess amount of (3*S*)- phosphonium salt **5b** in the presence of sodium methoxide in dichloromethane at room temperature and subsequent desilylation stereoselectively provided (3*S*,3<sup>ȝ</sup>*S*)- alloxanthin (**1b**) (Scheme 2). On the other hand, *meso*-alloxanthin (**1c**) was synthesized via condensation between (3*S*)-phosphonium salt **5b** and (3 *R*)-C25- acetylenic apocarotenal **<sup>8</sup>**, which was prepared by Wittig reaction of C10-dialdehyde **7** with (3 *R*)-phosphonium salt **5a** in the presence of sodium methoxide in dichloromethane at 0 °C. 

**Scheme 2.** Synthesis of three stereoisomers of alloxanthin (**1a** –**<sup>c</sup>**).

CD spectrum of (3*S*,3<sup>ȝ</sup>*S*)-alloxanthin (**1b**) showed an antisymetrical curve having week Cotton effects to that of previously synthesized [20] (3 *R*,3<sup>ȝ</sup>*R*)-alloxanthin (**1a**) as shown in Figure 2. 

*2.2. 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 (3*<sup>R</sup>*,3<sup>ȝ</sup>*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**–**<sup>c</sup>** (Table 1). 

Column: CHIRALPAK AD-H 0.46 × 25 cm (Daicel, Tokyo, Japan); eluent: 2- PrOH–*n*-hexane (4:96); flow rate: 0.6 mL/min; temperature: 23 °C; detection: 450 nm. 



n.d.: not detected.

Previously, one of the authors reported that zeaxanthin in plants, shellfishes, and tunicates consisted of only (3*<sup>R</sup>*,3<sup>ȝ</sup>*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 (3*<sup>R</sup>*,3<sup>ȝ</sup>*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 (3*S*,3<sup>ȝ</sup>*S*)- and *meso*alloxanthin (Table 1). Lake shrimp is a one of the major food of catfish and biwa trout. Therefore, (3*S*,3<sup>ȝ</sup>*S*)- and *meso*-alloxanthin in these fishes might be originated from lake shrimp. However, origin of (3*S*,3<sup>ȝ</sup>*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<sup>ȝ</sup>,8<sup>ȝ</sup>-tetradehydroastaxanthin originated from crustacean at C3 and C3<sup>ȝ</sup>-positions and subsequent reduction at C4 and C4<sup>ȝ</sup>-positions. Further studies are need to reveal the origin of (3*S*,3<sup>ȝ</sup>*S*)- and *meso*-alloxanthin in crustaceans and fishes. 

This is the first report of the occurrence of (3*S*,3<sup>ȝ</sup>*S*) and *meso*-alloxanthin in nature. 
