**Yumiko Yamano 1,\*, Takashi Maoka 2 and Akimori Wada 1**


Kyoto 606-0805, Japan; E-Mail: maoka@mbox.kyoto-inet.or.jp 

**\*** Author to whom correspondence should be addressed; E-Mail: yyamano@kobepharma-u.ac.jp; Tel./Fax.: +81-78-441-7562.

*Received: 20 March 2014; in revised form: 15 April 2014 / Accepted: 15 April 2014 / Published: 8 May 2014* 

**Abstract:** In order to determine the absolute configuration of naturally occurring alloxanthin, a HPLC analytical method for three stereoisomers **1a**–**<sup>c</sup>** was established by using a chiral column. Two authentic samples, (3*S*,3<sup>ȝ</sup>*S*)- and *meso*-stereoisomers **1b** and **1c**, were chemically synthesized according to the method previously developed for 

(3*R*,3<sup>ȝ</sup>*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 (3*R*,3<sup>ȝ</sup>*R*)-stereoisomer **1a**, and unexpectedly those from lake shrimp, catfish, biwa goby, and biwa trout are mixtures of three stereoisomers of **1a**–**c**. 

**Keywords:** carotenoid; alloxanthin; synthesis; chiral HPLC separation; absolute configuration 

## **1. Introduction**

Alloxanthin (**1**) (Figure 1) was first isolated from *Cryptomonas* algae [1] and its structure was determined to be 7,8,7<sup>ȝ</sup>,8<sup>ȝ</sup>-tetreradehydro-Ά,Ά-carotene-3,3<sup>ȝ</sup>-diol by MS, IR and 1H-NMR spectroscopies [2]. Additionally, cynthiaxanthin [3] from the tunicate *Cynthia rorezi* (*Halocynthia rorezi*) and pectenoxanthin [4] from giant scallop *Pecten maximus* were isolated by Japanese scientists. 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 3 *R*,3<sup>ȝ</sup>*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]. 

**Figure 1.** Structures of stereoisomers of alloxanthin (**1a** –**<sup>c</sup>**) and other related carotenoids. 

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<sup>ȝ</sup>-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** –**<sup>c</sup>**) 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. 
