A Novel Carotenoid with a Unique 2,6-Cyclo-ψ-End Group, Roretziaxanthin, from the Sea Squirt Halocynthia roretzi

A novel carotenoid with a unique 2,6-cyclo-ψ-end group, named roretziaxanthin (1), was isolated from the sea squirt Halocynthia roretzi as a minor carotenoid along with (3S,3′S)-astaxanthin, alloxanthin, halocynthiaxanthin, mytiloxanthin, mytiloxanthinone, etc. This structure was determined to be 3-hydroxy-1′,16′-didehydro-1′,2′-dihydro-2′,6′-cyclo-β,ψ-carotene-4,4′-dione by UV–VIS, MS, and NMR spectral data. The formation mechanism of roretziaxanthin in the sea squirt was discussed.


Introduction
Tunicates are marine invertebrates belonging to Protochordata that can show bright red colors due to containing various carotenoids such as (3S,3 S)-astaxanthin, alloxanthin, mytiloxanthin, mytiloxanthinone, halocynthiaxanthin, amarouciaxanthin A, and amarouciaxanthin B [1][2][3][4]. The sea squirt Halocynthia roretzi is an edible tunicate that is classified in the family Pyuridae in Protochordata, and has been consumed as sashimi, vinegared food, and salted food in East Asia, especially in South Korea and the northeast region of Japan. (3S,3 S)-astaxanthin, alloxanthin, diatoxanthin, zeaxanthin, diadinochrome, fucoxanthinol, halocynthiaxanthin, mytiloxanthin, and mytiloxanthinone have been reported as the principal carotenoids in H. roretzi ( Figure 1) [3]. In the present study, we isolated another new carotenoid, having a unique 2,6-cyclo-ψ-end group, named roretziaxanthin (1) from H. roretzi as a minor carotenoid. This manuscript reports the isolation and structural elucidation of this new carotenoid.

Results and Discussion
A new crimson-colored carotenoid (1) named roretziaxanthin (1.5 mg, 1% of the total carotenoids) was isolated from the tunic (4 kg) of the sea squirt H. roretzi. This carotenoid (1) showed visible light absorption maxima at (463), 482, and 505 nm in diethyl ether (Et 2 O) ( Figure S1). The molecular formula of 1 was determined to be C 40 H 50 O 3 by highresolution FAB MS and ESI MS data ( Figure S2). The 13 C-NMR spectrum, including DEPT experiment, showed forty carbon signals: nine CH 3 , two sp 3 CH 2 , one sp 2 CH 2 , two CH, fourteen alkenes CH, and twelve quaternary carbons. 13 C-NMR signals of this carotenoid at δ 200.42 (C4) and 207.79 (C4 ) indicated the presence of two carbonyl groups in the molecule. Similarly, the 13 C-NMR signal at δ 69.19 (C3) and 1 H-NMR signals at δ 4.32 (H3) and 3.69 (OH) showed the presence of a hydroxy group in 1 (Figures S3 and S4). 1 H-and 13 C-NMR signals of 1 were assigned by COSY, NOESY, HSQC, and HMBC experiments ( Table 1, Figures S5-S8). From the 1 H-and 13 C-NMR data, this carotenoid consisted of two end groups and a polyene chain with nine conjugated double-bond systems. The presence of a 3-hydroxy-4-keto-β-end group (C1 to C6, C16, C17, C18) in 1 was revealed by a comparison of 1 H-and 13 C-NMR data with those of astaxanthin [5]. The structure of the polyene chain with all E geometry was also confirmed by 1 H-and 13 C-NMR data and NOE correlations [5]. The remaining structural part consisted of two methyl groups (C17 , C18 ), one sp 3 methylene group (C3 ), one sp 2 methylene group (C16 ), one sp 3 methin group (C2 ), and four quaternary carbons (C1 , C4 , C5 , C6 ), including one carbonyl group (C4 , δ 207.79). The structure of this end group was elucidated by detailed analysis of HMBC correlations, as shown in Figure 2. The HMBC correlations from H2 to C3 and C6 , from H3 to C2 and C4 , and from H18 to C4 , C5 , and C6 revealed the existence of a cyclopentene-4 -one ring attached to a methyl group (C18 ) at C5 . Furthermore, HMBC correlations from H2 to C16 and C17 , from H16 to C2 and C17 , and from H17 to C2 and C16 indicated that the propylene group (C1 , C16 , C17 ) was attached at the C2 position of this cyclopentene-4 -one ring. Moreover, HMBC correlations from H7 to C2 and C6 , and from H8 to C6 showed that this end group was attached at the C7 position of the polyene chain, as shown in Figure 2. This novel end group could not be described from the ordinal IUPAC semi-systematic nomenclature of carotenoids [6]. However, according to the new nomenclature rule proposed for 2,6-cyclo-lycopene derivatives of carotenoids by Khachik et al. [7][8][9], this novel end group was regarded as a derivative of the 2 ,6 -cycloψ-end group and designated as 4 -keto-1 ,16 -didehydro-1 ,2 -dihydro-2 ,6 -cyclo-ψ-end group. The NOE correlations between H8 and H2 , between H8 and H16 , and between H8 and H17 indicated that the propylene group in this end group was located on the same side of the polyene chain, as shown Figure 2. Furthermore, the product ion of FAB MS/MS at m/z 425 (M-153, C 9 H 11 O) was in agreement with the elimination of this end group from the molecule due to the cleavage of a single bond between C6 and C7 . Therefore, the structure of roretziaxanthin (1) was determined as 3-hydroxy-1 ,16 -didehydro-1 ,2 -dihydro-2 , 6 -cyclo-β,ψ-carotene-4,4 -dione ( Figure 2). This carotenoid had two asymmetric carbons at C3 and C2 . However, the chiralities of these asymmetric carbons could not be determined by CD spectral data. Astaxanthin presented in sea squirts only shows (3S,3 S) chirality [3]; therefore, (3S) chirality was proposed for roretziaxanthin (1) ( Figure S9).

General
The UV-visible (UV-VIS) spectra were recorded with a Hitachi U-2001 (Hitachi High-Technologies Corporation, Tokyo, Japan) in diethyl ether (Et2O). The positive ion

General
The UV-visible (UV-VIS) spectra were recorded with a Hitachi U-2001 (Hitachi High-Technologies Corporation, Tokyo, Japan) in diethyl ether (Et 2 O). The positive ion fast atom bombardment mass spectrometry (FAB MS) spectra were recorded using a JEOL JMS-HX 110A mass spectrometer (JEOL, Tokyo, Japan) with m-nitrobenzyl alcohol as a matrix. The positive ion electro spray ionization time of flight mass (ESI-TOF MS) spectra were recorded using a Waters Xevo G2S Q TOF mass spectrometer (Waters Corporation, Milford, CT, USA). The 1 H-NMR (500 MHz) and 13 C-NMR (500 MHz) spectra were measured with a Varian UNITY INOVA 500 spectrometer (Agilent Technologies, Santa Clara, CA, USA) in CDCl 3 . The chemical sifts are expressed in ppm relative to tetramethyl silane (TMS) (δ = 0) as an internal standard for 1 H-NMR and CDCl 3 (δ = 77) as an internal standard for 13 C-NMR. J values are given in Hz. The CD spectra were recorded in EPA [Et 2 O-isopentane-ethanol (5:5:2)] at room temperature with a Jasco J-500C spectropolarimeter (Jasco corporation, Tokyo, Japan). Preparative high-performance liquid chromatography (HPLC) was performed on a Shimadzu LC-6AD with a Shimadzu SPD-6AV spectrophotometer (Shimadzu Corporation, Kyoto, Japan) set at 450 nm. The column used was a 250mmX10mm i.d. 10 µm Cosmosil 5SL-II and 5C18 II (Nacalai tesque, Kyoto, Japan).

Animal Material
The sea squirt H. roretzi, cultivated at Yamada Bay in Iwate Prefecture, was purchased at a local fish market in Kyoto in October.

Isolation of Roretziaxanthin from the Sea Squirt H. roretzi
The tunic (4 kg) of the sea squirt H. roretzi was extracted with acetone (Me 2 CO) at room temperature. The Me 2 CO extract was partitioned with hexane/Et 2 O (1:1) and water. The hexane/Et 2 O layer was evaporated to dryness and chromatographed on silica gel using increasing Et 2 O in hexane. The fraction eluted with hexane/Et 2 O (2:8) was subjected to HPLC on silica gel with Me 2 CO/hexane (3:7), with a retention time (RT) of 14.1 min. Further purification to remove lipid impurities from 1 was performed by HPLC on ODS with methanol. Finally, 1.5 mg (1% of the total carotenoids) of roretziaxanthin was obtained as a crimson-colored powder.

Carotenoids from the Sea Squirt H. roretzi
The following carotenoids were isolated from the sea squirt H. roretzi along with roretziaxanthin. The fraction eluted with hexane/Et 2 O (2:8) was subjected to HPLC on silica gel with Me 2 CO/hexane (3:7), with an RT of 12.2 min to yield (3S,3 S)-astaxanthin (3.5 mg), and an RT of 15.0 min for mytiloxanthinone (6.5 mg) ( Table S1). The fraction eluted with Et 2 O/Me 2 CO (1:1) was subjected to HPLC on silica gel with Me 2 CO/hexane  (Table S2); RT of 23.0 min, halocynthiaxanthin (3 mg) (Table S3); and RT of 25.0 min, fucoxanthinol (2 mg). The identification of these carotenoids was described in a previous paper [3].  Table 1. The spectrums please see the Figures S1-S9.
Author Contributions: T.M. isolated and characterized roretziaxanthin. C.T. analyzed NMR spectral data. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.