Total Synthesis of Gobiusxanthin Stereoisomers and Their Application to Determination of Absolute Configurations of Natural Products: Revision of Reported Absolute Configuration of Epigobiusxanthin

(3R)-Gobiusxanthin stereoisomers (1a–d) were synthesized by stereoselective Wittig reaction of the (3R)-C15-acetylenic tri-n-butylphosphonium salt 7 with C25-apocarotenal stereoisomers 5a,b and 14a,b bearing four kinds of 3,6-dihydroxy-ε-end groups. The validity of the reported stereochemistry of gobiusxanthin was demonstrated by the fact that the reported spectral data of natural gobiusxanthin were in agreement with those of synthetic (3R,3'S,6'R)-gobiusxanthin (1a). On the other hand, the reported CD spectral data of natural epigobiusxanthin, which has been assigned as (3R,3'R,6'R)-isomer (3'-epigobiusxanthin), were identical with those of synthetic (3R,3'S,6'S)-isomer 1d (6'-epigobiusxanthin) rather than those of the corresponding synthetic 3'-epi-isomer 1b. It was found that the stereochemistry at C3-position has little effect on the shape of their CD spectra. Thus, in order to reinforce the validity of the absolute configurations at C3-position of natural specimens, (3S,3'S,6'R)- and (3S,3'S,6'S)-stereoisomers 1e and 1f were also synthesized and a HPLC analytical method for four stereoisomers was established by using a column carrying a chiral stationary phase. The HPLC analysis has proven that the stereochemistry of the natural epigobiusxanthin is 3R,3'S,6'S.


Introduction
Gobiusxanthin (1a) (Figure 1), which bears a novel 3',6'-dihydroxy-ε-end group, was first isolated from the common freshwater goby Rhinogobius brunneus [1] and then from the salmon Oncorhynchus keta [2]. Its structure was determined to be 7,8-didehydro-β,ε-carotene-3,3',6'-triol by MS and 1 H-NMR spectroscopies and the absolute configuration was tentatively assigned as 3R,3'S,6'R from the resemblance of its CD spectrum to the calculated one of half (3S,6S,3'S,6'S)-tunaxanthin and half (3R,3'R)-alloxanthin according to the additivity rule of CD spectra [3]. From the salmon Oncorhynchus keta, salmoxanthin and deepoxysalmoxanthin, possessing the same 3',6'-dihydroxy-ε-end group, were also isolated together with gobiusxanthin [2]. Their absolute configurations were similarly postulated by comparing their CD spectra with those of analogous compounds. Recently, we accomplished the first total synthesis of these two carotenoids and consequently confirmed that their proposed configurations are correct [4]. The stereoisomer of gobiusxanthin, 3'-epigobiusxanthin (1b) was isolated from the crown-of-thorns starfish Acanthaster planci [5]. Its trans-configuration of the two hydroxy groups at C3' and C6' was determined by NOESY experiment and a 6'R configuration was estimated from the fact that it showed the negative Cotton effect around 280 nm in the CD spectrum [3]. In order to obtain an additional proof on the stereochemistries of gobiusxanthin (1a) and 3'-epigobiusxanthin (1b), we expected that efficient combination of a sterically-defined synthesis of authentic stereoisomers, spectroscopic analyses including NMR and CD, and a HPLC separation using a chiral column could be beneficial.

Synthesis of Gobiusxanthin (1a) and 3'-Epigobiusxanthin (1b)
We previously reported [6] that the C15-acetylenic tri-n-butylphophonium salt 7 (Scheme 1) is a useful tool for stereoselective synthesis of acetylenic carotenoids. In addition, the triethylsilyl (TES)-protected 3,6-syn-dihydroxydienoate 2a and the 3,6-anti-dihydroxydienoate 8 have already been prepared [4] in the course of synthesis of salmoxanthin. Thus, we synthesized gobiusxanthin (1a) and 3'-epigobiusxanthin (1b) by stereoselective Wittig reaction of the C15-phosphonium salt 7 with C25-apocarotenals 5a and 5b, which were derived from compounds 2a and 8, as shown in Scheme 1. The C15-syn-dihydroxydienoate 2a [4] was subjected to lithium aluminum hydride (LAH) reduction and subsequent MnO2 oxidation to afford the dienal 3a in 61% yield. This was then condensed with the previously reported C10-phosphonate 6 [7], and the resulting hexaenoate was reduced with LAH and followed by MnO2 oxidation to provide TES-protected all-E-apocarotenal 4a and its 13Z-isomer in 46% and 16% yield from 3a, respectively. The 13Z-isomer was converted (52%) into the desired all-E-isomer by isomerization using a palladium catalyst [4,8]. After treatment of compound 4a with tetrabutylammonium fluoride (TBAF), the resulting deprotected apocarotenal 5b was condensed with phosphonium salt 7 under previously reported [6] conditions (NaOMe in CH2Cl2) and then desilytated to provide gobiusxanthin in 69% over the 3 steps. After protection (quant.) of C15-anti-dihydroxydienoate 8 [4], the resulting TES-ether 2b was transformed into 3'-epigobiusxanthin (1b) via the stereoselective condensation of C25-apocarotenal 5b with the C15-acetylenic tri-n-butylphophonium salt 7, in the same procedure as synthesis of gobiusxanthin (1a). 1 H-NMR spectral data of synthetic 1a and 1 H-and 13 C-NMR spectral data of synthetic 1b were in good agreement with those of the reported data of goboiusxanthin [1] and 3'-epigobiusxanthin [5], respectively. As shown in Figure 2, the CD spectrum of synthetic 1a was basically identical with the reported spectrum of gobiusxanthin, except for the intensities. On the other hand, the CD spectrum of the synthetic 1b was opposite in sign to that of the reported 3'-epigobiusxanthin, indicating the reported absolute stereochemistry needs a correction. To make sure the stereochemistry of natural epigobiusxanthin, (6'S)-stereoisomer was also prepared as a reference sample.
The syn-configuration of the hydroxyl group at C3 and the epoxide oxygen was firstly identified in carotenoids, diadinoxanthin B and antheraxanthin B (Figure 1), isolated from the common freshwater goby Rhinogobius brunneus [1]. Diadinoxanthin B would be involved in a biosynthetic pathway of gobiusxanthin. On the contrary, epigobiusxanthin, which was found to be 6'-epi-form rather than 3'-epi-form, would be derived from common anti(α)-epoxy carotenoids such as diadinoxanthin A.

General
UV-VIS spectra were recorded on a JASCO V-650 instrument (JASCO, Tokyo, Japan), with ethanol solutions. IR spectra were measured on a Perkin-Elmer spectrum 100 FT-IR spectrometer (Perkin-Elmer, Yokohama, Japan), with chloroform solutions. 1 H-and 13 C-NMR spectra were determined on a Varian Gemini-300 or a VXR-500 superconducting FT-NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA), with deuteriochloroform solutions (tetramethylsilane as the internal reference). J Values are given in Hz. Mass spectra were 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). The concentrations were calculated using log ε = 5.0 at main λmax (in EPA). Optical rotations were measured on a JASCO P-2200 polarimeter (JASCO, Tokyo, Japan).
HPLC analyses were performed on Simadzu-LC-20AT instrument (Shimadzu, Kyoto, Japan) with a photodiode array detector (Waters 996, Tokyo, Japan) and column oven (GL Sciences Model 552, Tokyo, Japan). Flash column chromatography (CC) was performed on using Kanto Silica Gel 60 N. Preparative HPLC was carried out on a Shimadzu LC-6A with a UV-VIS detector (Shimadzu, Kyoto, Japan).
All operations were carried out under nitrogen or argon. Evaporation of the extract or the filtrate was carried out under reduced pressure. Ether refers to diethyl ether, and hexane to n-hexane. NMR assignments are given using the carotenoid numbering system.
Isomerization of 13Z-isomer of compound 4a. A solution (2 mL) prepared from PdCl2(MeCN)2 (13 mg), Et3N (7 mL) and water (1.2 mL) in MeCN (8.8 mL) was added to a solution of 13Z-isomer of compound 4a (103 mg) in MeCN (18 mL) and the mixture was stirred at rt for 3 h. The solvent was evaporated off to give a residue, which was purified by the same method described above to provide the all-E-isomer 4a (54 mg, 52%).
The present research has indicated that HPLC analysis can be a strong tool to determine the absolute stereochemistries of chiral compounds, especially those having multiple chirogenic centers. To do this, in a concerted manner, development of a total synthetic method is essential to supply a sterically-defined authentic sample.

Author Contributions
Basic idea of the research was proposed by Yumiko Yamano, Takashi Maoka, and Akimori Wda, collaboratively. The synthetic and analytical experiments were designed and performed by Yumiko Yamano, Kotaro Ematsu, and Hiromasa Kurimoto. The isolation of epigobiusxanthin was carried out by Takashi Maoka.