We previously reported [20] stereoselective total synthesis of (3R,3′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, (3S,3′S)-alloxanthin (1b) and its meso-stereoisomer 1c were newly synthesized using (3S)-phosphonium salt 5b, which was prepared from 3-epi-actinol 6 [21] in the same procedure [20] as preparation of (3R)-one 5a.

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

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

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.

IR spectrum was measured on a Perkin-Elmer FT-IR spectrometer (Perkin-Elmer, Yokohama, Japan), spectrum 100. ¹H and ¹³C 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).

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).

NMR assignments are given using the carotenoid nmbering system.

In the same procedure [20] as preparation of (3R)-phosphonium salt 5a and (3R,3′R)-alloxanthin (1a), (3S)-5b and (3S,3′S)-alloxanthin (1b) were prepared. Spectral data except for optical data of compounds 1b, 3b, 4b and 5b were identical with the corresponding previous reported [20] enantiomers 1a, 3a, 4a and 5a.

(3S,3′S)-Alloxanthin (1b): HRMS (ESI) m/z calcd for C₄₀H₅₃O₂ [M + H]⁺ 565.4040, found 565.4038.

Compound 3b: [α]_D²⁶ 102.9 (c 1.03, MeOH); HRMS (ESI) m/z calcd for C₁₁H₁₇O [M + H]⁺ 165.1274, found 165.1277.

Compound 4b: [α]_D²³ 68.1 (c 1.00, MeOH); HRMS (ESI) m/z calcd for C₁₇H₃₁OSi [M + H]⁺ 279.2139, found 279.2139.

Compound 5b: HRMS (ESI) m/z calcd for C₃₃H₆₂OPSi [M − Cl]⁺ 533.4302, found 533.4293.

meso-Alloxanthin (1c) was synthesized via condensation between 5b and (3R)-C₂₅-acetylenic apocarotenal 8, which was prepared by Wittig reaction of C₁₀-dialdehyde 7 with 5a as follows.

(2E,4E,6E,8E,10E)-2,7,11-trimethyl-13-[(R)-2,6,6-trimethyl-4-triethylsilyloxycyclohex-1-en-1-yl]trideca-2,4,6,8,10-pentaen-12-ynal (8). NaOMe (1 M in MeOH; 1.2 mL, 1.2 mmol) was added to a solution of the (3R)-phosphonium salt 5a (409 mg, 0.73 mmol) and C₁₀-dialdehyde 7 (100 mg, 0.61 mmol) in CH₂Cl₂ (10 mL) at 0 °C. After being stirred at 0 °C for 15 min, the mixture was poured into saturated aq. NH₄Cl and extracted with AcOEt. The extracts were washed with brine, dried over Na₂SO₄ and evaporated to afford a residue, which was purified by flash column chromatography (AcOEt–n-hexane, 1:4) to give the (3R)-C₂₅-acetylenic apocarotenal 8 (165 mg, 57%) as an orange viscous oil: UV-VIS λ_max (EtOH)/nm 420; IR ν_max (CHCl₃)/cm⁻¹ 2170 (C≡C), 1663 (conj. CHO), 1610 and 1599 (split) (C=C), 1552 (C=C); ¹H-NMR (CDCl₃, 300 MHz) δ 0.61 (6H, q, J = 8 Hz, SiCH₂ × 3), 0.97 (9H, t, J = 8 Hz, CH₂CH₃ × 3), 1.14 and 1.18 (each 3H, s, 1-gem-Me), 1.49 (1H, t, J = 12 Hz, 2-H_β), 1.74 (1H, ddd, J = 12, 3.5, 2 Hz, 2-H_α), 1.89 (3H), 1.91 (3H) and 2.03 (6H) (each s, 5-Me, 9-Me, 13-Me and 13′-Me), 2.11 (1H, br dd, J = 17.5, 9.5 Hz, 4-H_β), 2.30 (1H, br dd, J = 17.5, 5.5 Hz, 4-H_α), 3.94 (1H, m, 3-H), 6.32 (1H, br d, J = 12 Hz, 14-H), 6.37 (1H, d, J = 15 Hz, 12-H), 6.46 (1H, br d, J = 11.5 Hz, 10-H), 6.66 (1H, dd, J = 15, 11.5 Hz, 11-H), 6.70 (1H, dd, J = 14.5, 11.5 Hz, 15′-H), 6.96 (1H, br d, J = 11.5 Hz, 14′-H), 7.03 (1H, dd, J = 14.5, 12 Hz, 15-H), 9.46 (1H, s, CHO); ¹³C-NMR (CDCl₃, 75 MHz) δ 4.82 (C × 3), 6.83 (C × 3), 9.59, 12.96, 18.17, 22.53, 28.61, 30.45, 36.53, 42.11, 47.04, 65.01, 90.10, 98.16, 121.15, 123.84, 126.60, 127.73, 131.75, 134.51, 137.02, 137.07, 137.47, 138.70, 141.26, 148.75, 194.45; HRMS (ESI) m/z calcd for C₃₁H₄₇O₂Si (MH)⁺ 479.3340, found 479.3347.

Preparation of meso-alloxanthin (1c). NaOMe (1 M in MeOH; 0.24 mL, 0.24 mmol) was added to a solution of the (3S)-phosphonium salt 5b (113 mg, 0.20 mmol) and (3R)-C₂₅-acetylenic apocarotenal 8 (59 mg, 0.12 mmol) in CH₂Cl₂ (10 mL) at room temperature. After being stirred for further 15 min, the mixture was poured into saturated aq. NH₄Cl and extracted with AcOEt. The extracts were washed with brine, dried over Na₂SO₄ and evaporated to afford a residue, which was purified by flash column chromatography (AcOEt–n-hexane, 1:4) to give the TES-protected condensed product. Subsequently, to a solution of this condensed product in dry THF (5 mL) were added AcOH (1 M in THF; 0.20 mL, 0.20 mmol) and then tetrabutylammonium fluoride (TBAF) (1 M in THF, 0.40 mL, 0.40 mmol). After being stirred at room temperature for 2 h, the mixture was concentrated to give a residue, which was purified by flash column chromatography (AcOEt–n-hexane–MeOH, 50:45:5) to provide meso-alloxanthin (1c) (70 mg, quant.) as red solids. Its spectral data were identical with those of (3R,3′R)-alloxanthin (1a) [20]. HRMS (ESI) m/z calcd for C₄₀H₅₃O₂ [M + H]⁺ 565.4040, found 565.4033.