How is astaxanthin synthesized?

 Astaxanthin (containing 40 carbon atoms) is a non-vitamin A carotenoid and is widely used in the production of health products, pharmaceuticals, cosmetics, food and feed additives.  In food, it can not only color, but also effectively play a role in preserving freshness, preventing discoloration, tastelessness, and deterioration, and can also be used for coloring beverages, food, and seasonings.  Astaxanthin has brilliant color, and can be unintentionally bound to actin, and is widely used in aquatic feeds to improve the skin and muscle color of farmed fish and increase the disease resistance of fish and shrimp[1,2] .

 

The following methods for the total synthesis of astaxanthin have been reported in the literature. (1) Taking Canthaxanthin (containing 40 carbon atoms) as the starting material, treating the two carbonyl groups with lithium diisopropylammonium to form the dienophile anion, then protecting it with trimethylchlorosilane to form the dienophile silyl ether, and then oxidizing the dienophile silyl ether with peroxyacid to obtain astaxanthin selectively.  Although the reaction step is short, but keratine is very expensive, two-step yield 55% ~ 65%, so this route is not economical [3]; (2) C9 + C1 → C10, 2C10 + C20 → C40 route, 6-oxoisophorone (C9) as the starting material, after a multi-step reaction, and then in the strong alkali and chloromethyltrimethylsilane (C1) addition reaction to obtain C10 enol silicone ether, then with C20 bisacetal, and then with C20 bisacetal to obtain C10 enol silicone ether, and then with C20 bisacetal. C10 enol silyl ether was obtained by the addition reaction with chloromethyltrimethylsilane (C1) under the action of strong base, and then condensed with C20 acetal to form C40 skeleton under the catalytic reaction of Lewis acid, and then dealkoxylated to form a conjugated double bond under the action of strong base to obtain astaxanthin [4]; (3) C9 + C6 → C15, 2C15 + C10 → C40 Route, 6-oxoisophorone (C9) was used as starting material, and then hexacarbonyloxyalkynyl alcohol (C6) was used as starting material, and then reacted with 2,7-dimethylsilyl (2,7-DM) and hexacarboxylic alcohol (C6) in a multi-step reaction to form C15 triphenyl phosphonate, and then reacted with 2,7-DM to obtain C15 triphenyl phosphonate. C15 triphenylphosphine salt was formed by a multi-step reaction with hexadecynyl alcohol (C6), and then reacted with 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde (C10) in the presence of alkali to form astaxanthin.  Although the process is long and complicated, this route is the only one that has been industrialized so far[5-9] .

 

In this paper, the synthesis route of 2C15+C10→ C40 was adopted for the preparation of astaxanthin, in which a new route was designed for the synthesis of the key intermediate C15, i.e., C13+C2→ C15 (Scheme 1).  A total of 9 steps were carried out to obtain astaxanthin using α-violet ketone (C13) as the starting material.  On the basis of the literature[10] , the conditions for the preparation of compound 6 were optimized, so that the yield of each reaction step was above 85%.  In particular, the purification of compound 4, which was not solved in the literature, was accomplished, so that the whole route could be completed successfully.  The preparation of compounds 7-9 is also the key success or failure point of this route.  It was found that the synthesis reactions of compounds 7-9 were very selective and high in yield, and almost all of them were quantitative reactions.  In the selective epoxidation reaction of compound 7, it is known from chemical theory that the rate of addition of enolated double bonds to peroxyacid is 100,000 times higher than that of normal double bonds, and this is confirmed in our experiments, i.e., peroxyacid preferentially epoxidizes with enolated double bonds, and there is no heterogeneous point of oxidation of normal double bonds.  The key intermediate 6-hydroxy-3-(3-hydroxy-3-methyl-1,4-pentadienyl)-2,4,4-trimethyl-2-cyclohexen-1-one (9) was synthesized in 68% yield.  Astaxanthin was further synthesized according to the methodology of [5], with a total yield of 38% in 9 steps.  The present paper provides a new route for the synthesis of astaxanthin, which mainly refers to the synthesis of the key C15 intermediate compound 9 using the new route. The starting materials used are easily available, the reaction is highly selective and the overall yield is high.

 

1 Experimental component

1.1 Instruments and reagents

Main Instruments: Agilent 4890D Gas Chromatograph, GC/MS Mass Spectrometer (Agilent 6890N Network GC System, Agilent 5973 Network Mass Selective Detector), INOVA-400 Nuclear Magnetic Resonance Analyzer (TMS internal standard, CDCl3 solvent), PE PE-HPLC- 900 High Performance Liquid Chromatograph. ), PE-HPLC-900 high performance liquid chromatograph (PE, USA).

Reagents: α-Violanone (96% GC, industrial product), m-chloroperoxybenzoic acid (industrial product, 85%), 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde were self-manufactured, and the rest of the raw materials and solvents were reagent grade.

 

1.2 Synthesis

1.2.1 Preparation of 4-(2,6,6-trimethyl-2,3-epoxy-1-cyclohexyl)-3-buten-2- ketone (3)

Referring to the literature [10], α-violet ketone 202.0 g (1.0 mol, 95%) was dissolved in dichloromethane (500 mL), cooled to below 5 ℃, and m-chloroperoxybenzoic acid (207.0 g, 1.05 mol, 85%) was added dropwise into a dichloromethane solution (500 mL) at a controlled temperature below 5 ℃, and then the reaction was carried out at 10 ℃ for about 1 h. The endpoint of the reaction was reached when the raw material disappeared from the TLC and GC traces. The reaction was carried out at 10 ℃ for about 1 h, and the end point was reached when the TLC and GC traces disappeared.  The reaction was carried out at 10 ℃ for about 1 h. The end point was reached when the raw material disappeared by TLC and GC. The filter was filtered, the cake was washed with dichloromethane (500 mL), and the dichloromethane solution was combined and washed with aqueous sodium sulfite (10%), aqueous sodium hydroxide (5%), and water, and then concentrated to obtain the epoxide 3 (209.0 g), which was analyzed by GC with purity of 94% and yield of 95%.

 

1.2.2 Preparation of 4-(2,6,6-trimethyl-3-hydroxy-1-cyclohexen-1-yl)-3-buten-2-one (4)

Referring to the literature [10], epoxide 3 (209.0 g) was dissolved in methanol (800 mL), 30% of sodium methanol methanol solution (50 mL), reflux reaction for 3 h, cooled to room temperature, neutralized with ice acetic acid (5 mL), methanol solution was recovered under reduced pressure, water was added, and the solvent was evaporated after drying to obtain the crude (211.0 g), the purity of the GC analysis was 87%.

 

1.2.3 Purification of 4-(2,6,6-trimethyl-3-hydroxy-1-cyclohexen-1-yl)-3-buten-2-one (4)

The crude product of the above reaction (211.0 g) was added with succinic anhydride (110.0 g, 1.1 mol), refluxed for 5 h, cooled down and extracted with aqueous sodium carbonate, the organic layer was discarded, the aqueous layer was acidified with 10% hydrochloric acid to pH=1~2, then extracted with ethyl acetate, dried and evaporated to remove the solvent, then added with methanol (800 mL) and a 30% methanolic solution of sodium methanolate (170.0 g, 1.05 mol), refluxed for 3 h, cooled down to room temperature, recovered by methanol solution under reduced pressure, added with water, evaporated to remove the solvent, then isolated and reacted. 1.05 mol), refluxed for 3 h, cooled to room temperature, recovered under reduced pressure from methanol solution, added water, extracted with ethyl acetate, dried and evaporated to remove the solvent, to obtain the pure hydroxyketone compound 4 (177.4 g) after separation and removal of impurities, GC analysis of the purity of 98%, the yield of 85%.

 

1.2.4 Preparation of 5-(2,6,6-trimethyl-3-hydroxy-1-cyclohexen-1-yl)-3-methyl-3-hydroxy-1,4-pentadiene (5)

Referring to the literature [10], hydroxyketone compound 4 (192.3 g) was dissolved in tetrahydrofuran (1000 mL), cooled to below -30 ℃, and then dropwise added with tetrahydrofuran solution of vinyl chloride Grignard reagent (700 mL, 3.3 mol/L), and reacted for 1 h at 0 ℃. Ether (1000 mL) and saturated aqueous ammonium chloride (250 mL) were added. Add ether (1000 mL) and ammonium chloride saturated aqueous solution (250 mL), filter, dry the filtrate with anhydrous magnesium sulfate, evaporate the solvent to obtain the diol compound 5 (208.5 g), yield 96%.

 

1.2.5 Preparation of 5-(2,6,6-trimethyl-3-oxo-1-cyclohexen-1-yl)-3-methyl-3-hydroxy-1,4-pentadiene (6)

Referring to the literature [10], the diol compound 5 (208.5 g) was dissolved in a mixed solution of acetone and dichloromethane (2000 mL, V:V=1 ∶ 1), added aluminum isopropanol (450.0 g), refluxed for 5 h, cooled to room temperature, neutralized with sulfuric acid (w = 10%) and acidified to pH=3-4, the organic phase was dried with anhydrous magnesium sulfate, and evaporated the solvent, to obtain compound 6 (200.6 g), yield 96%. The organic phase was dried with anhydrous magnesium sulfate, and the solvent was evaporated to give compound 6 (200.6 g) in 96% yield.

 

1.2.6 Preparation of 5-(2,6,6-trimethyl-3-trimethylsilyloxy-1,3-cyclohexadien-1-yl)-3-methyl-3-trimethylsilyloxy-1,4-pentadiene (7)

Compound 6 (24.0 g) was dissolved in anhydrous tetrahydrofuran, 250 mL of 2 mol/L lithium diisopropylammonium (LDA) solution was added dropwise at -10 ℃, completed the addition, and stirred for 30 min at -10 ℃, 24.0 g of hexane solution of trimethylchlorosilane was added dropwise, completed the addition, and stirred for 1 h at 0 ℃, 200 mL of water was added, and 200 mL of hexane was added. After dropping and stirring for 1 h at -10 ℃, 200 mL of water was added, 200 mL of hexane was added, the organic layer was washed with 5% sodium bicarbonate solution, and the solvent was evaporated under pressure reduction to obtain 35.7 g of light yellow oily 7. Yield 97%.  1H NMR (CDCl3, 400 MHz) δ: 0.12-0.20 [m, 18H, Si(CH3)3], 0.98 (s, 6H, 2CH3), 1.45 (s, 3H, CH3), 1.76 (s, 3H, CH3), 2.03 (d, J=5.0 Hz, 2H, CH2), 4.85 (t, J=4.5 Hz, 1H, cyclohexene hydrogen). , 1H, cycloalkenyl hydrogen), 5.02 (dd, J=1.2, 11.0 Hz, 1H, side-chain-terminal cis-alkenyl hydrogen), 5.20 (dd, J=1.2, 17.0 Hz, 1H, side-chain-terminal trans-alkenyl hydrogen), 5.54 (d, J= 16.0 Hz, 1H, HC = CH), 5.99 (d, J= 16.0 Hz, 1H, HC = CH), 5.90 (dd, J= 5.0 Hz, 1H, CH 5.90 (dd, J=11.0, 17.0 Hz , 1H, = CH).

 

1.2.7 Preparation of 5-(2,6,6-trimethyl-3,4-epoxy-3-trimethylsilyloxy-1,3-cyclohexadien-1-yl)-3-methyl-3-trimethylsilyloxy-1,4-pentadiene (8)

Compound 7 (30.0 g) was dissolved in 200 mL of anhydrous dichloromethane, 18.0 g of m-chloroperoxybenzoic acid dissolved in 200 mL of dichloromethane was added dropwise at -20 ℃, the dropwise addition was completed at -20 ℃, then stirred for 30 min at -10 ℃, and mixed with 10% sodium dithionite solution 100 mL. Then mix with 10% sodium dithionite solution for 1 h. The organic layer was washed with 200 mL of water, dried with anhydrous sodium sulfate, and evaporated under pressure to remove the solvent to obtain 30.0 g of light yellow oil 8 in 95% yield.  1H NMR (CDCl3, 400 MHz) δ: 0.12-0.20 [m, 18H, Si(CH3)3], 0.98 (s, 6H, 2CH3), 1.45 (s, 3H, CH3), 1.73 (s, 3H, CH3), 1.92 (d, J=5.0 Hz, 2H, CH2), 2.85 (t, J=4.5 Hz, 1H, cyclopentamethylene), 1.85 (t, J=4.5 Hz, 1H, cyclopentamethylene). , 1H, last cyclic methyl group), 5.02 (dd, J= 1.2, 11.0 Hz, 1H, side-chain-terminal cis-alkenylhydride), 5.20 (dd, J=1.2, 17.0 Hz, 1H, side-chain-terminal trans-alkenylhydride), 5.54 (d, J=16.0 Hz, 1H, HC ＝ CH), 6.01 (d, J=16.0 Hz, 1H, HC ＝ CH) , 5.89 (dd, J = 11.0, 17.0 Hz, 1H, ＝CH).

 

1.2.8 Synthesis of 6-hydroxy-3-(3-hydroxy-3-methyl-1,4-pentadienyl)-2,4,4-trimethyl-2-cyclohexen-1-one (9)

 

30.0 g of oil 8 was mixed with 200 mL of methanol and 2.0 g of potassium carbonate, refluxed for 2 h. After cooling, the solid was filtered off, and the methanol was removed by evaporation under pressure to give compound 9 (20.0 g).  The yield was essentially quantitative.  1H NMR (CDCl3, 400 MHz) δ: 1.14 (s, 3H, CH3), 1.28 (s, 3H, CH3), 1.46 (s, 3H, CH3), 1.86 (s, 3H, CH3), 1.80 (t, J=15.0 Hz, 2H, CH2), 2.18 (s, 1H, OH), 3.68 (s, 1H, OH), 4.33 (q, 1H, OH), 2.18 (s, 1H, OH), 3.68 (s, 1H, OH). OH), 4.33 (q, J=6.0 Hz, 1H, cyclic methyl group), 5.13 (dd, J=1.2, 11.0 Hz, 1H, cis-alkenyl hydrogen at the end of the side chain), 5.27 (dd, J=1.2, 17.0 Hz, 1H, trans-alkenyl hydrogen at the end of the side chain), 5.75 (d, J=16.0 Hz, 1H, HC=CH), 6.24 (d, J=16.0 Hz, 1H, HC=CH), 6.24 (d, J=16.0 Hz, 1H, HC=CH), 6.20 (d, J=15.0 Hz, 1H, CH J= 16.0 Hz, 1H, HC = CH), 5.97 (dd, J=11.0, 17.0 Hz, 1H, CH =); ESI-MS m/z (%): 251 (M++1, 100), 249 (M+-1, 55), 233 (18). 1H NMR is in agreement with literature [5].

 

1.2.9 Synthesis of astaxanthin(1)

Referring to the synthesis method in the literature [5], 100.0 g of compound 9 was dissolved in 400 mL of dichloromethane, and added dropwise into 104.0 g of 48% HBr aqueous solution within 30 min at 0 ℃, the mixture was stirred at 0 ℃ for 30 min, 360 mL of water was added, the organic phase was separated, the aqueous phase was extracted with 50 mL of dichloromethane, the organic phase was combined and mixed with 360 mL of water, 47.0 g of solid NaHCO3 was added, and then each phase was stirred together briefly, the organic phase was separated, washed with 360 mL of water, and 3 mL of 1,2-solid NaHCO3 was added. The aqueous phase was extracted once with 50 mL of dichloromethane, the organic phase was combined and mixed with 360 mL of water, 47.0 g of solid NaHCO3 was added, the phases were briefly stirred together again, the organic phase was separated, washed with 360 mL of water, 3 mL of 1,2-butyl epoxide was added, and 105.0 g of triphenylphosphine was added while the mixture was cooled to ≤10 ℃. The mixture was stirred at room temperature for 18 h, and then 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde was added.  The mixture was cooled to 0 °C and 58 g of 30% sodium methanol solution was added at 0 °C. The mixture was stirred at 0 °C for 3 h. 500 mL of water was added and the organic phase was separated. The aqueous phase was extracted twice with 100 mL of dichloromethane, the organic phases were combined, the organic phases were washed once with 300 mL of water, and the dichloromethane was evaporated, with the addition of methanol up to a boiling point of 65 °C. The suspension was refluxed for 15 h and then the mixture was allowed to reach its boiling point.  The suspension was refluxed for 15 h, then cooled to 0 ℃, and the resulting crystals were filtered out, dissolved in 500 mL of dichloromethane, replaced the solvent with methanol again as described above, and the filter cake was dried to yield 62.0 g (75% of the theoretical value) of astaxanthin. The purity was 98% by HPLC. ESI-MS and 1H NMR confirmed that it was consistent with the control.

 

2 Results and Discussion

When m-chloroperoxybenzoic acid (MCPB) was used in the first step to reduce the oxidation of α-violet ketone, the amount of MCPB was slightly overdosed by 5%~10% to ensure the complete epoxidation of α-violet ketone.  In the early stage of the reaction, it is easy to follow the reaction progress by TLC, but in the late stage of the reaction, it is necessary to use gas chromatography (GC) to follow the reaction progress due to the presence of impurities of β-violanone, which may interfere with the judgment of the end point of the reaction.  When the α-violet ketone material disappeared, the gas chromatographic analysis showed that the target product was in the range of 94% to 95%, and then the reaction should be terminated in time.  If the reaction continues, an impurity peak close to the main peak will increase significantly, from 1% to 10%.  Therefore, tracking the reaction with gas chromatography is one of the key factors in controlling the reaction.

When the epoxide is rearranged to the target product hydroxyketone compound 4 under alkaline catalytic reaction, an impurity is generated, which is reported to be a diketone compound in the literature, and is separated by a chromatographic column.  We analyzed two impurity peaks by gas chromatography, the impurity at about 8% was bis(ketone)11 as reported in the literature, and the impurity at 2%-3% was the product of hydroxyl dehydration to alkene 12.

 

The purity of hydroxyketone compound 4 will greatly affect the reaction yield and purity of the following fifteen-carbon synthesis unit, and it is also one of the key control points of this route. The chromatographic column separation method adopted in the literature is unsuitable for industrial production, so we creatively proposed the following purification method, i.e., using the hydroxyl group on the hydroxyketone compound 4 to form an ester with succinic anhydride, and then the ester-forming product was dissolved in alkaline aqueous solution, and the impurities insoluble in water were extracted with a non-water solvent, and then the ester-forming product was esterified with methanol under alkaline catalysis to obtain pure hydroxyketone compound 4 in 85% yield. The ester-forming product was dissolved in alkaline aqueous solution, and the water-insoluble impurities were extracted by a non-water-soluble solvent, and then the ester-forming product was esterified with methanol under alkaline catalyst to obtain the pure hydroxyketone compound 4 in 85% yield. The above post-processing method has not been reported in the literature.

 

3 Conclusion

The industrialized α-violet ketone product was used as the starting material, and the key intermediate 6-hydroxy-3-(3-methyl-3-hydroxy-1,4-pentadienyl)-2,4,4-trimethyl-2-cyclohexen-1-one was obtained by a multistep reaction using the industrially available conventional isolation method, and the C15 triphenylphosphine salt was obtained by the reaction of hydrobromic acid and triphenylphosphorane, and condensation of the C15 triphenylphosphorane salt with 2,7-dimethyl-2,4,6-octatriene-1,8-diels-Alder in the presence of alkali. C15 triphenylphosphine salt was then reacted with hydrobromic acid and triphenylphosphine to obtain C15 triphenylphosphine salt, which was condensed with 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde in the presence of alkali, and then recrystallized to obtain astaxanthin with a purity of 98%.  HPLC, ESI-MS and 1H NMR confirm that it is consistent with the control. The total yield of the 9-step reaction was 38%.

 

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