The present invention relates to a method of synthesis of monocaffeoylquinic or dicaffeoylquinic acid isomer.

The methods of obtaining of monocaffeoyl and dicaffeoyl acid reported to date base on their enzymatic extraction from plant material, as reported in CN1687435 or solvent extraction, as reported in CN103214371 and US2011054022. The required monocaffeoylquinic or dicaffeoylquinic acid isomer is isolated via chromatography from a mixture of isomers of monocaffeoylquinic and dicaffeoylquinic acids, as reported in US2011054022. Such a nonspecific method of obtaining an isomer of monocaffeoylquinic or dicaffeoylquinic acid is burdened with a need of simultaneous production of other isomers of monocaffeoylquinic and dicaffeoylquinic acid, therefore leading to low yields. The aim of the invention was to develop a method of synthesis of an isomer of monocaffeoylquinic or dicaffeoylquinic acid, without the need to isolate them from a mixture.

The method for the synthesis of isomer of monocaffeoylquinic or dicaffeoylquinic acid according to the invention is that the vinyl ester of 0,0-diacetylcaffeic acid, which is the acyl donor, is subjected to enzymatic transesterification in the presence of quinic acid or a quinic acid methyl ester in a solvent selected from polar solvents. Preferably, the solvents are selected from solvents with ether characteristics, in particular from MTBE, tert-butanol, THF, acetonitrile, hexane, dichloroethane, or mixtures thereof. The enzyme used as a catalyst is a lipase, and is selected from Lipozyme RM IM, that is, lipase from Mucor miehei immobilized on an ion exchange resin, Lipozyme TL IM, that is lipase from Thermomyces lanuginosus immobilized on an acrylic resin, Novozym 435, that is Lipase B from Candida antarctica immobilized on acrylic resin, PPL, that is porcine pancreatic lipase, CCL, that is Candida rugosa lipase, Amano AK, that is Pseudomonas fluorescens lipase and Amano PS, that is Burkholderia cepacia lipase.

The names Lipozyme RM IM, Lipozyme TL IM, Novozym 435 are trademarks of Novozymes A/S, PPL is the trademark of Lee Biosolutions Inc., CCL is a trademark of Sigma- Aldrich Co.., Amano AK and Amano PS are trademarks of Amano Enzyme Inc.

Lipozyme RM IM and Lipozyme TL IM catalyze the acylation reaction at the 3' position of the benzene ring of quinic acid or quinic acid methyl ester, wherein the preferred enzyme is Lipozyme RM IM. Novozym 435 catalyzes the acylation reaction at the 4' position of the benzene ring of quinic acid or quinic acid methyl ester. PPL, CCL, Amano AK and Amano PS catalyze the acylation reaction in the 5' position of the benzene ring of quinic acid or quinic acid methyl ester, wherein the preferred enzymes are CCL and Amano PS.

3,4-Isomer of dicaffeoylquinic acid is obtained in a reaction with Novozym 435, and then, after filtration, a reaction with Lipozyme RM IM or Lipozyme TL IM, wherein the order of preparation is not critical, but the reactions are carried out separately. 3,5-Isomer of dicaffeoylquinic acid is obtained by reaction with Lipozyme RM IM or Lipozyme TL IM, and then, after filtration, a reaction with PPL or CCL or Amano AK or Amano PS. 4,5-Isomer of dicaffeoylquinic acid is obtained by reaction with Novozym 435, and then, after filtration, PPL or CCL or Amano AK or Amano PS.

Preferably the acylation is carried out in a molar ratio of substrate to the acyl group donor contained in the range of 2 to 10. For each 1 mmol of substrate from 100 to 1000 mg of enzyme and 5 ml of solvent is used. Acylation process is conducted at a temperature from 10 to 60° Celsius, preferably from 30 to 40° Celsius. The isolated product is subjected to deacetylation and possible demethylation, where the acyl group donor is a methyl ester of quinic acid.

An advantage of the invention is obtaining of only a selected isomer of monocaffeoylquinic or dicaffeoylquinic acid because of high specificity of the enzyme used in the reaction and reduction of the cost of production of enantiomerically pure material due to the lack of necessary separation of the mixture of isomers of monocaffeoylquinic and dicaffeoylquinic acids.

The invention is illustrated in the embodiments. Example 1.

Synthesis of 3-caffeoylquinic acid

100 mM of caffeic acid was dissolved in 25 mL of pyridine and treated with 120 mM acetic anhydride. The reaction was left on a magnetic stirrer at room temperature for 24 hours. To the mixture was added 50 mL of water and extracted with 6x50 mL of ethyl acetate. The organic layers were combined and washed with saturated sodium bisulfate until the dissapearance of odor of pyridine, then with saturated sodium bicarbonate solution until disappearance of foam and with water. The organic layer was dried over anhydrous sodium sulfate and then evaporated.

The resulting solid was air dried until constant weight. The obtained white solid was dissolved in 100 mL of dry THF, 150 mL of vinyl acetate and 1 g of mercury (II) acetate or 1.5 g of palladium (II) acetate were added. The suspension was stirred for 30 minutes, after which 3 mL of an ethereal solution of BF ₃ was added, and heated to 40° Celsius. After 4 hours and TLC control, sodium acetate was added to the solution to neutralize, the mixture was filtered, THF and excess vinyl acetate were evaporated, the residue was dissolved in 200 mL of methylene chloride and washed with saturated sodium bicarbonate until disappearance of foaming, saturated sodium bisulfate to a pH of about 6 and water. The organic layer was dried over anhydrous sodium sulfate and evaporated to give 92 mM of vinyl ester of 0,0-diacetylcaffeic acid. Palladium acetate was recovered by dissolving the solid isolated after vinylation reaction in water and evaporating the solvent.

1 mM of quinic acid methyl ester was suspended in 5 mL of MTBE in a conical flask. To the suspension 8 mM of vinyl ester of O, O- diacetylcaffeic acid and 200 mg Lipozyme RM IM were added. The reaction was performed at 30° degrees Celsius until the dissapearance of quinic acid methyl ester signal in HPLC. To isolate the product the solvent was evaporated and the residue was suspended in methylene chloride and then filtered. The remaining product residue was the desired product, and the unused vinyl ester of Ο,Ο-diacetylcaffeic acid was recovered quantitatively from the solvent. The isolated product was deacetylated and demethylated by treatment with 3N HCl in water under TLC control. The mixture was then neutralized with solid sodium bicarbonate, evaporated and purified using an ion exchange column. Obtained was (l ,3 ,4S,5 )-3-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2- enoyl]oxy}-l,4,5-trihydroxycyclohexanecarboxylic acid of a structural formula:

The product was identified via Chromatography and spectra analysis.

HPLC conditions: DIONEX system, P580 pump, ASI-lOO Automatic Sample Injector, STH 585 thermostatic oven, UVD 340S detector, controlled by Chromeleon 6.20 software, Thermo Scientific BDS Hypersil CI 8 column, mobile phase water-acetonitrile-THF-acetic acid (85: 10:2:3), flow 1,5 mL/min, detection at 300 nm wavelenght, injection volume 1 μΕ. Product retention time 2,2 min H NMR conditions: Bruker DPX 250 Advance apparatus, samples dissolved in CDC1 ₃ with TMS as an internal standard. Signals represent the protons of quinic acid.

Product spectra data: 1.47 (m, 2H), 1.98 (m, 2H), 3.74 (m, 2H), 5.75 (m, 1H)

Example 2.

Synthesis of 5-caffeoylquinic Vinyl ester of 0,0-diacetylcaffeic acid was obtained according to Example 1.

1 mM of quinic acid was suspended in a conical flask in 5 mL of MTBE:THF with a volume ratio 7:3. To the suspension 10 mM of vinyl ester of O, O-diacetylcaffeic acid and 500 mg Amano PS were added. The reaction was performed at 30° degrees Celsius until the dissapearance of quinic acid signal in HPLC. To isolate the product the solvent was evaporated and the residue was suspended in methylene chloride and then filtered. The remaining product residue was the desired product, and the unused vinyl ester of Ο,Ο-diacetylcaffeic acid was recovered quantitatively from the solvent. The isolated product was deacetylated and demethylated by treatment with 3N HC1 in water under TLC control. The mixture was then neutralized with solid sodium bicarbonate, evaporated and purified using an ion exchange column. Obtained was (l S,3R,4R,5R)-3-{[(2E)-3-(3,4- dihydroxyphenyl)prop-2-enoyl]oxy} -1 ,4,5- trihydroxycyclohexanecarboxylic acid of a structural formula:

Product retention time 3,1 min

Product spectral data: 1.38 (m, 2H), 2.00 (m, 2H), 3.71 (m, 1H), 3.95 (m, 1H), 5.44 (m, 1H) Example 3

Synthesis of 4-caffeoylquinic acid

Vinyl ester of 0,0-diacetylcaffeic acid was obtained according to Example 1.

1 mM of quinic acid methyl ester was suspended in 5 mL of MTBE in a conical flask. To the suspension 8 mM of vinyl ester of O, O- diacetylcaffeic acid and 500 mg Novozym 435 were added. The reaction was performed at 40° degrees Celsius until the dissapearance of quinic acid methyl ester signal in HPLC. To isolate the product the solvent was evaporated and the residue was suspended in methylene chloride and then filtered. The remaining product residue was the desired product, and the unused vinyl ester of Ο,Ο-diacetylcaffeic acid was recovered quantitatively from the solvent. The isolated product was deacetylated and demethylated by treatment with 3N HC1 in water under TLC control. The mixture was then neutralized with solid sodium bicarbonate, evaporated and purified using an ion exchange column. Obtained was (l S,3 ,4S,5 )-4-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}- 1,3,5-trihydroxycyclohexanecarboxylic acid of a structural formula:

Product retention time 2,7 min Product spectral data: 1.43 (m, 2H), 1.58 (m, 2H), 3.92 (m, 1H), 4.03 (m, 1H), 4.88 (m, 1H) Example 4

Synthesis of 3,4-dicaffeoylquinic acid

Vinyl ester of Ο,Ο-diacetylcaffeic acid was obtained according to Example 1.

1 mM of quinic acid methyl ester was suspended in 5 mL of MTBE in a conical flask. To the suspension 8 mM of vinyl ester of O, O- diacetylcaffeic acid and 500 mg Novozym 435 were added. The reaction was performed at 40° degrees Celsius until the dissapearance of quinic acid methyl ester signal in HPLC. To isolate the 4-caffeoylquinic acid the solvent was evaporated and the residue was suspended in methylene chloride and then filtered.

1 mM of 4-caffeoylquinic acid was suspended in 5 mL of MTBE in a conical flask. To the suspension 10 mM of vinyl ester of O, O- diacetylcaffeic acid and 200 mG Lipozyme RM IM were added. The reaction was performed at 40° degrees Celsius until the dissapearance of 4-caffeoylquinic acid signal in HPLC. To isolate the product the solvent was evaporated and the residue was suspended in methylene chloride and then filtered. The remaining product residue was the desired product, and the unused vinyl ester of Ο,Ο-diacetylcaffeic acid was recovered quantitatively from the solvent. The isolated product was deacetylated and demethylated by treatment with 3N HC1 in water under TLC control. The mixture was then neutralized with solid sodium bicarbonate, evaporated and purified using an ion exchange column. Obtained was ( 1 Pv,3Pv,4 S,5Pv)-3 ,4-di { [(2E)-3 -(3 ,4-dihydroxyphenyl)prop-2- enoyl]oxy}-l,4-dihydroxycycohexanecarboxylic acid of a structural formula:

Product retention time 10,1 min

Product spectra data: 1.61 (m, 2H), 2.05 (m, 2H), 4.02 (m, 1H), 5.02 (m, 1H), 5.87 (m, 1H)

Example 5

Synthesis of 4,5-dicaffeoylquinic acid

Vinyl ester of Ο,Ο-diacetylcaffeic acid was obtained according to Example 1.

1 mM of quinic acid methyl ester was suspended in 5 mL of MTBE in a conical flask. To the suspension 8 mM of vinyl ester of O, O- diacetylcaffeic acid and 500 mg Novozym 435 were added. The reaction was performed at 40° degrees Celsius until the dissapearance of quinic acid methyl ester signal in HPLC. To isolate the 4-caffeoylquinic acid the solvent was evaporated and the residue was suspended in methylene chloride and then filtered. 1 mM of 4-caffeoylquinic acid was suspended in 5 mL of MTBE in a conical flask. To the suspension 10 mM of vinyl ester of O, O- diacetylcaffeic acid and 800 mG CCL were added. The reaction was performed at 40° degrees Celsius until the dissapearance of 4- caffeoylquinic acid signal in HPLC. To isolate the product the solvent was evaporated and the residue was suspended in methylene chloride and then filtered. The remaining product residue was the desired product, and the unused vinyl ester of Ο,Ο-diacetylcaffeic acid was recovered quantitatively from the solvent. The isolated product was deacetylated and demethylated by treatment with 3N HC1 in water under TLC control. The mixture was then neutralized with solid sodium bicarbonate, evaporated and purified using an ion exchange column. Obtained was ( 1 S,3 ,4 ,5 )-3 ,4-di { [(2E)-3 -(3 ,4-dihydroxyphenyl)prop-2- enoyl]oxy}-l,5-dihydroxycycohexanecarboxylic acid of a structural formula:

Product retention time 14,2 min

Product spectral data: 1.43 (m, 2H), 2.03 (m, 2H), 4.12 (m, 1H), 5.00 (m, 1H), 5.54 (m, 1H)

Example 6

Synthesis of 3,5-dicaffeoylquinic acid

Vinyl ester of 0,0-diacetylcaffeic acid was obtained according to Example 1.

1 mM of quinic acid methyl ester was suspended in 5 mL of MTBE in a conical flask. To the suspension 8 mM of vinyl ester of O, O- diacetylcaffeic acid and 200 mG Lipozyme RM IM were added. The reaction was performed at 30° degrees Celsius until the dissapearance of quinic acid methyl ester signal in HPLC. To isolate the 3 -caffeoyl quinic acid the solvent was evaporated and the residue was suspended in methylene chloride and then filtered.

1 mM of 3-caffeoylquinic acid was suspended in a conical flask in 5 mL of MTBE:THF with a volume ratio 1 : 1. To the suspension 10 mM of vinyl ester of Ο,Ο-diacetylcaffeic acid and 800 mG CCL were added. The reaction was performed at 40° degrees Celsius until the dissapearance of 3-caffeoylquinic acid signal in HPLC. To isolate the product the solvent was evaporated and the residue was suspended in methylene chloride and then filtered. The remaining product residue was the desired product, and the unused vinyl ester of Ο,Ο-diacetylcaffeic acid was recovered quantitatively from the solvent. The isolated product was deacetylated and demethylated by treatment with 3N HCl in water under TLC control. The mixture was then neutralized with solid sodium bicarbonate, evaporated and purified using an ion exchange column. Obtained was (lR,3R,4S,5R)-3,5-di{[(2E)-3-(3,4- dihydroxyphenyl)prop-2-enoyl] oxy } - 1 ,4- dihydroxycycohexanecarboxylic acid of a structural formula:

Product retention time 22,0 min

Product spectral data: 1.99 (m, 4H), 3.70 (m, 1H), 5.51 (m, 1H), 5.83 (m, 1H)

Applications of the products obtained using the method according to the invention are anti-high blood pressure agents, potential laxative substances, psychostimulant compounds, weight loss supporting supplements, substances supporting regulation of liver and pancreas, and antioxidants.