IPC분류정보
국가/구분 |
United States(US) Patent
등록
|
국제특허분류(IPC7판) |
|
출원번호 |
UP-0922680
(2006-05-26)
|
등록번호 |
US-7759095
(2010-08-09)
|
우선권정보 |
IT-PD2005A0164(2005-05-30) |
국제출원번호 |
PCT/EP2006/005030
(2006-05-26)
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§371/§102 date |
20071219
(20071219)
|
국제공개번호 |
WO06/128639
(2006-12-07)
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발명자
/ 주소 |
- Zanellato, Anna Maria
- Pittarello, Mara
- Gambillara, Antonio
- Vaccaro, Susanna
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출원인 / 주소 |
- Fidia Farmaceutici S.p.A.
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대리인 / 주소 |
Porzio, Bromberg & Newman, P.C.
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인용정보 |
피인용 횟수 :
0 인용 특허 :
0 |
초록
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Process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X=OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transphospha
Process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X=OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transphosphatidylation reaction between phosphatidylcholine of the general formula wherein R1 and R2 and X have the above specified meanings, R3=CH2—CH2—NH2 o CH2—CH2—N+(CH3)3 and Serine in D, L or racemic form catalized by the phospholipase D enzyme (PLD), characterized in that said reaction is carried out in a hydroalcoholic medium containing an aliphatic alcohol and in the presence of bivalent metal oxide.
대표청구항
▼
The invention claimed is: 1. A process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X=OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner s
The invention claimed is: 1. A process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X=OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transphosphatidylation reaction between a compound of the general formula wherein R1 and R2 and X have the above specified meanings, R3=CH2—CH2—NH2 or CH2—CH2—N+(CH3)3 and Serine in D, L or racemic form catalysed by the phospholipase D enzyme (PLD), characterised in that said reaction is carried out in a hydroalcoholic medium containing an aliphatic alcohol and in the presence of bivalent metal oxide. 2. The process according to claim 1 wherein the aliphatic alcohols are selected from methanol, ethanol, n-propanol and isopropanol. 3. The process according to claim 2 wherein the aliphatic alcohol is isopropanol in a concentration between 0.1 and 50% expressed as a % by volume on the volume of the starting buffer. 4. The process according to claim 3 wherein isopropanol concentration is 10%. 5. The process according to claim 1 wherein the compound is phosphatidylcholine and the concentration of serine ranges between 1 and 5 gg/gg of phosphatidylcho line. 6. The process according to claim 5 wherein the compound is phosphatidylcholine and the concentration of serine ranges between 2 and 3 gg/gg of phosphatidylcho line. 7. The process according to claim 1 wherein the compound is phosphatidylcholine and the phosphatidylcholine is of animal and/or vegetal origin, natural or synthetic, present in purified form or as raw material, at an starting concentration of between 10 and 500 mg/ml. 8. The process according to claim 1 wherein the transphosphatidylation reaction occurs at a temperature of between 20° C. and 60° C. 9. The process according to claim 8 wherein the transphosphatidylation reaction occurs at a temperature of 45° C. 10. The process according to claim 1 wherein the enzyme PLD is of fermentative origin derived from the micro-organism Streptoverticillium hachijoense, used in purified, partially purified or non-purified form. 11. The process according to claim 10 wherein the compound is phosphatidylcholine and the concentration of PLD that is used varies between 1 and 100 units/g of phosphatidylcholine. 12. The process according to claim 11 wherein the compound is phosphatidylcholine and the concentration of PLD that is used varies between 1 and 10 units/g of phosphatidylcholine. 13. The process for the purification of phosphatidylserine (PS) produced according claim 1 and involving the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with the subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 14. The process for the purification of phosphatidylserine (PS) produced according to claim 1 comprising the following steps: I) adding a saline solution of sodium chloride to the PS that has been previously filtered, with the subsequent mixing and separation of the PS; II) collection and elimination of the supernatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 15. The process for the purification of phosphatidylserine (PS) produced according to claim 1 comprising the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) adding a saline solution of sodium chloride and performing ultrafiltration through a porous membrane; IV) drying the final product. 16. The process according to claim 10 wherein the enzyme PLD derived from the micro-organism Streptoverticillium hachijoense is purified involving the following steps: I) elimination of the producing agent by microfiltration with a pore size of 0.2 μm; II) ultrafiltration through filters with a molecular cut-off of 10,000 D; III) ultrafiltration through filters with membranes with a molecular cut-off of 300,000 D; IV) ultrafiltration through membranes with a molecular cut-off of 10,000 D to re-concentrate the enzyme and dialyse it against acidic buffer. 17. The process according to claim 7 wherein the starting concentration is between 200 and 300 mg/ml. 18. The process according to claim 8 wherein the transphosphatidylation reaction occurs at a temperature of 55° C. 19. A process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X=OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transphosphatidylation reaction between a compound of the general formula wherein R1 and R2 and X have the above specified meanings, R3=CH2—CH2—NH2 or CH2—CH2—N+(CH3)3 and Serine in D, L or racemic form catalysed by the phospholipase D enzyme (PLD), characterised in that said reaction occurs in a medium containing an aprotic polar solvent and in the presence of a bivalent metal oxide. 20. The process according to claim 19 wherein the aprotic polar solvents are selected from dimethylsulphoxide, acetonitrile, dimethylformamide and N-methyl-pyrrolidone. 21. The process according to claim 20 wherein the aprotic polar solvent is dimethylsulphoxide in a concentration between 0.1 and 50% expressed as a % by volume on the volume of the starting buffer. 22. The process according to claim 21 wherein dimethylsulphoxide concentration is 1.25%. 23. The process according to claim 20 wherein the bivalent metal oxide is calcium or magnesium or zinc oxide in a concentration between 0.1 and 1M. 24. The process according to claim 23 wherein the concentration of the selected oxides is 0.33 or 0.54 M. 25. A process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X=OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transphosphatidylation reaction between a compound of the general formula wherein R1 and R2 and X have the above specified meanings, R3=CH2—CH2—NH2 or CH2—CH2—N30(CH3)3 and Serine in D, L or racemic form catalysed by the phospholipase D enzyme (PLD), characterised in that said reaction is carried out in a medium consisting of a two-phase system formed by water/organic solvent and in the presence of bivalent metal oxide. 26. The process according to claim 25 wherein the organic solvents are selected from n-hexane, toluene, benzene and n-butanol. 27. The process according to claim 26 wherein the organic solvent is n-hexane in a concentration between 0.1 and 40% expressed as % by volume on the volume of the starting buffer. 28. The process according to claim 27 wherein n-hexane concentration is 1.25% or 2.5%. 29. The process according to claim 19 wherein the bivalent metal oxide is calcium or magnesium or zinc oxide in a concentration between 0.1 and 1M. 30. The process according to claim 19 wherein the compound is phosphatidylcholine and the concentration of serine ranges between 1 and 5 gg/gg of phosphatidylcholine. 31. The process according to claim 19 wherein the compound is phosphatidylcholine and the phosphatidylcholine is of animal and/or vegetal origin, natural or synthetic, present in purified form or as raw material, at an starting concentration of between 10 and 500 mg/ml. 32. The process according to claim 2 wherein the transphosphatidylation reaction occurs at a temperature of between 20° C. and 60° C. 33. The process according to claim 2 wherein the enzyme PLD is of fermentative origin derived from the micro-organism Streptoverticillium hachijoense, used in purified, partially purified or non-purified form. 34. The process for the purification of phosphatidylserine (PS) produced according claim 2 and involving the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with the subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 35. The process for the purification of phosphatidylserine (PS) produced according to claim 19 comprising the following steps: I) adding a saline solution of sodium chloride to the PS that has been previously filtered, with the subsequent mixing and separation of the PS; II) collection and elimination of the supernatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 36. The process for the purification of phosphatidylserine (PS) produced according to claim 19 comprising the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) adding a saline solution of sodium chloride and performing ultrafiltration through a porous membrane; IV) drying the final product. 37. The process according to claim 25 wherein the bivalent metal oxide is calcium or magnesium or zinc oxide in a concentration between 0.1 and 1M. 38. The process according to claim 25 wherein the compound is phosphatidylcholine and the concentration of serine ranges between 1 and 5 gg/gg of phosphatidylcholine. 39. The process according to claim 25 wherein the compound is phosphatidylcholine and the phosphatidylcholine is of animal and/or vegetal origin, natural or synthetic, present in purified form or as raw material, at an starting concentration of between 10 and 500 mg/ml. 40. The process according to claim 25 wherein the transphosphatidylation reaction occurs at a temperature of between 20° C. and 60° C. 41. The process according to claim 25 wherein the enzyme PLD is of fermentative origin derived from the micro-organism Streptoverticillium hachijoense, used in purified, partially purified or non-purified form. 42. The process for the purification of phosphatidylserine (PS) produced according claim 25 and involving the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with the subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 43. The process for the purification of phosphatidylserine (PS) produced according to claim 25 comprising the following steps: I) adding a saline solution of sodium chloride to the PS that has been previously filtered, with the subsequent mixing and separation of the PS; II) collection and elimination of the supernatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 44. The process for the purification of phosphatidylserine (PS) produced according to claim 25 comprising the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) adding a saline solution of sodium chloride and performing ultrafiltration through a porous membrane; IV) drying the final product.
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