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WO2000018945A1 - Transphosphatidylation catalized by phospholipase d in anhydrous organic solvents in the presence of ion-exchange resin - Google Patents

Transphosphatidylation catalized by phospholipase d in anhydrous organic solvents in the presence of ion-exchange resin Download PDF

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Publication number
WO2000018945A1
WO2000018945A1 PCT/US1999/022826 US9922826W WO0018945A1 WO 2000018945 A1 WO2000018945 A1 WO 2000018945A1 US 9922826 W US9922826 W US 9922826W WO 0018945 A1 WO0018945 A1 WO 0018945A1
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reaction
transphosphatidylation
enzyme
nucleophile
exchange resin
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PCT/US1999/022826
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French (fr)
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Joseph O. Rich
Yuri L. Khmelnitsky
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Albany Molecular Research, Inc.
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Priority to AU64082/99A priority Critical patent/AU6408299A/en
Publication of WO2000018945A1 publication Critical patent/WO2000018945A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen

Definitions

  • Enzymatic transphosphatidylation is an efficient and convenient tool for production of phosphorylated derivatives of hydroxyl-containing compounds such as alcohols.
  • the reaction is catalyzed by phospholipase D (PLD), which transfers the diacylphosphatidvi moiety from diacylphosphatidylcholine to an alcohol (R, and R 2 can be different and represent saturated or unsaturated hydrocarbon chain of 1 to 20 carbon atoms):
  • the reaction is carried out in biphasic water-organic systems composed of aqueous buffer and apolar organic solvent, such as ether or chloroform.
  • the phosphatidyl donor e.g. , diacylphosphatidylcholine
  • the acceptor alcohol is partitioned between the phases.
  • the transphosphaiidyiated product is preferentially dissolved in the organic phase, while the choline by-product stays in water.
  • U.S. Patent 5.700,668 to DeFerra et al. discloses preparation of phosphatidylserines.
  • U.S. Patent 5,516,662 to Singh discloses the synthesis of phosphatidylhydroxalkanols.
  • This reaction system contains water, which reacts as a nucleophile in the transphosphatidylation reaction. Water competes with the alcohol resulting in the formation of a mixture of products containing both the transphosphatidylated compound and the hydrolysis product phosphatidic acid. As a result, the yield of the desired transphosphatidylated product is greatly reduced, despite the fact that PLD generally prefers alcohols as nucleophiles.
  • One solution to this problem is to conduct the reaction in a dry organic solvent in the absence of water, and thus eliminate the interfering hydrolysis reaction. However, this approach does not work well because choline is not soluble in most dry organic solvents and quickly accumulates in the immediate vicinity of PLD, which is insoluble in the solvent and reacted in suspension. This results in the inhibition of the enzyme and early termination of the reaction.
  • the transphosphatidylation reaction is carried out in a nonaqueous organic solvent in the presence of a cation exchange resin.
  • the resin e.g. , Amberlite IRC-50
  • Dry systems provide several advantages over biphasic systems. First, it is not necessary to follow the reaction carefully in order to determine the point in time when the reaction should be stopped. Such monitoring must be done in biphasic systems, since once the phosphatidyl donor is consumed in the presence of water, the enzyme will eventually hydrolyze the product as well. In the dry system this will not happen, since water is not present in the system.
  • a further advantage of nonaqueous reaction systems is that it is not necessary to use a large excess of phosphatidyl donor in order to push the reaction towards transphosphatidylation versus competing hydrolysis, since the latter simply does not occur in dry reaction systems.
  • the transphosphatidylation reaction of the present invention is characterized in that it is carried out in a nonaqueous organic solvent in the presence of a cation exchange resin.
  • a nonaqueous organic solvent in the presence of a cation exchange resin.
  • an organic solvent is selected which dissolves the nucleophile/ phosphatidyl acceptor.
  • the preferred solvents are chloroform, ethyl acetate, and tert-butyl methyl ether (conversions > 75% after 24 h).
  • Solvents that yield smaller amounts of product are hexane, acetonitrile, hexane/acetonitrile (1: 1), tert-butyl alcohol, and toluene (conversions ⁇ 25% after 24 h). No reaction was observed in tert-amyl alcohol.
  • the solvent is dried, for example, using a molecular sieve, to remove residual water, and prevent undesired hydrolysis.
  • Any cationic exchange resin should be useful in the present invention.
  • cation exchange resins include AMBERLITE resins (for example. 200, IRP-69, or DP-1), DOWEX 50W resins (for example, 50X1-100), DIAION resins (for example 1-3561, 1-3581, or 1-3585), and DUOLITE resins (for example, D7416 or D5552).
  • the amount of resin to be added to the reaction can be estimated based on the ion-exchange capacity of the resin as reported by the manufacturer.
  • the amount of the resin must be sufficient to absorb essentially all the choline liberated in the reaction, i.e., the ion exchange capacity of added resin must be at least equal to the amount of choline formed. In many cases it will be convenient to use excess resin, e.g., a 50-fold excess of ion-exchange resin, calculated based on the binding capacity reported by manufacturer.
  • the phosphatidyl donor is typically a diacylphosphatidylcholine wherein the acyl group is derived from a saturated or unsaturated fatty acid having 2 to 22 carbon atoms such as stearic, oleic, lmoleic, and palmitic acids.
  • useful donors include dipalmitoylphosphatidylcholine, dioleylphosphatidylcholine, dimyristoylphosphatidylcholine, and naturally occurring lecithins, e.g., soybean and egg lecithins. Examples of commercially available lecithins are EPIKURON 200, EPIKURON
  • a common phospholipase D is isolated from Streptomyces sp. but other phospholipases D such as those isolated from cabbage, rice germ, and peanut should also be useful. The activity of these enzymes will vary with the nucleophiles with which they are reacted.
  • the amount of ihe enzyme used in the reaction is not particularly critical and tends to be determined principally by balancing reaction rate and cost considerations. In general higher amounts of enzyme will provide faster reactions than lower amounts.
  • the upper limit of the enzyme amount is determined simply by the capacity of the reaction system. A practical range of catalyst concentration is 20 to 500 mg/ml.
  • the enzyme is preferably prepared by lyophilization from buffer solution at a pH which maximizes enzyme activity, e.g., 4.2 to 8.5 with the optimal pH being about 5.6. It has been observed that the protonation state of enzymes in organic solvents is a function of the pH of the aqueous buffer solution from which the enzyme is dried. This phenomenon is known as pH memory. In this regard, buffers are used in preparing the enzyme.
  • Useful buffers with pH shown in parenthesis include: 100 mM sodium acetate (5.6. 5.5, 4.0), 200 mM sodium acetate (5.6). 200 mM CaCl 2 , 200 mM sodium acetate (6.0, 4.5), 100 mM CaCl 2 , 100 mM sodium acetate (5.6), 40 mM CaCl 2 , 100 mM sodium acetate (5.6). 50 mM CaCl 2 , 100 mM sodium acetate (6.5), 100 mM Tris-HCl (8.0), etc. Any buffer thai buffers at pH 4-8 would be acceptable including: citric acid/ sodium hydroxide, citric acid sodium citrate, cacodylic acid sodium salt/ HC1.
  • the enzyme can be iyophilized with an inorganic salt such as KCl as described in U.S. Patent 5,449,613, which is incorporated herein by reterence.
  • the reaction is typically carried out at temperatures ranging from 20 to 80°C with shaking (20-500 rpm) to overcome the diffussional limits of the suspended enzyme. Because the enzyme is not dissolved in the reaction system, the reaction is preferably performed under constant agitation. The reaction time will vary from about 2 hours to several days. The reaction is carried out at a temperature and time sufficient to ensure that the transphosphatidylation reaction is complete.
  • a wide range of hydroxy-containing compounds, including primary, secondary, and aromatic alcohols can be reacted as nucleophiles in the transphosphatidylation reaction in accordance with the present invention. Conversions obtained using dipalmitoylphosphatidylcholme (DPPC) with various alcohols are given below next to the corresponding structure.
  • DPPC dipalmitoylphosphatidylcholme
  • the reaction should also be useful in phosphatidylation of serine. The reaction does not occur in the absence of either the enzyme or the ion exchange resin.
  • the typical experiment consisted of 20 mM dipalmitoyl phosphatidylcholine (DPPC, Sigma) and 20 mM alcohol in 1 ml of CHCl 3 containing ca. 100 rag of Amberlite IRC-50 ion-exchange resin (Rohm and Haas) in a 2 ml screw-cap vial. The reaction was initiated by the addition of 20 mg PLD/KC1 enzyme preparation.

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Abstract

A method for transphosphatidylation which comprises reacting a phosphatidylcholine donor with a nucleophile in a nonaqueous solvent in the presence of a cation exchange resin, wherein the resin binds choline formed as a byproduct of the transphosphatidylation reaction and thereby prevents the byproduct from inhibiting the reaction.

Description

TRANSPHOSPHATIDYLATION CATALYZED BY PHOSPHOLIPASE D
IN ANHYDROUS ORGANIC SOLVENTS IN THE PRESENCE
OF ION-EXCHANGE RESIN
CROSS-REFERENCE TO RELATED APPLICATION(S)
This application claims priority from U.S. Provisional Patent Application Serial No. 60/102,700, filed October 1, 1998.
BACKGROUND OF THE INVENTION
Enzymatic transphosphatidylation is an efficient and convenient tool for production of phosphorylated derivatives of hydroxyl-containing compounds such as alcohols. The reaction is catalyzed by phospholipase D (PLD), which transfers the diacylphosphatidvi moiety from diacylphosphatidylcholine to an alcohol (R, and R2 can be different and represent saturated or unsaturated hydrocarbon chain of 1 to 20 carbon atoms):
Figure imgf000003_0001
Conventionally the reaction is carried out in biphasic water-organic systems composed of aqueous buffer and apolar organic solvent, such as ether or chloroform. The phosphatidyl donor (e.g. , diacylphosphatidylcholine) resides in the organic phase, whereas the enzyme resides in the aqueous phase. The acceptor alcohol is partitioned between the phases. The transphosphaiidyiated product is preferentially dissolved in the organic phase, while the choline by-product stays in water. U.S. Patent 5.700,668 to DeFerra et al. discloses preparation of phosphatidylserines. U.S. Patent 5,516,662 to Singh discloses the synthesis of phosphatidylhydroxalkanols.
The major drawback of this reaction system is that it contains water, which reacts as a nucleophile in the transphosphatidylation reaction. Water competes with the alcohol resulting in the formation of a mixture of products containing both the transphosphatidylated compound and the hydrolysis product phosphatidic acid. As a result, the yield of the desired transphosphatidylated product is greatly reduced, despite the fact that PLD generally prefers alcohols as nucleophiles. One solution to this problem is to conduct the reaction in a dry organic solvent in the absence of water, and thus eliminate the interfering hydrolysis reaction. However, this approach does not work well because choline is not soluble in most dry organic solvents and quickly accumulates in the immediate vicinity of PLD, which is insoluble in the solvent and reacted in suspension. This results in the inhibition of the enzyme and early termination of the reaction.
SUMMARY OF THE INVENTION
In accordance with the present invention the transphosphatidylation reaction is carried out in a nonaqueous organic solvent in the presence of a cation exchange resin. The resin (e.g. , Amberlite IRC-50) serves as a molecular sponge that absorbs choline accumulating during the reaction. This also shifts the equilibrium of the reaction towards formation of the desired transphosphatidylated product.
Dry systems provide several advantages over biphasic systems. First, it is not necessary to follow the reaction carefully in order to determine the point in time when the reaction should be stopped. Such monitoring must be done in biphasic systems, since once the phosphatidyl donor is consumed in the presence of water, the enzyme will eventually hydrolyze the product as well. In the dry system this will not happen, since water is not present in the system. A further advantage of nonaqueous reaction systems is that it is not necessary to use a large excess of phosphatidyl donor in order to push the reaction towards transphosphatidylation versus competing hydrolysis, since the latter simply does not occur in dry reaction systems.
DETAILED DESCRIPTION OF THE INVENTION The transphosphatidylation reaction of the present invention is characterized in that it is carried out in a nonaqueous organic solvent in the presence of a cation exchange resin. A wide range of polar and nonpolar nonaqueous organic solvents are useful in the invention. Preferably, an organic solvent is selected which dissolves the nucleophile/ phosphatidyl acceptor. For example, in the phosphatidylation of glycerol, the preferred solvents are chloroform, ethyl acetate, and tert-butyl methyl ether (conversions > 75% after 24 h). Solvents that yield smaller amounts of product are hexane, acetonitrile, hexane/acetonitrile (1: 1), tert-butyl alcohol, and toluene (conversions <25% after 24 h). No reaction was observed in tert-amyl alcohol. Preferably, the solvent is dried, for example, using a molecular sieve, to remove residual water, and prevent undesired hydrolysis.
Any cationic exchange resin should be useful in the present invention. Several examples (out of the large number of commercially available cation exchange resins) include AMBERLITE resins (for example. 200, IRP-69, or DP-1), DOWEX 50W resins (for example, 50X1-100), DIAION resins (for example 1-3561, 1-3581, or 1-3585), and DUOLITE resins (for example, D7416 or D5552). The amount of resin to be added to the reaction can be estimated based on the ion-exchange capacity of the resin as reported by the manufacturer. The amount of the resin must be sufficient to absorb essentially all the choline liberated in the reaction, i.e., the ion exchange capacity of added resin must be at least equal to the amount of choline formed. In many cases it will be convenient to use excess resin, e.g., a 50-fold excess of ion-exchange resin, calculated based on the binding capacity reported by manufacturer.
The phosphatidyl donor is typically a diacylphosphatidylcholine wherein the acyl group is derived from a saturated or unsaturated fatty acid having 2 to 22 carbon atoms such as stearic, oleic, lmoleic, and palmitic acids. Examples of useful donors include dipalmitoylphosphatidylcholine, dioleylphosphatidylcholine, dimyristoylphosphatidylcholine, and naturally occurring lecithins, e.g., soybean and egg lecithins. Examples of commercially available lecithins are EPIKURON 200, EPIKURON
135 and OVOTHIN 160 from Lucas Meyer.
A common phospholipase D is isolated from Streptomyces sp. but other phospholipases D such as those isolated from cabbage, rice germ, and peanut should also be useful. The activity of these enzymes will vary with the nucleophiles with which they are reacted.
The amount of ihe enzyme used in the reaction is not particularly critical and tends to be determined principally by balancing reaction rate and cost considerations. In general higher amounts of enzyme will provide faster reactions than lower amounts. The upper limit of the enzyme amount is determined simply by the capacity of the reaction system. A practical range of catalyst concentration is 20 to 500 mg/ml. The enzyme is preferably prepared by lyophilization from buffer solution at a pH which maximizes enzyme activity, e.g., 4.2 to 8.5 with the optimal pH being about 5.6. It has been observed that the protonation state of enzymes in organic solvents is a function of the pH of the aqueous buffer solution from which the enzyme is dried. This phenomenon is known as pH memory. In this regard, buffers are used in preparing the enzyme. Useful buffers with pH shown in parenthesis include: 100 mM sodium acetate (5.6. 5.5, 4.0), 200 mM sodium acetate (5.6). 200 mM CaCl2, 200 mM sodium acetate (6.0, 4.5), 100 mM CaCl2, 100 mM sodium acetate (5.6), 40 mM CaCl2, 100 mM sodium acetate (5.6). 50 mM CaCl2, 100 mM sodium acetate (6.5), 100 mM Tris-HCl (8.0), etc. Any buffer thai buffers at pH 4-8 would be acceptable including: citric acid/ sodium hydroxide, citric acid sodium citrate, cacodylic acid sodium salt/ HC1. In another embodiment of the invention, the enzyme can be iyophilized with an inorganic salt such as KCl as described in U.S. Patent 5,449,613, which is incorporated herein by reterence.
The reaction is typically carried out at temperatures ranging from 20 to 80°C with shaking (20-500 rpm) to overcome the diffussional limits of the suspended enzyme. Because the enzyme is not dissolved in the reaction system, the reaction is preferably performed under constant agitation. The reaction time will vary from about 2 hours to several days. The reaction is carried out at a temperature and time sufficient to ensure that the transphosphatidylation reaction is complete.
A wide range of hydroxy-containing compounds, including primary, secondary, and aromatic alcohols can be reacted as nucleophiles in the transphosphatidylation reaction in accordance with the present invention. Conversions obtained using dipalmitoylphosphatidylcholme (DPPC) with various alcohols are given below next to the corresponding structure. The reaction should also be useful in phosphatidylation of serine. The reaction does not occur in the absence of either the enzyme or the ion exchange resin.
Figure imgf000007_0001
92% 72% 100%
Figure imgf000007_0002
100% 78% 100%
Figure imgf000007_0003
The invention is illustrated in more detail by the following non-limiting examples.
Example
2 mg of PLD (Asahi) was added to 1 ml of 200 mM sodium acetate buffer (pH 5.6) containing 80 mM CaCl2 and 38 mg KCl. This solution was immediately flash frozen using liquid nitrogen and lyophilized for 24 h on a Labconco freeze-drier.
The typical experiment consisted of 20 mM dipalmitoyl phosphatidylcholine (DPPC, Sigma) and 20 mM alcohol in 1 ml of CHCl3 containing ca. 100 rag of Amberlite IRC-50 ion-exchange resin (Rohm and Haas) in a 2 ml screw-cap vial. The reaction was initiated by the addition of 20 mg PLD/KC1 enzyme preparation. The vial was shaken at 250 rpm at 45 °C for 48 h Aliquots were periodically removed and analyzed by electrospray mass spectrometry in both positive and negative modes and by thin layer chromatography (CHCl3:MeOH:H2O 65:25:2, visualization by staining with phosphomolybdic acid and heating).
After 48 h reaction time, the enzyme was removed by centrifugation and the resulting supernatant was evaporated under vacuum leaving an oily residue. The product was then redissolved in CHC13 for analysis.
This approach was successfully tested for transphosphatidylation catalyzed by freeze-dried PLD from Streptomyces sp. suspended in 10 organic solvents using the previously listed alcohols, including primary, secondary, and aromatic (phenols). Virtually no undesired hydrolysis side reaction was detected in these reactions, with yields of the desired product ranging from 70 to 100% .
Having described the invention in detail and by reference to specific embodiments thereof it will be apparent that numerous variations and modifications thereof are possible without departing from the spirit and scope of the invention as defined by the following claims:
What is claimed is:
- /-

Claims

1. A method for transphosphatidylation which comprises reacting a phosphatidylcholine donor with a nucleophile in a nonaqueous solvent in the presence of a cation exchange resin, wherein said resin binds choline formed as a byproduct of the transphosphatidylation reaction and thereby prevents said byproduct from inhibiting said reaction.
2. The method of claim 1 wherein said reaction is conducted in the presence of an enzyme.
3. The method of claim 2 wherein said enzyme is a phospholipase D.
4. The method of claim 3 wherein said nucleophile is a hydroxy group containing compound.
5. The method of claim 4 wherein said donor is a diacylphosphatidylcholine.
6. The method of claim 1 wherein said nonaqueous solvent is selected from the group consisting of chloroform, ethyl acetate and alkyl ethers.
7. The method of claim 4 wherein said nucleophile is an alcohol.
8. The method of claim 7 wherein said nucleophile is a primary, secondary or aromatic alcohol.
9. The method of claim 8 wherein said nucleophile is glycerol.
10. The method of claim 3 wherein the enzyme is isolated from Streptomyces sp.
1. The method of claim 3 wherein the enzyme is isolated from cabbage or rice germ.
PCT/US1999/022826 1998-10-01 1999-09-30 Transphosphatidylation catalized by phospholipase d in anhydrous organic solvents in the presence of ion-exchange resin WO2000018945A1 (en)

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US60/102,700 1998-10-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6878532B1 (en) 2003-04-28 2005-04-12 Sioux Biochemical, Inc. Method of producing phosphatidylserine
CN114214377A (en) * 2021-12-24 2022-03-22 中国海洋大学 Phosphatidyl-agaropectin oligosaccharide and preparation method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5516662A (en) * 1993-07-30 1996-05-14 The United States Of America As Represented By The Secretary Of The Navy Process for the preparation of headgroup-modified phospholipids using phosphatidylhydroxyalkanols as intermediates
US5700668A (en) * 1995-12-08 1997-12-23 Italfarmaco Sud S.P.A. Process for the industrial preparation of phosphatidylserine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5516662A (en) * 1993-07-30 1996-05-14 The United States Of America As Represented By The Secretary Of The Navy Process for the preparation of headgroup-modified phospholipids using phosphatidylhydroxyalkanols as intermediates
US5700668A (en) * 1995-12-08 1997-12-23 Italfarmaco Sud S.P.A. Process for the industrial preparation of phosphatidylserine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE CHEMICAL ABSTACT TAKAMI ET AL.: "Transphosphatidylation Reaction of Phosphatidylcholine to 4-Methoxyphenol in Water-Immiscible Organic Solvents with Immobilized Phospholipase D" *
J. FERMENT. BIOENG., vol. 79, no. 4, 1995, pages 316 - 316 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6878532B1 (en) 2003-04-28 2005-04-12 Sioux Biochemical, Inc. Method of producing phosphatidylserine
US7049107B1 (en) 2003-04-28 2006-05-23 Sioux Biochemical, Inc. Method of producing phosphatidylserine
CN114214377A (en) * 2021-12-24 2022-03-22 中国海洋大学 Phosphatidyl-agaropectin oligosaccharide and preparation method thereof
CN114214377B (en) * 2021-12-24 2024-03-08 中国海洋大学 Phosphatidyl-agar oligosaccharide and preparation method thereof

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