# US 5,700,668 A · Process for the industrial preparation of phosphatidylserine

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- Number: US 5,700,668 A
- Document ID: US5700668A
- Status: Granted
- Kind: A
- Application number: 08/570,000
- Filed: 1995-12-08
- Granted: 1997-12-23
- Earliest claimed: 1995-12-08
- Assignees: Italfarmaco Sud S.p.A.
- Inventors: Lorenzo De Ferra; Pietro Massardo; Oreste Piccolo; Stefano Servi
- Web page: https://patentgrep.com/patent/US5700668A

## CPC

- C12P 13/06: Alanine; Leucine; Isoleucine; Serine; Homoserine
- C07F 9/103: {Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure}
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- Y10S 435/886: Streptomyces
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The invention relates to a process for the preparation of phosphatidylserines by reacting racemic or enantiomerically pure serine, preferably (L)-serine, with natural phosphatides, such as soybean or egg lecithin, or with synthetic phosphatides, in the presence of a phospholipase D, having transphosphatidylating activity, in an aqueous/organic diphasic system.

## Claims

### Claim 1

A process for the preparation of phosphatidylserines compounds of formula (I) ##STR2## wherein: R.sup.1 and R.sup.2, which are the same or different, are a C.sub.10 -C.sub.30 acyl; X.dbd.OH or OM, wherein M is an alkali, alkaline-earth metal, ammonium or alkylammonium; which process comprises reacting phosphatides of general formula (II) ##STR3## wherein R.sup.1, R.sup.2 and X have the above defined meanings and R.sup.3 .dbd.CH.sub.2 --CH.sub.2 NH.sub.2 or CH.sub.2 --CH.sub.2 N.sup.+ (CH.sub.3).sub.3, with racemic or enantiomerically pure serine, in a water/organic solvent diphasic system, in the presence of crude phospholipase D from centrifuged fermentation broths of microorganisms strains producing extracellular PLD having a high transphosphatidylation activity.

### Claim 2 (depends on 1)

A process according to claim 1, wherein the organic solvent is toluene.

### Claim 3 (depends on 1)

A process according to claim 1, wherein the phosphatides of general formula (II) are mixtures of phosphatidyl-choline and/or -ethanolamine of natural origin, selected from soy and egg lecithins, having a phospholipids content ranging from 20% to 95%.

### Claim 4 (depends on 1)

A process according to claim 1, wherein the reaction pH ranges from 4 to 4.5.

### Claim 5 (depends on 1)

A process according to claim 1, wherein the reaction temperature is 25.degree. C..+-.5.degree. C.

### Claim 6 (depends on 1)

A process according to claim 1, wherein the crude phospholipase D is produced by Streptomyces strain ATCC 55717.

### Claim 7 (depends on 1)

A process according to claim 1, wherein the phosphatidylserine is phosphatidyl-(L)-serine and wherein R.sup.1 and R.sup.2 are acyl chains of palmitic, stearic, oleic, linoleic or linolenic acids in proportions substantially the same as that of soy lecithin.

### Claim 8 (depends on 1)

A process according to claim 1, wherein the phosphatidylserine is phosphatidyl-(L)-serine and wherein R.sup.1 and R.sup.2 are acyl chains of palmitic, stearic, palmitoleic, oleic, linoleic, arachidonic acids in proportions substantially the same as that of egg lecithin.

### Claim 9 (depends on 1)

A process according to claim 1, wherein the phosphatidylserines in an alkali, alkaline-earth, ammonium or alkylammonium salts form, are selectively extracted from PS-containing phospholipid mixtures in a dipbasic system of organic solvents.

### Claim 10 (depends on 9)

A process according to claim 9, wherein the phosphatidylserine is selectively extracted from a corresponding calcium salt in a heptane/methanol mixture.

### Claim 11 (depends on 10)

A process according to claim 10, wherein the phosphatidylserine is crystallized from heptane/acetone, in the form of a calcium salt, and subsequently converted into another salt.

### Claim 12 (depends on 1)

A process according to claim 1, wherein the serine is L-serine.

### Claim 13 (depends on 10)

The process of claim 10, wherein phospholipids are extracted in a purified form.

### Claim 14 (depends on 13)

The process of claim 13, wherein the phospholipids are one or more of PC, PE, PA and corresponding lysophospholipids.

### Claim 15 (depends on 1)

The process of claim 1, wherein R.sup.1 and R.sup.2 are mono- or polyunsaturated acyl.

### Claim 16 (depends on 1)

The process of claim 1, wherein M is an inner salt of ammonium or alkylammonium.