CA2045305A1 - Mono-n,n-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate, its production and use in preparation of phosphatidic acids - Google Patents

Abstract

Abstract Provided is a novel salt of sn-glycerol-3-phosphate comprising mono-N,N-dimethyl-4-aminopyridine along with methods for making same and using same for the production of diacyl phosphatidic acids.

Description

~3 ~ c k~ r Q~ nd Q ~ I nv ~n t i o n ~d `;, The present invention pertains to the production of a crystalline salt of sn-glycerol-3-phosphate and to its subsequent use in the direct preparation of diacyl phosphatidic acids. More specifically, this invention relates to the production o~ the mono-N,N-dimethyl~4-aminopyridinium salt of sn-glycerol-3-phosphate ~G-3-P(~MAP)l), a uniquely crystalline and anhydrous form of the phosphate. In addition, this invention relates to the method of direct acylation oE the aforementioned N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate salt with fatty acid anhydrides so as to provide new methods for production of diacyl phosphatidic acids.

Diacyl phosphatidic acids are useful precursors for the synthesis o phospholipids which are major co~ponents of cellular membranes. Diacyl phosphatidic acids may be prepared by the acylation of various salt forms of sn-glycerol 3-phosphate. Fos example, Y. Lapidot and Z.
Selinger in J. Am. Chem. Soc., Vol 87, 5522 5523 (1965) described the synthesis of diacyl phosphatidic acids Vicl the acylation of pyridinium sn-glycerol-3-phosphate. The Lapidot et al. method gave acceptable yields (70% - 80%) of the desired diacyl phosphatidic acids without significant formation of by-products, but on a small scale (0.4 mmol). Gupta et al. in Proc. Nat. Acad. Sci., Vol. 74, 4315-4319 (1977) described an improvement on the method of Lapidot et al., wherein the basic -atalyst, N,N-dimethyl-4-aminopyridine, was added to enhance the reactivity of the fatty acid anhydride towards nucleophilic attack by pyridinium sn-glycerol-3-phosphate. According to Gupta et al., pyridinium sn-glycerol-3-phosphate gave superior yields (87~) of diacyl phosphatidic acid when reacted with fatty acid anhydride (3 equiv) and N,N-dimethyl-4-aminopyridine ~4 equiv). While the method of Gupta et cll. is amenable for production of diacyl phosphatidic acids on a millimolar scale, there are serious limitations in the large-scale synthesis of~
diacyl phosphatidic acids using this method.

It i5 one aspect of the present invention to provide new methods for the synthesis of diacyl phosphatidic acids which are useful for large-scale application.

Another limitation to these conventional methods for diacyl phosphatidic acid production is the required use of pyridinium sn-glycerol-3-phosphate. As described by Gupta et al., pyridinium sn-glycerol-3-phosphate salt is a hygroscopic and gummy oil which consequently poses several disadvantages in large-scale acylation reactions. As an intractable and hygroscopic oil, the pyridinium sn-glycerol-3-phosphate is difficult to accurately weigh thereby making quantification of reaction stoichiometry problematic. The difficulty in completely removing water and alcohols from the pyridinium sn-glycerol-3-phosphate poses another significant disadvantage.

~ecause the reaction conditions for diacyl phosphatidic acid production must be free of water and alcohol, large-scale diacyl phosphatidic acid synthesis using pyr;diniurn sn-qlycerol-3-phosphate poses substantial disadvantages in order to render the salt anhydrous and solvent-free. Both the Lapidot et al. and the Gupta et al. methods are labor intensive and require repeated addition of dry pyridine and subsequent evaporation of the solvent to render the pyridinium sn-glycerol-3-phosphate salt anhydrous.

It is another aspect o the present invention to avoid th~ use of pyridinium sn-glycerol-3-phosphate in the synthes;s of diacyl phosphatidic acid.

Another serious drawback to the methods of Lapidot et al. and Gupta et al. is the indirect formulation of the pyridinium sn-glycerol-3-phosphate itself. Conventional methods known to date provide for the derivation of pyridinium sn-glycerol 3-phosphate from other sn-glycerol-3-phosphate salt forms. The other known crystalline salts of sn-glycerol-3-phosphate are the barium, calcium, sodium, monocyclohexylammonium and the dicyclohexylammonium sn-glycerol-3-phosphates. Both the barium and dicyclohexylammonium salts are generally prepared solely as a means to isolate the sn-glycerol-3-phosphate (C.F. Crans et al.
in J. Am. Chem. Soc., Vol 107, 7019 (1986)). However, neither salt may be used directly for diacyl phosphatidic acid production since the barium salt is insoluble in the requisite ; ?s ~ 2 ; ~ ~
organic reaction medium and the dicyclohexyla~nonium salt is not compatible with the coreactant fatty-acid anhydrides. To make either the barium or cyclohexylammonium salts cornpatible it is necessary to convert them to the pyridinium salt of sn-glycerol-3-phosphate if an acylation reactlon is to be conducted. Such a conversion is a laborious process requiring exchange o~ the counter-ion for pyridine via the use of an ion-exchange resin. The solvent used to convert the salt to the pyridinium sn-glycerol-3-phosphate must be aqueous pyridine. The subsequent isolation of the pyridinium sn-glycerol-3-phosphate from the aqueous solution and the drying of the salt is, however, too expensive in terms of time and effort to make this procedure practical on a k;logram scale.

It is yet another aspect of the present invention to avoid the use of barium and dicyclohexylammonium salts in the preparation of diacyl phosphatidic acids.

It is still yet another aspect of the present invention to provide a method to readily isolate a salt of sn-glycerol-3-phosphate in a crystalline and anhydrous form which can be subsequently directly acylated to economically form phosphatidic acids, and to provide a process for the synthesis of diacyl phosphatidic acids and other phospholipids from sn-glycerol-3-phosphate.

S~,mm~ry Q___nven~i~n In accordance with the various principles and aspects of the present invention, there is described a convenient and practical large scale production of N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate and the direct use of this salt for the production of diacyl phosphatidic acids. In general the invention features, in one aspect, the production of a crystalline and anhydrous salt of sn-glycerol-3-phosphate. In another aspect the invention features the use of the crystalline and anhydrous salt, N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate, shown in the Figure, in the direct formation of diacyl phosphatidic acids, themselves usef~l as intermediates in the synthesis of other phospholipids, most especially diacyl phosphatidy:L
esters. The preparation of such phospholipids using the foregoing is also contemplated by the present invention.

The N,N-dirnethyl-4-aminopyridinium sn-glycerol-3-phosphate salt of the present invention, when compared to the conventional pyridinium sn-glycerol-3-phosphate salt, e~hibits enhanced solubility and reactivity in organic solvents such as methylene chloride and chloroform. The enhanced solubility and reactivity greatly facilitates and now makes practical for the first tirne, the large scale production of diacyl phosphatidic acids and their derivatives.

B_ie~f_De_~i~tion _f the_Drawinq Furthur understanding of the present invention may be had by reference to the accompanying figure showing a structural presentation of the novel composition of the present invention, N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate.

De~ailed ~eSçript~ t~lL~ ~e~On and Bes~ Mode The crystalline and anhydrous N,N-dimethyl-4-am;nopyridinium sn-glycerol-3-phosphate is ideally obtained as follows. The free acid form of sn-glycerol-3-phosphate is preerably mixed with an equimolar amount of the base N,N-dimethyl-4-aminopyridine in an alcoholic solvent until both reactants are in solution. The most preferred solvent is methanol. Other possible solvents which may be used include ethanol or n-propanol. One may also substitute for sn-glycerol-3-phosphate analogues thereof wherein, for example, the Cl hydroxyl may be replaced by -Cl, -Br, -SH, -OCH3, -CH20H or -CH2CH3.

The preferred method of isolating the crude N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate comprises removal of the methanol via in vacuo concentration. The resulting oil contains residual methanol and N,N-dimethyl-4-aminopyridinium hydrochloride as the major contaminants.

The preferred method for removing the major contaminants comprises dissolution of the oil in isopropyl alcohol and, following addition of an organic solvent~ precipitation of the N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate as an off-white crystalline solid. The preferred organic solvent used for effecting the precipitation of the N,~-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate is acetone. The preferred ratio of acetone to isopropyl alcohol, while not crucial, should be 10 volumes to 1 volume respectively. The range of acetone to isopropyl alcohol volumes can vary from approxiametely 5-to-1 to about 100-to-1 respectively.

The resulting suspension of precipitated salt is then vigorously stirred so as to ensure no forMation of oily residue. Isolation of the sn-glycerol-3-phosphate salt is ideally effected by filtration.

To ensure complete removal of volatile solvent contaminants it is preferred that the crystalline salt be dried in vacuo prior to both analyses. The analysis of the resulting N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate is best done by using either one or most preferably both of the two following procedures~

The first preferred analytical procedure is an enzymatic assay for sn-glycerol-3-phosphate. The enzymatic assay, according to Bergmeyer et al. in Biochem. Z, Vol 33, 471 (1961), depends on the quantitative conversion of sn-glycerol-3-phosphate to l-phospho-3-hydroxyacetone by an NADH-requiring dehydrogenase, preferably sn-glycerol-3-phosphate dehydrogenase. A second preferred analytical procedure for the N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate is the high-resolution proton magnetic resonance spectrum of the salt. This analytical test allows for the accurate quantification of the equivalents of dimethylaminopyridine per equivalent of sn-glycerol-3-phosphate in the salt. The preferred ratio of N,N-dimethyl-4-aminopyridine to sn-glycerol-3-phosphate is 1:1. It is preferred that the purity of the N,N-dimethyl-4-aminopyridinium sn-~lycerol-3-phosphate salt as determined by both analyses be greater than 98%.

If the aforementioned analyses indicate that th~ salt does not meet the preferred purity criteria, a second purification step should be performed. The preferred method is to resuspend the crystalline salt in isopropyl alcohol and stir the suspension vigorously overnight. The volume of isopropyl alcohol is not critical but the preferred volume will be about 20 ml per gram of salt. Filtration of the suspension after prolonged stirring gives material of greater than 98% purity when reassayed using the two aforementioned analytical procedures. The major contaminant, N,N-dimethyl-4-aminopyridinium hydrochloride, is completely soluble in the isopropyl alcohol while the sn-glycerol-3-phosphate salt is only minimally soluble.

~cylation of the crystalline and anhydrous N,N--dimethyl-9-aminopyridinium sn-glycerol-3-phosphate is effected as follows:

The N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate salt is dissolved in an organic solvent and the base N,N-dimethyl-4-aminopyridine and a fatty-acid anhydride are added to the solution. The organic solvent used may be either dichloromethane, chloroform, or carbon tetrachloride with chloroform being most preferred. The ra-tio of sn-glycerol-3-phosphate salt to base and the ratio of sn-glycerol-3-phosphate salt to fatty-acid anhydride is not crucial but the preferred ratios are 1 molar equiYalent of sn-glycerol-3-phosphate salt to 9 molar equivalents of N,N-dimethyl-4-aminopyridine, and 1 molar equivalent of sn-glycerol-3-phosphate salt to 5 equivalents o~ fatty-acid anhydride.

While the reaction ~ime is also not crucial, it has been found preferable to employ a reaction time of approximately 72 hours. The reaction temperature may vary from room temperature (25C) to solvent reflux temperatures and is not crucial. For reactions containing myristic anhydride ~C=14) and palmitic anhydrids (C=16), the preferred reaction temperature is 25C, while for longer-chain atty-acid anhydrides, which tend to be less soluble, such as stearic anhydride (C=18~, the preferred reaction temperature is on the order of 60C so as to aid in dissolution of the fatty-acid anhydride.

G`3 ~
he reactio~ may be conveniently terminated by quenching with a methanol: H20 mixture. The ratio of methanol to water can be varied from 1 to 10 parts methanol to 1 part water. It is most preferred to use a ratio of 1 part methanol to 4 parts water, although this ratio is not critical, for quenching the acyla~ion reaction. The resultant diacyl phospha~idic acid products may be purified from the reaction mi~ture by a preferred sequence of 1) an acid wash to remove residual dimethylaminopyridine and 2) chromatography to remove the fatty acid hydrolysis by-products. It is preferred to wash an organic solution of the hydrolyzed reaction mixture with an acidic aqueous solution, most preferably lN HCl, so as to remove the excess base. Subsequently, the organic layer, containing product phosphatidic acid and residual fatty acids, may then be advantageously fractionated by a variety of chromatographic techniques such as those employing silica gel ~upta et al.).

Once isolated, the diacyl phosphatidic acid may be further purified by recrystallization from organic solvents, wikh the preferred solvents being methanol and ethanol.

Further understanding of the invention will he had by a detailed study of the following examples. These examples are given by way of illustration and are not intended to limit the invention.

xam~lel : Preparation of N,N-Dimethyl-4-aminopyridinium sn-Glycerol-3-Phosphate (G-3-P (DMAP)l) To 100 g of sn-glycerol-3-phosphate, enzymatically prepared from sn-glycerol and A~P according to Whitesides et al. in J.
Am. Chem. Soc., Vol 107, 7019-7023 (1985), and dissolved in 500 ml of methanol, was added 66 g of N,~-dimethyl-4-aminopyridine ~DMAP) obtained rom Reilly Tar Co. The methanolic reaction mixture was vigorously stirred at room temperature until all the DMAP was in solution.

The methanol was removed in vacuo to yield a heavy oil. The oil was dissolved in 150 ml of isopropyl alcohol and 1.5 liters of acetone was added to the solution. An oily precipitate was formed upon addition of the acetone. The precipitate was dispersed in solution by vigorous stirring for 18 hours.
Filtration of the precipitate and in vacuo drying yielded a fine crysta1line, off-white solid ~49, 60% molar yield).

The crystalline G-3-P (D~AP)l so produced was analyzed by enzymatic assay and found to be 88% G-3-P by weight. A
H-NMR spectrum indicated the contaminating material to be ~MAP-HCl salt. The impure material was resuspended in 500 ml of isopropyl alcohol and stirred overnight at room temperature. The suspended material was collected by conventional paper filtration and dried in vacuo to yield G-3-P
(DMAP)l (rn.p. 128-130C) which was of 100% purity as judged by enzyrne assay. A lH-NMR confirmed the structure of the salt: (D20~ppm 7.9 ~d)2~, 6.~(d~2H, 3.82(m) and 3.64(m)5H, 3.1(s)6H.

Example 2: Preparation of Dipalmitoyl Phosphatidic Acid (DPPA) by Acylation of N,N-Dimethyl-4-Aminopyridinium sn-Glycerol-3-Phosphate (G-3-P
(DMAP)l~

In this example the acylation of G-3-P (DMAP)l was carried out at elevated ternperatures (60C3. G-3-P (DMAP)l ~5y, 15.2mmol) fro~ Example 1, palmitic anhydride (37.5g, 75.9mmol) ~Aldrich) and DMAP (16.7, 137mmol) (Reilly Tar Co.) were suspended in 150 ml of dry chloroform. The reaction mixture was heated at reflu~ with vigorous stirring for 2 hours. At this time all reactants were in solution. The reaction mixtura was then stirred at room temperature for 18 hours. The reaction mixture was then hydrolyzed by addin~ an equal volume of MeOH:H2O ~4:1). The resultant aqueous phase was acidi~ied to pH 1 with the addition of concentrated HCl.

The organic layer was separated and washed three times with egual volumes of MeOH:H2O:CHC13 (48:47:3). The organic layer was collected and reduced in vacuQ to one hal~ its original volume. This volume contains the desired dipalmitoyl phosphatidic acid and the fatty acid hydrolysis byproducts.
Such byproducts may be advantageously removed using silica gel chromatography techniques as described by Gupta ~ al . The ~ractions containing the dipalmitoyl phosphatidic acid are then ideally combined and then concentrated in vacuo to remove the chromatography solvent. The resulting solid may then be further purified by acetone precipitat.ion or methanol recrystallization. Characterization of the resultant product can be expected to yield a chemical purity of about 99% with a melting point of 155-156C. ~ipalmitoyl phosphatidic acid thusly prepared will ideally be characterized by the following Rf = 0.39, silica gel 60F254 (Merck), ~Cl3-MeOH-H2~
(65:25:4); 400 MHZ ~H NMR (D20) ppm 5.2 (d3 lH, 4.38 (d) lH, 4.16 (dd) lH, 3.92 (m) 2H, 2.29 (dd) 4H, 1.20 (m3 56H, 0.87 (t) 6H.

Following a detailed study of the foregoing, those skilled in the art will readily recognize that numerous changes may be made with respect to reaction conditions ~e.g. buffers, temperatures, reaction times) without departing from the substance, spirit or scope of the present invention.

Claims (13)

1. A method of making crystalline N,N-dialkyl-4-aminopyridinium salts from sn-glycerol-3-phosphate or an analogue thereof comprising the steps of a) reacting a free acid form of sn-glycerol-3-phosphate or an analogue thereof with an N,N-dialkyl-4-aminopyridine to form a salt solution;
b) obtaining the N,N-dialkyl-4-aminopyridinium sn-glycerol-3-phosphate salt from the salt solution of step a) by precipitation with an organic solvent, and optionally;
c) repeatiny step b) as required to obtain a predetermined purity.

2. The method of claim 1 wherein said analogue of sn-glycerol-3-phosphate reacted in step a) comprises the structure wherein X is selected from the group consisting of -Cl, -Br, -SH, -OCH3, -CH2OH and -CH2CH3.

3. The method of claim 1 wherein sn-glycerol-3-phosphate is reacted in step a).

4. The method of claim 2 wherein the N,N-dialkyl-4-aminopyridine reacted in step a) comprises N,N-dimethyl-4-aminopyridine.

5. The method of claim 3 wherein the N,N-dialkyl-4-aminopyridine reacted in step a) comprises N,N-dimethyl-4-aminopyridine.

6. The method of claim 2 wherein said N,N-dialkyl-4-aminopyridine reacted in step a) comprises the structure wherein Y is (i) wherein R1 and R2 each represent a hydrocarbon group having from 1 to 6 carbon atoms, or (ii) wherein m is 2-6.

7. The method of claim 3 wherein said N,N-dialkyl-4-aminopyridine reacted in step a) comprises the structure wherein Y is (i) wherein R1 and R2 each represent a hydrocarbon group having from 1 to 6 carbon atoms, or (ii) wherein m is 2-6.

8. The N,N-dialkyl-4-aminopyridinium salt produced by the method of claim 2.

9. The N,N-dialkyl-4-aminopyridinium salt produced by the method of claim 3.

10. A method of making phosphatidic acids comprising the steps of reacting a) a compound represented by the following formula:

wherein X is selected from the group consisting of -OH, -Cl, -Br, -SH, -OCH3, -CH2OH and -CH2CH3 and ;

wherein Y is (i) wherein R1 and R2 each represent a hydrocarbon group having from 1 to 6 carbon atoms or (ii) ; wherein m is 2-6;

and wherein n is 1 or 2 with;

b) a fatty acid anhydride represented by the following formula wherein R3 and R4 are identical or different and each represents a saturated or unsaturated aliphatic hydrocarbon group having from 4 to 21 carbon atoms;

c) in the presence of a basic catalyst comprising N,N-dimethyl-4-aminopyridine.

11. The method of claim 10 wherein X is -OH.

12. The method of claim 11 wherein n is 1.

13. The method of claim 12 wherein Y is structure (i) and wherein R1 is -CH3 and R2 is -CH3.