XI. SYNTHESIS OF PHOSPHONOLIPID METABOLITES. L- ~~L-GLYGERYL- (2-TRIMETH'IILAMMONIUMETHYI,]PHOSPHQNATE1

ERICEH BAER~ AND RANGA ROBINSON The Subdegartmeat of Synthetic Chemistry i n Relatiota to Medical Research, Banting and Bes%

Department of Medical Research, Utaiversity of Tormtcp, Toronto, Ontario Received March 27, 198'9

The synthesis of 2 possible intermediate in phssphsnolipid metabolism, viz. ~-a-glyceryl-(2-trimethylan~moniurnethyl)phosphonate, a phosphonic acid ana- logue of L-a-glycerylp%aosphorylc%aoline, ie $ecribed. The compound was obtained by condensation of macetone glycerol with (&bromwthyl)metaphosphonak in boiling benzene, removal of the acetone group of the condensation product by acid hydrolysis, treatment of the hydrolysis product in dimethylformamide with trimethylarnime a t 60-62" for 3 days, isolation of E-a- Iyceryl-(2-trimethylam- moniumethy1)phosphate as cadmium chloride complex. kernoval of the cadrniunl chloride was effected with a mixture of Amberlites IR-45 and IRC-50. A procedure for the preparation of (2-bromwthyl)metaphmphonate is reported.

The recent discovery of the existence in nature of phosphsnic acid am(~Log~es of phospholipids (1-5) has aroused speculation as to the pathways of their biosynthesis, metabolism, and biological functions. The synthesis of phos- phonolipids likely to occur in nature, and of their possible biological inter- mediates is being undertaken in this laboratory to provide pure, individual phosphonolipids of known structure, both for a study of their biological functions and as reference compounds for the elucidation of the chemical structure and configuration of the natural compounds. We have already described the synthesis of phosphonic acid analogues sf L-a-cephalins (6, a) , diether L-a-cephalins ($) , L*-lecithins (9, lo), and diether L-a-lecithins (1 1, 12). Here we report the synthesis of a phssphonic acid analogue of L-a-glyceryl- phmphoaqrlcholine, viz. ~-a-glycery%-(2-trimethylammoniumethyl)phosphonate (Reaction Scheme 1, formula V). This compound possesses further interest as reference compound for the identification of naturally occurring phosphonolipids (131, as a potential inhibitor of phospholipid metabolism, and as starting material for the chemical or enzymatic synthesis of phosphsnic acid analogues of saturated and unsaturated L-a-lecithins. The chemical synthesis of these via the cadmium chloride complexes of L-a-glyceryl-(2-trimethylammoniumethyl) phosphonate is in progress in this laboratory.

Methods Several methods for the synthesis of L-a-glycery1-(2-trimethylammoniun1-

ethy1)phosghonate can be envisaged. The one appearing most promising to us involved the following series of reactions: phosphonylation of D-acetone glycerol with (2-brornoetkyI)phosphonic acid monochloride in the presence of

IAbbreviation, L - ~ - G P ~ C . This work was supported by a grant (HE-08'980-81) from the National Heart Institute,

United States Public Health Services, Betheda, Md., U.S.A.

Canadian Journal of BiochemistryaP. Volume 45 (1967)

I 02

P-C

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-H I

BAER AND ROBINSON: PHOSPHONOLIPEDS. XH 1749

an organic base, e.g. trimethylamine or triethylamine; removal of the acetone group of the phosphonylation product by acid hydrolysis; quarternization of the hydrolysis product with trirnethylamine; and isolation of L-a-glyceryl-(2- trimethylammoniumethy1)phosphonate as cadnniurn chloride consplex. This procedure gave some cadmium chloride complex of L-a-GPnC but in low yields ('7-12yo) and of unsatisfactory purity . Various changes in the phosphonyla tion procedure, including the use of other organic bases, e.g. pyridine or quinoline, improved neither the yield nor the quality of the cadmium chloride connplexes of L-~-C;PXI C.

The observation that (2-bromoethy1)phosphonic acid in the presence of an organic base loses readily some of its organicaHIy bound bromine, prompted us to attempt its esterification with acetone glycerol in the absence of a base. T o save the time of other workers, we wish to mention tw~3 of the procedures that were investigated but found to be of limited value: (a) an equirnolecular ~nixture of acetone glycerol and (2-hromoethj7l)phosphsnlc acid monochloride was kept in a desiccator under reduced pressure over potassium hydroxide to absorb released hydrogen chloride; ( b ) a solution of acetone glycerol and (2-brsn~oethyl)phosphonHc acid in a mixture of petroleum ether (b.p. 30-60") and acetone (I: B v/v) containing toluene #-sulfonie acid tvas refluxed, and the water of condensation was removed by azeotropie distillation. Subsequent removal of the acetone group of the phosphonylation products of both pro- cedures, quaternization of the acetone-free compounds with trimethylamine, and treatment of the reaction products ~erith cadmium chloride gave cadmium chloride complexes of ~-a-glyceryl-(2-trimethylammoniufnethy~)phospRonate in overall yields of 20 and 26%, respectively. The analytical values, however, remained urssatisfactory .

LVe succeeded finally in obtaining fair yields of analytically pure cadmium chloride complexes sf ~-a-glyceryl-(2-trimethylammoniui~~ethyl)phosphonate (see Reaction Scheme 1) by condensing D-acetone glycerol with (2-bromo- ethyl) metaphosphonate in boiling benzene, removing the acetone group of the condensation product by acid hydrolysis, quaternizing the hydrolysis product in dimethylformamide with trimethylamide a t 60-62O for 3 days, and treating the reaction product with an alcoholic solution of cadmium chloride. Two types of cadmium chloride complexes were obtained depending ow the manner of their preparation: one with a ratio of phosphonate to cadmium chloride of 1 : 1 , the other with a ratio of 2: 3. Removal of the cadmium chloride with a mixture of Amberli tes I R-45 and I WC-50 gave L-a-glyceryl-(2-trimethyl- anlmoniumethyl)phosphonate in overall yields ranging from 30 to 34% of theory. The D-isomer and the racemate of a-GPnC are obtainable by the same procedure with 1,-acetone glycerol or IIL-acetone glycerol, respective1 y, as starting materials.

The (2-bromoethy1)metaphosphonate was prepared by one of two proced- ures described by Michaelis and Wothe (14) for the synthesis of p-methyl- phenylmetaphosphonate, i.e. by heating a solution of equimolecular amounts

1%50 CANADIAN JOURNAL OF BIOCHEMISTRY. VOE. 45, 1967

of (2-bromoethy1)phosphoslic acid and its dichloride in benzene to 75" until the evolution of hydrogen chloride ceased. The hydrogen chloride was removed in a stream of nitrogen.

Experimental Part Ma terials

D-Acetone glycerol was prepared by the method of Baer and Fischer (15, 16). If the D-acetone glycerol was to be kept for more than several weeks, i t was stored as a soIution in dry ether over anhydrous sodium sulfate. Shortly before use, the ether was distilled off under reduced pressure, and the remaining acetone glycerol was distilled in zacuo, the fraction distilling from 70 to 73" a t 9 mm pressure being collected. The 2-bromoethylphosphonic acid was prepared via its anilinium salt as described by Baer and Stanacev (101, except that the 2-bromoethylphospkonic acid was crystallized once more from boiling chloro- form in the presence of charcoal. The acid was obtained in colorless, eentimeter- long, narrow prisms, m.p. 94.5-95.5".

(2-Bromse~~k)mehphosphonate Into a thoroughly dried, three-necked, 250 m% round-bottomed flask with

ground joints, and equipped with a calcium chloride tube were placed 3.78 g (20 mmoles) of 2-bromoethylphosphornic acid, 50 ml of thionyl chloride, and 6.3 ml of anhydrous dimethylformamide. The flask was closed, and the mixture was stirred magnetically for 16 h. Then the flask was attached to a receiver, and the thionyl chloride was distilled off under anhydrous conditions a t reduced pressure from a bath a t 35-40' temperature. The last traces of thionyl chloride were removed a t a pressure of 0.1 mm Hg. T o the crude (2-bromoethy1)phos- phonic acid dichloride were added 110 ml of anhydrous benzene and 3.78 g (26 mmoles) of 2-bromoethylphosphonic acid. The flask was provided with a dropping funnel and a gas inlet tube, attached to a water-cooled Eiebig conden- ser, protected by a calcium chloride tube, and immersed in an oil bath a t 7&75O The reaction mixture was kept a t this temperature for 6 h. T o facilitate the removal of the hydrogen chloride, the solution was stirred magnetically and a stream of thoroughly dried nitrogen (concentrated sulfuric acid) was passed through the apparatus. In time, a precipitate formed which, when isolated gave the correct analysis for (2-bromoethyl)metaphssphonate. For preparative purposes it is not necessary to isolate the metaphosphonate.

Anal. Caled. for C2H402PBr (171): P, 18.12; Br, 46.75. Found: P, 18.08; Br, 46.41.

~-a-Glymry1-(2-brommthyd)~kos~honic Acid To the suspension of (2-bromoethy1)metaphosphonate in benzene, freshly

prepared as described above, mPas added in the course of 80 min a solution of 5.29 g (40 mmles) of D-acetone glycerol in 50 ml of anhydrous benzene. The temperature of the oil bath then was raised to 95', and the mixture was refluxed for 6 h while nitrogen was passed through the apparatus. The clear, slightly

BAER AND ROBINSON: PHOSPHONOLIPI[DS. XI 1751

br~wrrish solution was evaporated under reduced pressure f r ~ m a bath a t 30- 35", and the residue was freed of traces of dimethylformamide a t a pressure of 0.1 rnm Hg. The viscous oil was dissolved in 75 ml of water, and to the solution was added cautiously a 10% aqueous solution of lead acetate until no further precipitate of the lead salt of (2-bromoethyl)phosphonic acid was formed. The precipitate was centrifuged down, washed with two 25-ml portions of water, and the combined aqueous solutions were freed of lead ions with hydrogen sulfide. The lead sulfide was removed by filtration, and the filtrate was freed of hydrogen sulfide in the vacuum ~f a water aspirator. The solution, after standing for 16 h a t room temperature, was evaporated under reduced pressure a t a bath temperature of 30-35", and the yellow viscous oil was dried in a vacuum desiccator over sodium hydroxide and phosphorus pentoxide a t a pressure of 0.1 mm Hg for 36 h. The remaining material, consisting mainly of L-a-glyceryl- (2-bromoethyl) phosphonic acid and some free glycerol, the latter the hydrolysis product of unreacted acetone glycerol, without further purifica- tion was converted into ~-ae-glyceryl-(2-trimethylammoniumetkyl)phosphonate as described below.

T o obtain pure L-a-glyceryl-(2-brornoethy1)pkaosphonic acid, a solution of the crude material (1.7 g) in chloroform was passed through a column (2.5 cm width) of 5 3 g of silicic acid, and the column was eluted with 250 ml of chloro- form (fraction I) , 250 ml of cklor~form containing 5y0 of methanol (fraction 21, 488 ml of chloroform containing 10% of methanol (fraction 3), and 600 ml of chloroform containing 20y0 methanol (fraction 4). Evaporation of fractions 3 and 4 under reduced pressure, and drying of the residues a t a pressure of 0.1 mm Hg, gave 1.1 g of L-a-glywryl-(2-bromoethy1)phosphonic acid and 8.27' g of glycerol, respectively.

Anal. Calcd. for CgHa206PBr (263.1): P, 11.77 ; Br, 30.98. Found: P, 11.58; Br, 29.82.

~-a-Glyceryl-(~-trime~hylammoniumethyl)pho~phon~~t - Cadmium Chloride Comfilexes

The crude L-a-glyceryl-(2-bromoethy1)phosphonic acid was dissolved in 30 ml of anhydrous dimethylformamide, and the solution was transferred to a pressure bottle, into which was distilled, with cooling, 30 ml of trimethylamine, with the gas passing through a column of soda lime. The bottle was sealed, and kept for 3 days in an oil bath a t 60-62'. The initially clear solution became turbid after about 30 min and a heavy, viscous oil formed within 2 days. At the end of the third day, the reaction mixture was brought to room temperature and the dimethylformamide solution was decanted. The remaining oil was washed with a small amount of dimethylformamide, and dissolved in 25 ml of methanol. The solution was evaporated under reduced pressure a t 30-35', and the residue was kept for 30 min a t a pressure of 0.1 mm Hg. The strongly acidic material, dissolved in a mixture of 20 ml of water and 10 ml of methanol, was stirred for 10 min with 3 g of silver carbonate. The silver salts were removed by

1752 CANADIAN JOURNAL OF BIOCHEMISTRY. VQL. 45. 1967

centrifugatiora, washed with 30 ml of the solvent rnixture, and the combined supernatant solutions, now weakly basic, were stirred for 10 min with 28 rnl of Amberlite IW-120. The mixture was filtered, the filtrate n-as evaporated under reduced pressure (bath 30-35"), and the residue was dried a t a pressure of 8.1 mm Hg.

Isslafion of ~-~-Glycery%-(~-t~imefhyIawm~nZz~anefh~~I)phospBzo~ate crs 2:S-Cadmium Chloride Complex

The above material was dissolved in 30 n ~ l of 99% ethanol by adding 3-4 drops of water. T o the solution was added slowly and with stirring, a saturated solution of cadmium chloride in 95y0 ethanol until rao further precipitate formed; then the mixture was kept a t 6" for 1 h. The dense, white precipitate was centrifuged off, washed with ice-cold 99% ethanol, and dried a t room tem- perature over phosphorus pentoxide a t a pressure of 0.1 mrn Hg for 15 11. The L-a-glyceryl-(2-trime thylammoniumethyl) phosphonate cadmium chloride @om- plex weighed 8.0 g. Overall yield calculated for macetone glycerol was 37.5% ; [alD -0.6" (c, 7 in water).

Anal. Calcd. for [CJIaz06NP]z[CdC12]a (1868.5): C, 17.98; H, 4.15; N, 2.62; P, 5.79; Cd, 31.56. Found3: C, 16.92, 18.79; H, 4.11, 4.25; N, 2.57, 2.63, 2.52; I?, 5-82, 5.74, 5.98; Cd4 31.49.

Vicinal-Glycol Titration with Periodi~ Acid6 A sample of the 2:3-cadmium chloride connplex in aqueous solution was freed

sf cadmium chloride by the addition of potassium carbonate. The cadmium carbonate was removed by centrifugation and washed with snlall amounts of water. The combined aqueous solutions were made up to a known volun-ne, and the content of the monoester was ascertained by a phosphorus determination. Bar aliquot of the solution containing 8.0124 mmole of the monoester consumed 0.0137 mmole (110%) of theory of periodic acid.

Isolation of ~-a-G~y~e~yd-(2-trimethylammo~i~~methyl)~hos~fioaaa~te as f :l -Cadmia~m Chloride Complex

To 6.73 g of crude L-a-glyceryl-(2- trimethylam~noniumethyl) phosphonate dissolved in 125 nil of 99YQ ethanol was added slowly and with stirring an ethmolic solution of cadmium chloride, which had been prepared by dissolving 9.80 g of cadmium chloride (2.5HaO) in 6.7 ml of water and adding 110 ml of 99y0 ethanol. After standing for I h a t +6", the dearse, white precipitate was filtered off with suction, washed on the filter successively with small amounts of ice-cold 95% ethanol and ether, and dried a t room temperature over phos- phorus pentoxide a t 0.1 m n ~ Hg for 8 h. The cadmium chloride complex weighed 7.94 g (69.1% of theory). Overall yield calcuIated was 44.8%, based on ace tone glycerol.

%,$nalytical values sf several independent preparatisms. 4Deterrmimd as cadmium carbonate. T h e vicignal-glycol determinations were carried out by the rne thd of Fleury as modified by

B'orris, Ellis, and Maynard (17). See footnote 42 of a publication by Baer and Stancer (18).

BAER AND RBBINSOK: PHOSPHBNBLIPIBS. XI 1753

Anal. Calcd. for [C8Hz206NP][CdCla] (442.6): C, 21.71; H, 5.01; N, 3.16; P, 7.00; Cd, 25.40. Founds: C, 22.11; 22.08; H, 5.09, 4.85; N, 3.04; P, 7.10; Cd, 25.03.

Bricinal-Glycol Debrminalioaz An aqueous solution of the 1: 1-cadmium chloride complex was freed of cad-

mium chloride by the addition of potassium carbonate. An aliquot of the solution containing 0.049 mmole of L-a-glyceryl-(2-trimethylamm~niumeth~l) - phosphonate, on the basis of a phosphorus determination, consumed 0.050 mmole (102% of theory) of periodic acid.

L-a-G&yceryL- (5'-trimeth ylammoaziumetTzyl)phosphonab A solution of 2.13 g of the 2:3-cadmium chloride complex in 100 ml of water,

to which 20 g each of the Amberlites IR-45 and IRC-50 had been added, was stirred for 1 h. The resins were filtered off and washed with a small amount of water. If the filtrate still contained cadmium chloride, it was treated once more for 15 min with a mixture of 10 g of each of the Amberlites. The filtrate was cleared by centrifugation and evaporated under reduced pressure from a bath a t 35". The remaining colorless viscous oil, on drying a t room temperature over phosphorus pentoxide a t a pressure of 0.01 mm Hg, solidified gradually. The ~-~-~l~cer~l-(2-trimethylammoniumethyl)phosphonate, a highly hygroscopic white solid, weighed 0.82 g ('79.7yc of theory). Overall yield was 29.9% calcula- ted for D-acetone glycerol ; [a]B - 1.5" (c 3.6 in water). A 0.1 M solution of the compound in water free of carbon dioxide had a pH of 4.3 a t 24". The com- pound is readily soluble in water, methanol and 99Tc ethanol, but insoluble in acetone, ether, chloroform, or benzene. For analytical purposes the axsaterial was dried a t 56O (boiling acetone).

Anal. Calcd. for CsH,2OeNP (259.2): C, 37.06; H, 8.55; N, 5.40; P. 11.95. Found: C, 37.13; H , 8.61; N, 5.42; P, 11.99.

Vicinal-Glycol Titration with Periodic Acid In the titration, 0.240 mmole of the glycerol ester consumed 0.237 mmole

(9$.'77c of theory) of periodic acid. In a similar manner, a solution of 0.550 g of the 1: 1-cadmium chloride com-

plex of ~ - a - G P n c in 30 ml of water, on treatment with 10 g each of Amberli tes IR-45 and IRC-50, evaporation of the filtrate under reduced pressure a t 35", and drying the remaining material over phosphorus pentoxide a t room tem- perature and a pressure of 0.01 mm Hg, gave 0.249 g (77.3% of theory) of L-a-glyceryl-(2-trimeth ylammoniumethyl) phosphonate.

Anal. Calcd. for CsHaeO6NP (259.2): N, 5.40 ; P, 11.95. Found: N, 5.34; P, 11.91.

The compound is fairly stable in aqueous solution. If, however, the solution is being kept for longer than 6 weeks, even a t a temperature as low as 6", traces of a second compound may be found.

CANADIAN JOURNAL OF BIOCHEMISTRY. ITOE. 45. 1967

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