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  1. 1. THE JOURNALOF BIOLOGICAL CHEMISTRY Vol. 236,No. 9, September 1961 Printed in U.S.A. N , IV-Dimethylcephalins * I. SYNTHESIS OF DISTEAROYL L-(r-GLYCERYLPHOSPHORYL- (N ,iV-DIMETHYL)ETHANOLAMINE ERICH BAER AND SRIPADA K. PAVANARAM From the Subdepartment of Synthetic Chemistry in Relation to Me %a1Research, Bunting and Best Department of Medical Research, University of Toronto, Tore. to 5, Ontario, Canada (Received for publication, May 4, 1961) Studies in recent years by Horowitz (l), Wolf and Nyc (2), N , N-dimethylcephalins, unable to perform the biological func-Hall and Nyc (3), Artom (4), Crowder and Artom (5), Artom, tions of the normal end products of synthesis, are perhaps re-Lofland, and Oates (6), and Bremer and Greenberg (7) have pro- moved and may thus contribute to the demyelination of thevided strong evidence for the biological formation of structural nervous tissue. Similar considerations may also apply to corre- Downloaded from www.jbc.org by guest, on December 27, 2010analogues of cephalins in which one or both hydrogen atoms sponding sphingomyelin precursors.of the amino group are replaced by a methyl group. The N- To make available reference compounds for the elucidation ofmethyl derivatives of cephalin have been observed both as the structure and configuration of naturally occurring N-methyl-end products of an incomplete synthesis of lecithin by the mu- substituted cephalins, and as substrates for biological investiga-tant strain 47904 of Neurospora crassa, and as short lived inter- tions, the synthesis of N-monomethyl- and N ,N-dimethyl-mediates in the synthesis in viva and in vitro of lecithin in the cephalins of assured structure and configuration was undertaken.presence of N-monomethyl- and N ,N-dimethylethanolamine or The present publication, the second in this series, reports thetheir phosphate esters. Except for the isolation of an N ,N- synthesis of an optically active N , N-dimethylcephalin.dimethylcephalin by Artom (4), most of the earlier evidence forthe natural occurrence of N-methyl derivatives of cephalin was EXPERIMENTAL PROCEDUREcircumstantial. It has been reviewed by us in more detail in a As far as the authors are aware, the synthesis of optically ac-recent publication reporting a chemical synthesis of an N-mono- tive CY-N,N-dimethylcephalins has not yet been reported.methylcephalin (8). Since then, several other publications have However, in a recent publication by Bremer and Greenbergappeared confirming the role of the N-monomethyl- and N ,N- (11) on the methyl-transferring enzyme system of microsomesdimethylcephalins as normal products of cell metabolism. Hall in the biosynthesis of lecithin, it has been announced that Dr.and Nyc (9) identified as N-monomethyl- and N ,N-dimethyl- D. Shapiro has succeeded in synthesizing racemic (N ,N-di-cephalins two phospholipids which they had isolated from the methyl)distearoyl-ar-cephalin. Details of the procedure are notmutant strain 47904 of N. crassa. Artom and Lofland (lo), yet available. A dl-(N ,N-dibenzyl)cephalin has been reportedby means of a (Y-labeled phosphatidyl dimethylethanolamine, by Verkade and Stegerhoek (15, 16) as an intermediate in theshowed that it is converted directly to lecithin by methylation synthesis of racemic cr-cephalins. It was obtained by theof the intact phospholipid, and thus is an immediate precursor condensation of silver benzyl diacyl-glycerol-a-phosphate withof lecithin. Bremer and Greenberg (11) confirmed their former 2-(dibenzylamino)ethyl bromide in boiling benzene. It is un-conclusion (12) that choline is synthesized by a progressive likely, however, that the N ,N-dibenzylcephalin is a physiologi-methylation of the amino group of cephalin. Work reported cal compound.by Gibson, Wilson, and Udenfriend (13) essentially corroborates A series of investigations in this laboratory have culminatedtheir findings, and offers further support for the reaction mecha- in the synthesis of the ~-a isomers of glycerophosphoric acidnism proposed by Bremer and Greenberg. (17, 18), saturated and unsaturated lecithins (19, 20) and The N-methyl derivatives of cephalin are of interest for still cephalins (21, 22), a saturated phosphatidyl serine (23), and theother reasons. For instance, in multiple sclerosis it is known on basic structural units of these phosphatides, viz. glycerylphos-histochemical grounds that there is a disturbance of the lipid phorylcholine (24)) glycerylphosphorylethanolamine (25) andmetabolism. According to a hypothesis advanced some years glycerylphosphorylserine (26). Comparison of the syntheticago by Sperry and Waelsch (14), the disturbance is the result of products with the corresponding natural products establishedan imbalance in the anabolism and catabolism of the normal that naturally occurring glycerophosphatides possess the a!lipids of the nervous tissue. An alternative mechanism has structure and L configuration. Naturally occurring N-mono-been suggested recently by us (8). It assumes that the synthe- methyl- and N , N-dimethylcephalins, whether considered assis of lecithin does not go to completion for the lack of certain derivatives of cephalin or as precursors in the biosynthesis ofenzymes responsible for the stepwise methylation of cephalin lecithin, would be expected to possess the same structure andto lecithin. The intermediates, viz. the N-monomethyl- and configuration. Hence, the N-methyl and N, N-dimethyl deriva- * Alternative name, phosphatidyl (N,N-dimethyl)ethanola- tives of cephalin were prepared possessing the a! structure and Lmine. configuration. In deciding on stearic acid as substituent for the
  2. 2. September 1961 E. Baer and X. K. Pavanaram 2411 CH,(CH&,COO-CHz CH3(CHz)&OO-CHz CH3(CH&,COO-i-H 0 CICHg-CHZN(CH& CH@H&COO-C-H 0 , H&-O-;-O Ag in boiling benzene H&-O-k-O-CHz-CHzN(CH& I OCHPC~,HS 0 CH#sHb XI XII CH3(CHz)&OO-CHz Pd Hz CH3(CH&COO-C-H 0 --I H,C-O-h-O-CH,-CH,~7H~)p !H XIII Distearoyl n-a-glycerylphosphoryl-N,N-dimethylethanolamine SCHEME 1 Synthesis of N,N-Dimethylcephalins N , N-dimethylcephalin to be synthesized, we took into considera- The unsaturated lecithin was obtained by heating a solution Downloaded from www.jbc.org by guest, on December 27, 2010 tion that a considerable part of the saturated as well as unsatu- of the barium salt of n-Lu-dioleoylglycerylphosphoryl ethylene rated fatty acids of naturally occurring glycerophosphatides chlorohydrin and of trimethylamine in benzene to 60” for 4 have unbranched chains of 18 carbon atoms, and, thus, on reduc- days. However, on treating n-ar-distearoylglycerylphosphoryl tion would yield phosphatides with stearic acid as the main sub- ethylene bromohydrin with dimethylamine under similar experi- stituent. mental conditions, a mixture of reaction products was formed In a recent publication (S), we described the chemical synthe- that contained little or none of the desired N ,N-dimethylcepha- sis of an L-cr-(monomethyl)cephalin. The particular procedure lin. chosen for its preparation was one that experience has shown We finally succeeded in obtaining the distearoyl L-~-N, N-di- can be relied upon to give structurally and optically pure phos- methylcephalin by condensing the silver salt of distearoyl pholipids, an important consideration if it is impossible to check n-a-glycerophosphoric acid monobenzyl ester with N ,N-di- the optical purity of the synthetic compound by comparison methylaminoethyl chloride, and removing the protective benzyl with natural material. The N-monomethylcephalin was group of the phosphoric acid by catalytic hydrogenolysis (Reac- obtained by phosphorylating ~-a ,@-distearin with phenylphos- tion scheme: XI ---f XII -+ XIII). Our procedure for the syn- phoryl dichloride and pyridine, esterifying the resulting distea- thesis of optically active ac-(N ,N-dimethyl)cephalins, which re- royl L-cr-glycerylphenylphosphoryl chloride with N-carbobenz- sembles in its latter stages that of Verkade et al. (15, 16) for oxy-N-(methyl)ethanolamine, and removing the protective the preparation of racemic or-cephalins, is outlined in totality by phenyl and benzyl groups by catalytic hydrogenolysis. Unex- the following sequence of intermediate compounds: D-IDannitOl pectedly, however, synthesis of the N , N-dimethylcephalin (I) + 1,2,5,6-diacetone n-mannitol (II) --) acetone D-&Ceral- proved much more difficult. When we attempted to prepare dehyde (III) -+ n-acetone glycerol (IV) -+ c&-toluenesulfonyl)- the distearoyl L-~-N, N-dimethylcephalin by the procedure that n-acetone glycerol (V) -+ cr-iodo-acetone-n-propylene glycol gave n-a-(N-methyl)cephalin, using N , N-dimethylethanolamine (VI) -+ cu-iodo-n-propylene glycol (VII) --) distearoyl n-a-iodo- instead of N-carbobenzoxy-(N-methyl)ethanolamine, we were propylene glycol (VIII) -+ distearoyl n-a-glycerophosphoric acid unable to obtain the intermediate, viz. distearoyl n-a-glyceryl- dibenzyl ester (IX) -+ distearoyl n-or-glycerophosphoric acid phenylphosphoryl-N , N-dimethylethanolamine in a practical monobenzyl ester sodium salt (X) -+ distearoyl n-a-glycero- yield. The mixture of reaction products after removal of pyri- phosphoric acid monobenzyl ester silver salt (XI) -+ distearoyl dine contained barely one-third of the theoretical amount of L-cu-glycerylphosphoryl-N, N-dimethylethanolamine benzyl ester nitrogen. Attempts to isolate the pure intermediate by column mu + distearoyl n-a-glycerylphosphoryl-N , N-dimethyl- chromatography of the reaction mixture on silicic acid were not ethanolamine (XIII). The last two steps of the synthesis are successful. We were equally unsuccessful in obtaining N ,N- shown in greater detail by reaction Scheme 1.dimethylcephalin by reversing the phosphorylation procedure, The silver salt of the distearoyl n-a-glycerophosphoric acidi.e. phosphorylating first the N , N-dimethylethanolamine with monobenzyl ester (IX) was prepared from cr-iodo-n-propylenephosphorus oxychloride and triethylamine, and esterifying the glycol (28)) via distearoyl n-Lu-glycerophosphoric acid dibenzylreaction product with ~-a JLdistearin. The failure of both pro- ester (29), as described by Stanacev and Kates (30) for the cor-cedures to give appreciable amounts of the desired phosphoryla- responding palmitoyl compound. The distearoyl n-ar-glycero-tion products of N , N-dimethylethanolamine may be the result phosphoric acid dibenzyl ester (IX) can also be obtained fromof phosphorus oxychloride acting as a chlorinating agent rather n-acetone glycerol (IV) via the following route: n-acetone glyc-than as a phosphorylating agent. The ease with which N ,N- erol-a-benzyl ether (Vu) --) n-a-benzyl glycerol ether (Via)dimethylethanolamine reacts with thionyl chloride to form di- + D-CY,@distearin-cr-benzyl ether (VIIa) + ~-a ,&distearinmethylaminoethyl chloride (27) would seem to support this sup- (VIIIa) -+ distearoyl L-cr-glycerophosphoric acid dibenzyl esterposition. (IX). Both routes, viz. IV -+ IX and IV + Vu -+ Via --+ We then tried to prepare distearoyl L-~-N, N-dimethylcepha- VIIu + VIIIu + IX, require the preparation of the same num-lin by a procedure which had yielded L-ar-(dioleoyl)lecithin (20). ber of intermediate compounds.
  3. 3. 2412 N ,N-Dimethylcephalins. I Vol. 236, Tu’o. 9 Materials-a-Iodo-n-propylene glycol was prepared by the [a]E4 +3.28” in anhydrous and ethanol-free chloroform (6.4);method of Baer and Fischer (28) from n-mannitol via the fol- MD +24.1”lowing intermediates: 1,2,5,6-diacetone n-mannitol (18, 31) -+ acetone n-glyceraldehyde (18) + n-acetone glycerol (18) -+ C&H75041 (734.9) Calculated: C 63.74, H 10.29, I 17.27cY-(p-toluenesulfonyl)-n-acetone glycerol (28) --+ ac-iodoacetone Found : C 63.36, H 10.00, I 17.39L-propylene glycol (28). Silver dibenzyl phosphate was ob- C 64.00, H 10.37, 117.16tained from sodium dibenzyl phosphate (32), by acidifying theaqueous solution of the sodium salt with sulfuric acid, isolating Distearoyl L-cY-Glycerophosphoric Acid Dibenzyl Ester (IX)-L4the dibenzyl phosphate, and converting it to the silver salt as solution of 7.35 g (10 mmoles) of distearoyl ac-iodo-L-propylenedescribed by Sheehan and Frank (33). The p-toluenesulfonyl glycol and 4.24 g (11 mmoles) of silver dibenzyl phosphate inchloride was purified by recrystallization from low boiling pe- 70 ml of anhydrous and thiophene-free benzene, while beingtroleum ether. The sodium iodide mas dried by heating to 120” stirred, was boiled under reflux in the dark for 3: hours. Thefor 6 hours. mixture was filtered while hot, the silver iodide was washed with N , N-Dimethylaminoethyl Chloride-The dimethylaminoethyl hot benzene, the filtrates were combined, and the benzene waschloride was prepared from its hydrochloride (34) by the method distilled off under reduced pressure from a bath at 35”. Theof Knorr (27). Inasmuch as the procedure is described but colorless, solid residue on recrystallization from 100 ml of lowbriefly, and the nitrogen values reported by Knorr for N , N- boiling petroleum ether gave 5.3 g of distearoyl n-a-glycerophos-dimethylaminoethyl chloride are not very satisfactory (Calcu- phoric acid dibenzyl ester. $n additional 0.8 g of the ester waslated: N 13.02, Found: N 11.85 and 12.05), we give a more de- obtained by concentrating the mother liquor to about one-halftailed description of its preparation. To a cold solution of 14.0 of its volume. Total yield of distearogl L-a-glycerophosphoric Downloaded from www.jbc.org by guest, on December 27, 2010g of N , N-dimethylaminoethyl chloride hydrochloride in 15 ml acid dibenzyl ester 6.1 g (68.9% of theory); m.p. 58.5-59.0”;of water was added a solution of 10 g of potassium hydroxide in [alo $2.8” in ethanol-free chloroform (c 5.7) ; M. +24.8”.10 ml of water, and the mixture was extracted with two 25 ml C53H8908P (885.3)portions of ether. The combined ethereal extracts were dried Calculated: C 71.91, H 10.14, P 3.50with anhydrous magnesium sulfate for 15 minutes, and the Found : C 71.31, H 10.04, P 3.36ether was removed by distillation under normal pressure. The C 71.82, H 10.08faintly yellow, oily residue on distillation under normal pressure Distearoyl L-oc-Glycerophosphoric Acid-A solution of 960 mggave 6.5 g (62.1% of theory) of colorless N ,N-dimethylamino- of distearoyl L-a-glycerophosphoric acid dibenzyl ester in 20ethyl chloride: b.p. 109-110” (bath temperature 135”); b.p. ml of glacial acetic acid,’ together with approximately 200 mg ofreported (27) 109-110”. The N ,N-dimethylaminoethyl chloride palladium black (35) were shaken in an atmosphere of pure hy-was prepared for immediate use only, because it has the tend- drogen at an initial pressure of 50 cm of water until the absorp-ency to dimerize, forming a piperazine derivative. tion of hydrogen ceased. The hydrogen was replaced with CdH,,NCl (107.6) nitrogen, the reaction mixture was warmed slightly until clear, Calculated: N 13.02 and was freed from catalyst by centrifugation. The catalyst Found: N 12.80 was extracted with three 5 ml portions of lukewarm acetic acid, and the extracts were added to the main solution. The acetic SYNTHESIS acid was distilled off under reduced pressure from a bath at 30- Distearoyl cr-Iodo-L-propylene Glycol (VIII)-Into a 250-ml 35”, and the last traces of acetic acid were removed by dryingthree-necked round flask equipped with an oil-sealed stirrer, the substance thoroughly at 30” in a vacuum of 0.1 mm. Thiscalcium chloride tube, and dropping funnel, were placed 2.5 g material, on reprecipitation from its solution in 15 ml of luke-(12.5 mmoles) of a-iodo-n-propylene glycol, 20 ml of dry and warm benzene by the addition of 45 ml of petroleum etherethanol-free chloroform, and 3.5 ml of anhydrous quinoline. To (b.p. 3560”), and drying of the precipit.ate at room tempera-the solution were added with stirring 7.57 g (25 mmoles) of ture over phosphorus pentoxide in a vacuum of 0.1 mm gavefreshly distilled stearoyl chloride dissolved in 30 ml of anhy- 460 mg (60.2% of theory) of distearoyl n-a-glycerophosphoricdrous chloroform, and the mixture was kept in the dark at room acid; m.p. 76.5-77.5”; [o(lo +3.54” in anhydrous and ethanol-freetemperature (20-22”) for 6 days. At the end of this period, the chloroform (c 10). Reported (36): m.p. 75.5-76.5”; [CL]” +3.7”clear solution was diluted with 150 ml of anhydrous ether, the in chloroform (c 9.2).quinoline hydrochloride was filtered off, and the filtrate was Distearoyl L-a-Glycerophosphoric Acid Monobenzyl Ester So-washed successively with two 25 ml portions of ice-cold 0.5 N dium Salt (X)-A solution of 5.31 g (6 mmoles) of distearoylsulfuric acid, 25 ml of water, two 25 ml portions of a half-satu- L-cr-glycerophosphoric acid dibenzyl ester and 1.35 g (9 mmoles)rated solution of sodium bicarbonate, and then was dried with of sodium iodide in 50 ml of anhydrous acetone was boiled underanhydrous sodium sulfate. The ether was distilled off under reflux for 3 hours. The clear solution was brought to roomreduced pressure from a bath at 30-35”, and the solid residue temperature and kept overnight at $8”. The mixture waswas redissolved in 30 ml of anhydrous ether. The solution was filtered with suction, and the material on the filter was washedcleared, and to the filtrate were added 45 ml of 99% ethanol. with ice-cold anhydrous acetone. For purification, the sodiumOn standing overnight at +8”, the solution deposited 6.0 g salt was recrystallized by dissolving it in 200 ml of boiling anhy- (65.9% of theory) of distearoyl cr-iodo-L-propylene glycol, which drous acetone containing 15 ml of 99y0 ethanol, filtering theon recrystallization under the same conditions as above gave 1 The glacial acetic acid was refluxed for 6 hours over potassium5.5 g of a colorless, crystalline product melting at 54.555.0”; dichromate and distilled with the exclusion of moisture.
  4. 4. September 1961 E. Baer and S. K. Pavanaram 2413solution while hot, and keeping the filtrate at $8”. The sodiumsalt was filtered off, washed with three 10 ml portions of coldacetone, and was dried over phosphorus pentoxide in a vacuumof 0.2 mm for 6 hours. The distearoyl L-a-glycerophosphoricacid benzyl ester sodium salt weighed 4.3 g (87.7% of theory) ;m.p. 164”; [o,]:’ +2.44” in chloroform (c 5). C46H8208PNa (817.1) Calculated: C 67.61, H 10.12, P 3.79 Found : C 67.86, H 10.50, P 3.85 Lhtearoyl L-ar-Glycerophosphoric Acid Monobenzyl Ester SilverSalt (XI)-To the hot solution pf 3.9 g (4.8 mmoles) of thesodium salt of distearoyl L-oc-glycerophosphoric acid monobenzylester in a mixture of 300 ml of acetone, 15 ml of 99% ethanoland 5 ml of water, was added a solution of 0.81 g (4.8 mmoles) Ot-,,,,,,,,,,,,,I,,~,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,I,,~,i,,~~,,,,,,,,,,J 2 3 4 5 6 7 8 9 10 11 12 13 14 15of silver nitrate in 20 ml of water and 40 ml of acetone, and the WAVELENGTH IN MICRONSmixture was kept overnight at +S”. The silver salt was filtered FIG. 1. Infrared spectra of (A) distearoyl L-a-N,iV-dimethyl-off, and dried over phosphorus pentoside in a vacuum of 0.2 cephalin; (B) distearoyl L-a-N-methylcephalin. Beckmanmm. The silver salt of distearoyl L-cY-glycerophosphoric acid IR-5 infrared spectrophotometer. Solvent: ethanol-free chloro-monobenzyl ester weighed 4.0 g (92.4”/, of theory). M.p. 113- form. Concentration of phosphatides, 5.6. Path of cell, 0.093 mm. Downloaded from www.jbc.org by guest, on December 27, 2010114”; [(Y],, $4.5” in dry, ethanol-free chloroform (c 4). C~H8208PAg (902) and 200 mg of palladium black (35), and the mixture was shaken Calculated: C 61.25, H 9.16, P 3.44 vigorously in an atmosphere of pure hydrogen at an initial pres- Found: C 61.81, H 9.10, P 3.50 sure of 50 cm of water for 1 hour. The reductive cleavage ap- Distearoyl L-oc-Glycerylphosphoryl-(A’ , N-dimethyl)ethanolamine peared to be complete at the end of 20 minutes. After replacingBenzyl Ester (XII)-Two and one-half millimoles (2.25 g) of the hydrogen with nitrogen, the contents of the reduction vesselthe silver salt of distcaroyl L-oc-glycerophosphoric acid mono- were transferred to a centrifuge tube, and the mixture wasbenzyl ester (XI) were dissolved in 25 ml of boiling, anhydrous warmed slightly to redissolve the dimethylcephalin. Theand thiophene-free benzene, and to the solution was added drop- catalyst was centrifuged off, the supernatant solution was de-wise a solution of 0.36 g (3.36 mmoles) of freshly prepared N , N- canted, and the catalyst was extracted with three 10 ml portionsdimethylaminoethyl chloride in 15 ml of benzene, and the solu- of chloroform. The combined acetic acid solution and chloro-tion was boiled under reflus with the exclusion of light for 12 form extracts were evaporated to dryness under reduced pres-hours. The solution then was cooled to room temperature, sure at a bath temperature of 3&40”, and the residue was freedcleared by centrifugation, and the supernatant solution was of acetic acid by drying at 40” for 4 hours in a vacuum of 0.1evaporated to dryness under reduced pressure (rotary evapo- mm. This material was dissolved in 12 ml of chloroform, to the rator) from a bath at 35”. The solid residue, after drying over solution were added 20 ml of 99% ethanol, and the mixturephosphorus pentoxide in a vacuum of 0.1 mm, weighed 2.1 g after being kept for 12 hours at +8” was filtered with suction. (97.2% of theory). It was dissolved in 20 ml of low boiling The N, N-dimethylcephalin on drying weighed 608 mg (84% of theory). For further purificat,ion, it was dissolved in 65 mlpetroleum ether, the solution was filtered, and the filtrate wasevaporated under reduced pressure, and the residue was dried of hot 99% ethanol, the hot solution was filtered, and the filtrate,as described above. The recovered material, weighing 2.05 g, after gradually attaining room temperature, was kept for 6 hours at f8”. The mixture was filtered with suction, and thewas dissolved in 13 ml of petroleum ether (b.p. 3560”), and the distearoyl L-ar-glycerylphosphoryl-N , N-dimethylethanolamine,solution was kept overnight at +8”. The precipitate was fil-tered off in the cold, washed with small portions of ice-cold a colorless, microcrystalline substance was dried for 8 hours atpetroleum ether, and was dried over phosphorus pentoxide at 56” (boiling acetone) in a vacuum of 0.1 mm. Yield, 526 mgroom temperature in a vacuum of 1 mm. The distearoyl L-W (72.8% of theory); m.p. 169-170°;2 [(Y], $5.4” in anhydrous, andglycerylphosphoryl-N , N-dimethylethanolamine benzyl ester, a ethanol-free chloroform (5.7). At room temperature, it ischromatographically homogeneous substance, weighed 1.38 g readily soluble in chloroform, fairly soluble in acetic acid or(64% of theory) ; [o(lo +3.75” in anhydrous and ethanol-free benzene, slightly soluble in 99% ethanol or acetone, and insolu-chloroform (c 6.4); m.p. 60-61”. The substance is readily solu- ble in ether or petroleum ether (Fig. 1).ble at room temperature in petroleum ether, benzene, chloro- CaaHs600sNP(776.1)form, and ether, but insoluble in water. Calculated: C 66.54, H 11.17, N 1.80, P 3.99 Found : C 66.18, H 11.04, N 1.70, P 3.89 CsoH,?O,NP (866.3) Calculated: C 69.32, H 10.71, N 1.62, P 3.57 DISCUSSION Found : C 68.42, H 10.81, ?u’ 1.46, P 3.58, 3.44 The use of two interchange reactions (VIII --f IX, XI + Distearoyl L-wGlycerylphosphoryl-(N , N-dimethyl)ethanolamine XII), both at high temperatures (SO”), in the preparation of(XIII)-In an all-glass hydrogenation vessel of 200 ml capacity 2 The melting point is uncorrected, and was determined in awere placed 806 mg of distearoyl L-a-glycerylphosphoryl-N ,N- capillary tube with an electrically heated bath of n-butyl phthal-dimethylethanolamine benzyl ester, 25 ml of glacial acetic acid,1 ate.
  5. 5. 2414 N , N-Dimethylcephulins. I Vol. 236, No. 9 distearoyl L-~-N, N-dimethylcephalin raised some doubts in SUMMARYour minds whether or not the synthesis had been achieved with- The first chemical synthesis of an ol-N ,N-dimethylcephalin out racemization. with the spatial arrangement of naturally occurring glycero- The optical purity of the distearoyl L-a-glycerophosphoric phosphatides, viz. distearoyl L-cY-glycerylphosphoryl-N , N-di-acid dibenzyl ester (IX) was readily checked by removing the methylethanolamine, has been accomplished by condensing thebenzyl groups by catalytic hydrogenolysis, and comparing the silver salt of distearoyl L-cu-glycerophosphoric acid monobenzylspecific rotation of the phosphatidic acid with that of authen- ester with N , N-dimethylaminoethyl chloride, and removingtic distearoyl n-a-glycerophosphoric acid. The value found the protective benzyl group of the reaction product by catalytic (~r3.54”) was practically identical with that reported for the hydrogenolysis. The distearoyl L-~-N , N-dimethylcephalin andauthentic compound (+3.7”). Since Stanacev and Kates (30) distearoyl L-ar-N-monomethylcephalin, also recently synthesizedhave made a similar observation for a homologous compound, by us, are representative members of two newly discoveredviz. dipalmitoyl L-cY-glycerophosphoric acid dibenzyl ester, it groups of phosphatides which have aroused interest in recentappears that the synthesis of L-rw-phosphatidic acid dibenzyl years as intermediates in the biosynthesis of lecithin.esters by the condensation of diacyl L-cr-iodopropylene glycolswith silver dibenzylphosphate proceeds without racemization. A check of the structural and optical purity of the synthetic Acknowledgment-This study was made possible by a grantdistearoyl n-a-N, N-dimethylcephalm (XIII), and implicitly of from the Multiple Sclerosis Society of Canada, whose supportits benzyl ester (XII), was not possible for lack of the necessary is gratefully acknowledged.reference compounds, either synthetic or from natural sources.We thus report the specific rotations of compounds XII and REFERENCES Downloaded from www.jbc.org by guest, on December 27, 2010XIII with some reservation. However, the fact that the spe- 1. HOROWITZ, N. II., J. Biol. Chem., 162, 413 (1946).cific rotation of distearoyl L-~-N ,N-dimethylcephalin is of an 2. WOLF, B., AND NYC, J. F., J. Biol. Chem., 234, 1068 (1959);order of magnitude one would expect for this compound by Biochim. et Biowhvs. Acta. 31. 208 (1959).analogy with L-cr-distearoylcephalin and the corresponding 3. HALL, M. O., AN’D “NYC, J.’ F.; J. Am. Chem. Sot., 81, 2275 (1959).lecithin, rules out the possibility that racemization has taken 4. ARTOM, C., Federation Proc., 19, 233 (1960).place to any significant extent during the condensation of the 5. CROWDER, M., AND ARTOM, C., Federation Proc., 8,180 (1949);silver salt of distearoyl L-cr-glycerophosphoric acid monobenzyl 11, 199 (1952).ester (XI) with N , N-dimethylaminoethyl chloride. 6. ARTOM, C., LOFLAND, H. B., AND OATES, J. A., JR., J. Biol. In general, phosphatides with two dissimilar fatty acid sub- Chem., 233, 833 (1958).stituents prevail in nature. However, a few phosphatides with 7. BREMER, J., AND GREENBERG, D. M., Biochim. et Biophys. Acta, 36, 287 (1959); 37, 137 (1960).two identical fatty acid substituents, saturated or unsaturated, 8. BAER, E., AND PAVANARAM, S. K., J. Biol. Chem., 236, 1269have been isolated from natural sources (3741). It is there- (1961).fore quite possible that the distearoyl n-a-N, N-dimethylcepha- 9. HALL, M. C., AND NYC, J. F., Federation Proc., 20, No. 1, Partlin which we have prepared may be found to be a natural prod- 1. 279 (19611.uct. Furthermore, oleic acid and stearic acid being two of the 10. ARTOM, c., A&D LOFLAND, H. B., JR., Biochem. and Biophys. Research Communs.. 3. 244 (1960).more common fatty acid substituents of natural glycerophos- 11. BREMER, J., AND GREENBERG, D.’ M., Biochim. et Biophys.phatides, natural N , N-dimethylcephalins containing these two Acta, 46, 205 (1961).fatty acids or other more highly unsaturated %-fatty acids 12. BREMER, J., FIGARD, P. H., AND GREENBERG, D. M., Biochim.would be expected on reduction to yield distearoyl L-(Y-N, N-di- et Biophys. Acta, 43, 477 (1960).methylcephalin. In either case, the synthetic distearoyl 13. GIBSON, K. D., WILSON, J. D., AND UDENFRIEND, S., J. Biol.L-~-N ,N-dimethylcephalin should prove useful as reference Chem., 236, 673 (1961). 14. SPERRY, W. M., AND WAELSCH, H., in Research Publications,compound for the unambiguous elucidation of the structure Association for Research in Nervous and Mental Diseases,and configuration of natural N, N-dimethylcephalins. Vol. 28, The Williams and Wilkins Co., Baltimore, 1948, The homologues of distearoyl L-~-N, N-dimethylcephalin p. 255.should be obtainable by the same procedure on use of the ap- 15. VERKADE, P. E., AND STEGERHOEK, L. J., Koninkl. Ned. Akad.propriate homologue of distearoyl a-iodo-L-propylene glycol. Wetenshap. Series B, 61, 155 (1958). 16. HOEFNAGEL, M. A., STEGERHOEK, L. J., AND VERKADE, P. E.,The N , N-dimethylcephalins are the last in a series of five closely Rec. Trav. Chim., 79, 605 (1960).related groups of naturally occurring glycerophosphatides to 17. BAER, E., AND FISCHER, H. 0. L., J. Biol. Chem., 128, 491become accessible by synthesis. The series includes the L-U- (1939).cephalins, L-a-N-methylcephalins, L-~-N ,N-dimethylcephalins, 18. BAER, E., in E. G. BALL (Editor), Biochemical preparations,L-a-lecithins, and L-a-phosphatidyl-L-serines (Scheme 2, A + Vol. ZZ, John Wiley and Sons, Inc., New York, 1952, p. 31.- 19. BAER, E., AND KATES, M., J. Am. Chem. Sot., 72, 942 (1950).Iii). 20. BAER, E., BUCHNEA, D., AND NEWCOMBE, A. G., J. Am. Chem. RI Sot., 78, 232 (1956).R. COO-CHz A = -CH2--CH2NH2 21. BAER, E., MAURUKAS, J., AND RUSSELL, M., J. Am. Chem. B = -CHz--CHzNH(CH,) Sot., 74, 152 (1952).R.COO-C-H 0 22. BAER, E., AND BUCHNEA, D., J. Am. Chem. Sot., 81, 1758 C = -CH2--CH2N(CH& (1959). H&-O-$-O-R, D = -CH2-CH&(CH& 23. BAER, E., AND MAURUKAS, J., J. Biol. Chem., 212, 25 (1955). bH 24. BAER, E., AND KATES, M., J. Am. Chem. Sot., 70,1394 (1948). E = -CHYCH(NH~)COOH (L) 25. BAER, E., AND STANCER, H. C., J. Am. Chem. Sot., 76, 4510 SCHEME 2 (1953).
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