The synthesis of phospholipids by direct amination

~emistry and Physics of Lipids, 22 (1978) 1-8 © Elsevier/North-HollandSctenlificPublishers Ltd.

THE SYNTHESIS OF PHOSPHOLIPIDS BY DIRECT AMINATION Hansj/Srg EIBL and Alfar NICKSCH Max PlanckInstitut flit biophysiicalischeChemie,Postfach 968, 3400 GSttingen (GFR) Received September 12th, 1977

acceptedNovember9th, 1977

The bromoethylesters of phosphatidicacids and their analoguesare generalintermediatesin the synthesis of phosphollpids.A direct amination with different amines such as ammonia, methylamine, dimethylamineand trimethylamine results in the correspondingphosphatidylethanolamines and -cholines. In addition to the well elaborated reactions of bromoethylesters with trimethylamine and dimethylamine,the synthesis of phosphatidylethanolaminesand -(N-methyl)-ethanolamines by amina~ionwith ammonia and methylamine is now possible in high yields (> 90%) without the need of the usual protecling groups.

1. Introduction The conversion of 1,2.diacyl,~n-glycerol-3-phosphoric acid bromoethylesters to phosphatidylethanolamines and phosphatidylcholines by amination with trimethylamine, dimethylamine, methylamJne and ammonia is of interest as a general and simple method for the synthesis of phospholipids. Unfortunately the use of ammonia or methylamine does not result in the expected phosphatidylethanolamines due to decomposition I1,2]. One purpose of the work reported in this paper was to learn if a re-examination of the amination reaction with properly selected model compouricls f~ight lead to the desired products. The instability of the fatty acid ester bond in the 2-position of the glycerol molecule is a serious problem, which is already known from earlier work on the isomerisation of fatty acids in 2-acyl.glycerols [3] and 2-acyl,sn-gtycerol.3-phosphorylcholines and .phosphorylethanolamines [4]. Therefore base-stable intermediates (see fig. l) such as 1,2.dipantadecylmethylidene-glycerol-3.phosphoric acid bromo. ethylesters (I) and 1,2-dihexadecyl,~n-glycerol analogues (II) were chosen to study the amination reaction in greater detail. These compounds do not decompose and have permitted the use of a variety of solvents, including mixtures containing water. With the experience gained from these experiments it was hoped finally to find conditions for the synthesis of acyl-propanediol-(1,3)-phosphorylethanolamines (III) a-c or IV a--c) and diacyl-glycerol-3-phosphorylethanolamines (V a - c or VI a-c) from the respective bromoethylesters (III-VI) which also may contain unsaturated fatty acid residues. Phospholipids with stepwise modifications of the polar part are useful tools for studies in model membranes. For instance the series of 1,2-dipentadecylmethylidene.

2

H. Eibl, A. Nicksch, Amination ofphosphatidic acids bromoethylesters

O .

O.

R-O-P-O-CH2-CH2-Br

.

NX 3

)

@ R-O.-P-O-CH2-CH2-NX 3 • NQBI

ONu

R: (I)

Oe

H2C-O ..(CH2)IcCH3 '

(ll) H2C-O-(CH2)ls-CH 3

;C

NX3: ((1) NH3

HC-O "(CH2)14-CH3

HC-O-(CH2)ls-CH 3

(b) NH2CH3

H2C-

H2C-

(c) NH (CH3)2 (d) N(CH3)3

(III) H2C-O-CO-(CH2)I~ - CH3

(IV)

H2C-O-CO-{CH2I~cCH3

H2C

H3C-C-CH3

H2C-

H2C-

(V) H2.C-O-CO-ICH2)12-CH3 HC-O-CO- (CH2)~2-CH3 H2C-

(VI) H2C-O-CO-(CH2)~{-CH3 HC -O-CO-(CH2) 7 -C H=CH-(CH2)7-CH 3 H2C-

Fig. 1. Direct amination of phosphatidic acid bzomoethylesters. The numbers I to VI characterize the apolar parts of the bromoethylesters as described in the text.'The letters a-d indicate the amine used in the reaction and thus refer to phosphatidylethanolamines, the reaction products, differing in the degree of methylation. (a) Stands for phosphatidylethanolamine, Co) for phosphatidyl-(N-methyl)-ethanolamine,(c) for phosphatidyl-(N,N-dimethyD-ethanolamineand (d) fol phosphatidylcholine. glycerol-3-phosphorylcholine and -ethanolamines was successfully applied to demonstrate the influence of polar groups on Grammicidin channels [5]. In addition information about the surface requirement of polar groups in phospholipids can be derived from model compounds, where the polar part determines the surface area of the molecule. This is the case for 1-palmitoyl-sn-glycerol-3.phosphorylcholine and 1-palmitoylpropanediol-(1,3)-phosphorylcholine as described earlier [6], but also for the series of palmitoyl-propanediol(1,3)-phosphorylethanolamines (A. Nicksch and H. Eibl, in prep.), which are described in this paper.

II. Materials and Methods

Propanediol-(1,3), dipentadecylketone, 1,2-dihexadecyl-sn-glycerol, ammonia and the methylated amines were products of Fluka (Buchs, Switzerland). Palmitoylpropanediol-(1,3) [7], palmitoyl-2,2-dimethyl-propanediol-(1,3) and 1,2-dimyristoyl sn-glycerol [8] were synthesized as described earlier. The preparation of 1-palmitoyl2-oleoyl-sn-glycerol-3-phosphoric acid bromoethylester is described in a separate paper (H. Eibl, in prep.).

H. Eibl, A. Nicksch, Amination of phosphatidic acids bromoethylesters

3

The crude products of the amination were purified by column chromatography using MaUinckrodt Silic AR (100-200 mesh). For elution mixtures of chloroform, methanol and aqueous ammonia (25%) were used. Starting with CHC13/CH3OH/ ammonia in a ratio of 200 : 15 : 1 (v/v/v) the polarity of the solvent mixture was increased stepwise up to 65 : 30 : 3 to elute the phosphatidylethanolamines or -cholines.

III. Experimental

1. 1,2-Dipentadecylmethylidene-glycerol Glycerol (60 g; 0.64 mol), dipentadecylketone (180 g; 0.4 mol) and p-toluenesulfonic acid (6 g) were added to 1 I of benzene. The mixture was stirred and heated in a circulation distillation apparatus with a magnesium perchlorate water trap for water elimination from the circulating solvent. More than 90% of the dipentadecylketone was converted to 1,2-dipentadecylmethylidene-glycerol as judged by thin layer chromatography (TLC) after 16 hr of reaction. The mixture was shaken with 500 ml of potassium carbonate (5% in water) to neutralize the acid. The benzene layer was evaporated to.dryness and the oily residue recrystallized from 1.8 1 of acetone/methanol (1 : 1). The yield was 130 g of 1,2-dipentadecylmethylidene-glycerol (64% based on the ketone). The purity of the compound was checked by TLC and elemental analysis (calculated for Ca4I-I6803(mol. wt. 524.91): C, 77.80%;H, 13.06%; found: C, 77.94%, H, 13.05%). 2. Bromoethylphosphoric acid dichloride To a thoroughly stirred solution of 200 g (1.3 mol) of phosphorusoxychloride (distilled before use) dissolved in 50 ml of trichloroethylene in a three necked round bottom flask fitted with a dropping funnel and reflex condensor were added 95 g (0.76 mol) of 2-bromoethanol (distilled before use). A continuous stream of nitrogen (20 ml/min.) was directed through the reaction mixture to remove the hydrogen chloride formed. After 12 h of reaction at 25°C 50 ml of toluene were added and the excess phosphorusoxychloride was removed by evaporation at 40°C. The residue, 152 ~ of bromoethylphosphoric acid dichloride (83% based on 2-bromoethanol) was used for the phosphorylation of alcohols without further purification, which was achieved by a variation of the method described by Hirt and Berchthold [9]. 3. Preparation of bromoethylesters o f phosphatidic acid analogues (I-II1) A solution of 44 g of bromoethyl phosphoric acid dichloride (0.18 mol) in 250 ml of trichloroethylene was cooled in an icebath. After the addition of 36 g of triethylamine (0.36 mol) the phosphorylation mixture was kept at 25°C by a water bath and the primary alcohol (0.1 mol of 1,2-dihexadecyl-sn-glycerol, 1,2-dipentadecylmethylideneglycerol, palmitoyl-propanediol-(1,3), palmitoyl-2,2-dimethylpropanediol-(1,3) or 1,2-diacyl-glycerol) in 250 ml of trichloroethylene was added dropwise with stirring.

4

H. Eibl,A. Nicksch,Amination ofphosphatidic acids bromoethylesters

As judged by the disappearance of starting material (TLC) the reaction was completed after 1 hr. The precipitate (triethylammonium hydrochloride) was removed by filtration and the filtrate was evaporated to dryness. The residue was dissolved in 300 ml of tetrahydrofuran. 300 ml 0.5 M Sodium acetate (pH 8.5) and 20 ml 0.5 M ethylenediaminetetraacetate (pH 10.5) were added with stirring. The pH dropped and reached a constant value of about 8 after 2 hr of hydrolysis. The product was extracted with 300 ml of diisopropylether. To accelerate phase separation 100 ml of methanol were added. The diisopropylether phase was evaporated to dryness. The residue, a yellow oil, was dissolved in 100 ml of chloroform and precipitated by the addition of 700 ml of acetone at 0°C. The precipitate was stored at 0°C and used directly for the aminations. 4. Direct amination of bromoethylesters of phosphatidic acids and analogues to yieM phosphorylethanolamines and phosphorylcholines (a) Amination with methylated amines (I b - d and VI b-d): The crude reaction product, 0.01 mol, was dissolved in 30 ml of chloroform at 50°C. 2-propanol (50 ml) and acetonitrile (50 ml) were added followed by the amine in water (70 ml of a 40% solution of methylamine, dimethylamine or trimethylamine). The reaction was completed after 10 h and the solvents were removed by rotary evaporation. (b) Amination with ammonia (Ia-VIa): The crude intermediate, 0.01 tool, was dissolved in 100 ml of chloroform at 40°C. 2-Propanol (100 ml) and.dimethylformamide (400 ml) were added followed by 300 ml of ammonia (25% solution in water). The reaction was completed after 6 hr and the solvents were removed by rotary evaporation. The residues of the reactions (a) or (b) were dissolved in 150 ml of water, 150 ml ot chloroform and 200 ml of methanol. After phase separation the chloroform layer was taken to dryness. The residue was dissolved in chloroform and precipitated by the addition of a 7-fold excess of acetone the precipitate at 0°C was collected, purified by silica gel chromatography (100 g silica gel were used for the purification of 7-10 g of product) and identified by elemental analysis. The yield of chromatographically pure product, phosphatidylethanolamine or phosphatidylcholine, was 80-90% based on the starting/~-bromoethylester. Analytical data for the synthesized compounds are summarized in table 1.

IV. Results and Discussion

It has been proposed that the impossibility of preparing phosphatidylethanolamines and N-methylethanolamines from phosphatidic acid bromoethylesters by direct arnination is due to: (a) the unreactivity of ammonia and methylamine in apolar and carefully dried solvent systems such as benzene and toluene and (b) the formation of byproducts such as fatty acid amides [ 1,2]. We have found it possible to overcome

Table 1 Analytical data for phosphatidylethanolamines and phosphatidylcholines obtained by direct amination. The numbers characterize the apolar part of the molecule, the letters a-d indicate the degree of methylation as shown in the figure. The mol. wts. of the phosphatidylcholines (Id-VId) are calculated for the monohydrates. Substance No.

Formula (mol. wt.)

Ia

C36H~4NO6P (647,98) C37H~6NO6P (662,0) C3sHTsNO~P (676,03) CagHs2NOTP (708,07)

Ib Ic Id

lla IId

Ilia lifo Illc IIId

IVa IVb IVc IVd

Va Vb VC Vd

Via Vld

%C

%H

%N

~P

calcd.: found: calcd.: found: calcd.: found: calcd.: found:

66.73 67.33 67.13 66.46 67.52 67.93 66.15 66.12

11.51 11.42 11.57 1153 11.63 11.85 11.67 11.66

2.16 2.11 2.12 2.12 2.07 2.13 1.98 2.09

4.78 4.63 4.68 4.49 4.58 4.55 4.37 4.35

C3~HTsNO~P (663,98) C4oH86NOTP (724,07)

calcd.: found: ealcd.: found:

66.92 66.51 66.35 64.96

11.84 11.33 11.97 12.29

2.11 2.14 1.93 i .85

4.66 4.75 4.28 4.02

C2 t H44NO6 P (437,57) C~mH¢6NO 6P (451,59) C23H4sNO6P (465,62) C24Hs2NOTP (497,67)

calcd.: found: calcd.: found: caled.: found: calcd.: found:

57.64 58.21 58.51 57.89 59.33 58.91 57.92 56.66

10.14 10.23 10.27 10.30 10.39 10.41 10.13 10.18

3.20 3.28 3.10 3.12 3.01 3.05 2.81 2.94

7.08 6.71 6.86 6.82 6.65 6.57 6.22 6.08

C2aH,sNO6P (465,62) C2,,HsoNO~P (479,65) C2 ~Hs 2NO6P (493,67) C2~Hs6NOTP (525,72)

calcd.: found: caled.: found: calcd.: found: calcd.: found:

59.33 58.97 60.10 59.85 60.82 58.32 59.40 59.30

10.39 10.45 10£1 1058 10.62 10.42 10.74 10.89

3.01 3.07 2.92 2.95 2.84 2.70 2.66 2.94

6.65 6.49 6.46 6.35 6.27 5.62 6.89 622

C33H6~NOsP (635,84) C34H~aNOsP (661,88) C~sH~oNOsP (687,91) C36HT~NOgP (695,94)

calcd.: found: calcd.: found: calcd.: found: calcd.: found:

62.33 60.82 62.83 61.97 63.32 63.46 62.13 62.42

10.46 10.27 10.55 10.95 10.63 10.87 10.72 10.71

2.20 2.25 2.16 2.27 2.11 1.98 2.01 2.04

4.87 5.55 4.77 452 4.66 4.57 4.45 4.41

C39HT~NOsP (717,98) C~2Ha4NOgP (7?8,08)

calcd.: found: calcd.: found:

65.24 64.86 64.83 64.11

10.67 10.61 10.88 10.79

1.95 2.01 1.80 1.69

4.31 4.20 3.98 3.82

6

H. Eibl, A. Nicksch, A mination ofphosphatidic acids bromoethylesters

formation of amides by using phosphatidic acid bromoethylesters of glycerols, which contained base stable residues like ketals or ethers instead of fatty acid esters. Especially useful in this connection are the phosphoric acid bromoethylesters of 1,2dipentadecyhnethylidene-glycerol and 1,2-dihexadecyl.sn-glycerol due to the similarity of the apolar part in these molecules with that of natural phospholipids. The conditions for amination with ammonia were most critical. The discussion therefore is concentrated on the reaction of phosphatidic acid bromoethylesters with ammonia. Surprisingly the base stable intermediates did not form phosphatidylethanolamine analogues in apolar solvents but compounds with RF-Values larger than that expected for phosphatidylethanolamine. These compounds were isolated and identified as reaction products of the phosphatidylethanolamines with bromoethylesters as described in a following paper (H. Eibl, in prep.). The observation that pbosphatidylethanolamine acts as aminating agent and competes for the bromoethylesters with ammonia is the result of the very limited amounts of ammonia dissolved in the waterfree and apolar solvent systems. The main reason to exclude water in earlier studies [ 1,2] was probably to avoid artefacts, for instance hydrolysis or aminolysis of fatty acid esters. The course of the amination is conveniently followed by TLC using the respective phosphorylethanolamines and phosphorylcholines as reference compounds. The yield of cephalin analogues from.base stable bromoethylesters after 12 hr of incubation at 40°C increased with increasing potarity of the solvent systems used. It was less than 5% in waterfree solvent systems such as benzene and toluene as described by Baer and Pavanaram [l ] and it amounted to more than 90% in a mixture of chloroform, methanol, acetonitril and water in a ratio of 30 : 50 : 50 : 60 (v/v/v/v). Similar results were observed with methylamine. The amination of palmitoyl-propanediol-(1,3)-phosphoric acid bromethylester and of the palmitoyl-2,2-dimethyl-propanediol-(1,3) analogue in a mixture of chloroform, methanol, acetonitrile and water was also possible without the formation of byproducts indicating that fatty acid esters of primary alcohols are stable during the amination step. However, in the case of 1,2-diacyl derivatives decomposition was observed and the products formed were identified as fatty acid methylesters rather than amides as noticed by others [ 1,2]. We propose that this is due to the presence of methanol and water, the fatty acid methylesters thus originating from a transesterification of fatty acids from the 2-position of glycerol to methanol in the solvent mixture. This is reminiscent of the instability of 2-acylglycerol in alkaline or acidic conditions, where an equilibrium of 90% in favour of the 1-position has been observed [3]. In this case an intramolecular rearrangement of fatty acids from the 2 to the 1-position in the glycerol molecule occurs. A Similar event, but intermolecular, leads to the formation of the fatty acid methylesters in the amination step. Therefore there was a good chance to reduce or avoid decomposition by substituting methanol in the solvent system for a secondary alcohol, for instance 2-propanol or 2-butanol. With this modification 1,2-diacyl-glycerol-3-phosphorylethanolamines were synthesized in excellent yields without the formation of unwanted byproducts. This

H. Eibl, A. Nicksch, A ruination of phosphatidic acids bromoethylesters

7

is demonstrated by the synthesis of 1,2.dimyristoyl,~n-glycerol- and 1-palmitoyl-2oleoyl-sn-glycerol-3-phosphorylethanolamine from the respective bromoethylesters in yields amounting to 80 and 90%. These results clearly reflect the different concentrations of ammonia in the solvents or solvent mixtures. The molar ratio of ammonia to bromoethylester was found to be about 3 : 1 in benzene or toluene in comparison to values of about 500 : ! for solvent systems including water. In the latter case the large excess of ammonia over phosphatidylethanolamine suppresses the competition of the amines for the bromoethylester and leads to a weil-defmed reaction product. Some remarks should be added concerning the chromatography of the end products. The amination of the bromoethylesters results in the formation of bromides with trimethylamine or hydrobromides with dimethylamine, methylamine or ammonia. The phosphatidylcholine bromides of the phosphatidylethanolamine hydrobromides are converted into the zwitterionic form by chromatography on silica gel using elution systems consisting of chloroform/methanol and ammonium hydroxide. Ammonium bromide is formed and is adsorbed to the silica gel. The previously described treatment with silveracetate [6] can be omitted. An unusual behaviour of the phosphatidyl41N, N-dimethyl)ethanolamines was observed by TLC. The RF-values in acidic solvent systems from 1,2-dipentadecylmethylidene-glycerol-3-phosphorylethanolamines to the choline decrease continuously with the degree of methylation; for instance in chloroform/methanol/formic acid (25% in water) 65 : 30 : 3 (v/v/v) values of 0.6,0.5,0.4 and 0,3 were found. However, in alkaline systems with ammonia (25% in water) instead of formic acid, the RF-value of the (N,N-dimethyl)ethanolamine derivative changed markedly from 0.4 to 0.8, indicating a deprotonation of the molecule in the presence of ammonia as a result of the low pK-value of this nitrogen group. The RF-values of the ethanolamine-, (N-methyl)ethanolamine- or the choline derivative are the same in both solvent systems. Two solvent systems are now routinely used by us for the amination of bromoethylesters: (a) chloroform, 2-propanol, acetonitril and a 40% solution of methylated amines in water in a ratio of 30 : 50 : 50 : 70 (v/v/v/v), and (b) chloroform, 2-propanol, dimethylformamide and a 25% solution of ammonia in water in a ratio of 100 : 100 : 400 : 300 (v/v/v/v). These solvent systems allow the reaction to complete in 6 - 1 2 hr without the formation of byproducts. The easy preparation of bromoethylesters in connection with our amination procedure is a useful tool for the synthesis of phospholipid molecules with small but well defined changes in the polar part of the molecule. To our knowledge there is no other method, which allows the synthesis of phosphatidylethanolamines and the phosphatidylcholines from one common intermediate by one additional, reaction step.

Acknowledgements We are grateful to Dr. D. Henderson and Dr. W. Vaz for critical remarks. We thank Mrs. Gudrun Daude for carefully writing the manuscript.

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H. Eibl, A. Nicksch, A ruination ofphosphatidic acids bromoethylesters

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E. Baer and S.J. Pavanaram, J. Biol. Chem. 236 (1961) 1269 D. Shapiro and Y. Rabinsohn, Biochemistry 3 (1964) 603 F.H. Mattson and R.A. Volpenhein, J. Lipid Res. 3 (1962) 281 H. Eibl and W.E.M. Lands, Biochemistry 9 (1970) 423 E. Neher and H. Eibl, Biochim. Biophys. Acta 464 (1977) 37 H. Eibl, R. Demel and LL.M. van Deenen, J. Colloid and Interface Sci. 29 (1969) 381 H. Eibl and O. Westphal, Liebigs Ann. Chem. 709 (1967) 244 H. Eibl, D. Arnold, H. Weltzien and O. Westphal, Liebigs Ann. Chem. 709 (1967) 226 R. Hirt and R. Berchthold, Pharm. Acta Helv. 33 (1958) 349