A versatile, flexible synthesis of 1,3-diglycerides and triglycerides

Chemistry and Physics o f Lipids 16 (1976) 107-114 © North-Holland Publishing Company

A VERSATILE, FLEXIBLE SYNTHESIS OF 1,3-DIGLYCERIDES A N D TRIGLYCERIDES A.P.J. MANK, J.P. WARD and D.A. VAN DORP Unilever Research, Vlaardingen, The Netherlands

Received October 7, 1975,

accepted November 10, 1975

A flexible method for synthesising 1,3-diflycerides and triglycerides is described. Glycidol esters, prepared by a known route from epichlorohydrin and the sodium salt of a fatty acid, were heated with another or with the same fatty acid and a quaternary ammonium salt. This resulted in a fast, mild reaction and higher yields and greater purity of the diglycerides than hitherto obtained in this synthesis. The mixture of 1,3- and 1,2-diglycerides obtained was isomerised by heating while still in the solid phase to 1,3-diglycerides. Triglycerides were prepared from the diglycerides by acylation using a fatty acid chloride and pyridine in hexane.

I. Introduction The present state of glyceride synthesis has recently been reviewed [1 ]. In view of the need in our laboratory for pure triglycerides, particularly for analytical, physico-chemical and nutritional studies, we devised a versatile, fl~xible method for synthesizing racemic 1,3-diglycerides and hence triglycerides. II. Synthesis The process of synthesizing 1,3-diglycerides and hence triglycerides is outlined in Scheme I. R1COONa

I

O

•

O ~

CH2 CI R2COONa

.~

CH2OCOR1 "IT O

I

- -

•

HO L_OCOR1 - -

--

R3COCI

IV

OCOR1

-- OCOR3 L OCOR

R1COOH P

CH2OCOR2

(OCOR2

R2COOH

HO

- -

L--OCOR2

'm

v

107

m"

108

A.P.J. Mank et al., 1,3-Di- and triglyceride synthesis

The preparation of glycidol esters (II and III) by the reaction of epichlorohydrin (I) with the sodium salts of carboxylic acids in the presence of catalytic amounts of a quaternary ammonium salt has been amply described [2]. We found that when a stirred mixture of, for example, sodium stearate and epichlorohydrin in a molar ratio of 1 : 16, containing 0.04 mol/mol tetraethylammonium bromide (TEAB), was heated to reflux temperature (about 115°C) within 15 min and kept at this temperature for 15 min, reaction was practically complete. The unpurified products, obtained in practically quantitative yield after some modifications of the method described in the literature [2], had a glycidol ester content of 90-93% by GLC. The glycidol esters could be purified by molecular distillation and crystallization to 99% purity, but this was not essential to the success of the following step. The second stage of the synthesis, the 1,3-diglyceride preparation, differed in two important aspects from the literature on the uncatalysed addition of fatty acids to glycidol esters. Inclusion of a quarternary ammonium salt as catalyst allowed the reaction to proceed faster and under milder conditions than those described hitherto [3]. In a simplified procedure the crude but degassed esters (If and III) still containing TEAB were heated with a fatty acid at 100°C. The diglyceride formed was a mixture of the 1,3-isomer (IV) and the 1,2-isomer in varying proportions, which in the least favourable cases were about 60 : 40, that is the equilibrium position [4]. Although kinetic studies (using GLC analysis) showed that 1,3-diglyceride was formed preferentially from the reaction of a glycidol ester with a fatty acid, thermally induced intramolecular isomerization occurred concurrently. The negative influence on the yield of 1,3-diglyceride caused by this isomerization in the molten state was offset by the observation that in solid mixtures of 1,3- and 1,2-diglycerides the latter isomerizes to the former [5]. In this way yields of 99% pure 1,3-diglyceride were obtained after two crystallizations in 70-75% of theory. The final stage, acylation of pure 1,3-diglyceride, was carried out with a 20% molar excess of the appropriate acid chloride in boiling hexane containing pyridine as HC1 scavenger. An improvement was the addition of water to the hot mixture at the end of the reaction to hydrolyse the excess acid chloride (see Experimental). The triglycerides were purified by chromatography and crystallization. They were characterized either collectively or, whenever appropriate, individually by the following techniques: IR spectroscopy, Roentgen, differential thermal, GLC, TLC and lipase analysis.

111. Discussion

The isomerization of 1,2- to 1,3-diglycerides proceeds faster at higher temperature, as long as the mixture remains solid [5]. Since a solid mixture of 1,2- and 1,3dipalmitate, and 1,2- and 1,3-distearate isomerizes to only 1,3-dipaimitate and 1,3distearate, without interesterification, the isomerization process must be intramole-

A.P.J. Mank et al., 1,3-Di- and triglyceride synthesis

109

cular. The role of catalysis in the solid phase isomerization is at present under investigation. The synthesis described derives its flexibility from this combination of fatty acid addition to a glycidol ester followed by solid phase isomerization. Glycidol esters, for example, can be prepared in bulk from more readily available fatty acids, and reacted with more sensitive fatty acids, for example, polyunsaturated fatty acids, in the second stage. Furthermore, our work currently in progress has shown that by a suitable choice of compounds the method can be used to synthesize chiral glycerides [6]. Limitations to the technique appear to be twofold: the increased reactivity of the lower fatty adds, acetic, butyric, etc., with the glycidol ester, whereby mixtures of possibly transesterified products are obtained, and the impracticability of isomerizing low melting diglycerides such as dioleate in the solid phase, because isomerization occurs too slowly at temperatures appreciably under 50°C. The difficulties can, however, partly be overcome, for example, by using glycidol acetate, butyrate etc., when permissible, or in all cases by separation and purification of the reaction products.

IV. Experimental A. Starting materials

Most of the fatty acids were obtained commercially. If necessary they were purified by fractional distillation of their methyl esters, followed by saponification and recrystallisation of the fatty acids to more than 99% (GLC). Solvents were purified and dried by standard procedures. Wherever possible Analytical Reagent grades of solvents and reactants were used. B. Acid chlorides

To a mixture of 1.3-1.4 mol (115 ml) freshly distilled oxalyl chloride and 250 ml dry hexane or benzene was added at 30-35°C dropwise while stirring 1 mol fatty acid dissolved in anhydrous hexane, benzene or alcohol-free chloroform. After 3 - 4 hr refluxing, the solvent was distilled off and the residual acid chloride was either degassed in vacuum or molecularly distilled. The presence of anhydride was checked by TLC (table 1). C. Glycidol esters

A mixture of 1 mol sodium salt of the appropriate fatty acid, 8.4 g (0.04 mol) TEAB and 1250 ml (16 mol) freshly distilled epichlorohydrin was stirred and heated under reflux for 15 min, cooled and diluted with 200-300 ml chloroform. The pre-

110

A.P.J. Mank et al., 1,3-Di- and triglyceride synthesis

Table 1 Rf-values determined from chromatograms over 10 cm in length (=100).a Compounds

Eluent A

Eluent B

Monoglycerides 1,2-Diglycerides

0 5 5 27 27 35 50 85

15 30 40

1,3-Diglycefides Acid chlorides Glycidol esters Triglycerides Fatty acid methyl esters Acid anhydrides a For details see Section G.

75 90 90

cipitated NaC1 was filtered off*, the filtrate was evaporated in vacuo and the residue dissolved in CHC13 (1 : 3, v/v), washed with water to remove the catalyst, dried (MgSO4), evaporated and degassed at 50-60°C/0.1 mmHg. Glycidol esters up to the palmitate were purified by distillation in vacuo; the higher saturated terms were crystaUised from acetone or hexane. D. 1,3-Diglycerides

A mixture of 1 mol pure glycidol ester, 1 tool pure fatty acid and 0.04 mol quatemary ammonium salt, e.g. TEAB, was heated and stirred for 2.5-3 hr at 100°C until hardly any glycidol ester remained (TLC, table 1). Tile reaction mixture of 1,3- and 1,2-diglyceride, which usually still contained about 2% free fatty acid, was dissolved in CHC13 and washed with water to remove catalyst. The solution was dried (Na2SO 4) and evaporated. The solid diglyceride mixture was then isomerised at a temperature of about 5-10°C below its melting point for 3 to 10 days in a drying cupboard until the conversion of 1,2- to 1,3-diglyceride had proceeded as far as possible (TLC and GLC). The product was crystallised from acetone and hexane. Lower melting diglycerides such as diolein were washed with water, dried, evaporated and crystallised at low temperature from acetone. In such cases improvements in yield of 10-15% 1,3-diglyceride could be obtained by evaporating the mother liquors, isomerising the residue at about 50°C and recrystaUising the 1,3-isomers directly from the mixtures. E. Ttqglycerides

To a mixture of 1 mol 1,3-diglyceride, 1.3 mol dry pyridine and dry benzene or hexane as solvent in the ratio 1 : 4, the appropriate acid chloride (1.2 mol) was quickly added dropwise while stirring at 25-40°C. The acid chloride was usually * Removal of NaCI by aqueous extraction caused relatively more monoglyceride to form.

A.P.J. Mank et aL, 1,3-Di- and triglyceride synthesis

111

diluted with an equal volume of benzene or hexane. After 2.5-3 hr heating under reflux the acylation was practically complete (TLC). The mixture was allowed to cool slightly to under its boiling point and 10 ml water was carefully added dropwise to decompose the excess acid chloride. The mixture was again heated under reflux for 5-10 min, cooled, and more water added. The lower layer was removed, and the organic layer was washed with dilute H2SO4, and water, dried over MgSO4 and evaporated. The free fatty acid (5-6%) was removed by a modified Borgstrom method (see below) for those triglycerides melting up to 60°C. After this deacidification triglycerides with a m.p. between 25-60°C were purified by recrystallization from acetone (1 : 5) at - 5 ° C - 0 ° C and sometimes further purified by column chromatography. For higher melting compounds (above 60°C) direct purification and removal of free fatty acid by recrystallization from benzene/ ethanol (1 : 1, v/v) at 10-20°C using 10 ml/g triglyceride was preferred. Yields varied from 70-90%, depending on the purification procedure.

F. Purification of triglyeerides 1. Deacidification by a modified BorgstrOm procedure [ 7] The triglyceride containing some free fatty acid was dissolved in the ratio of not more than 1 g/6 ml of a mixture of methanol/CHC13/hexane (40 : 33 : 27, v/v/v). The solution was extracted once with one third of its volume of aqueous ammonia (1 tool/l) or KOH (lmol/1). The upper layer containing the fatty acid salts was separated, the lower layer was diluted with an equal volume of CHC13 or hexane, and then this solution was washed to neutrality with water or aqueous Na2SO4 solution (50 g/l). The dried organic solution was evaporated. The yield of residual triglyceride was almost quantitative. 2. Column chromatography Merck silica gel was used, particle size 0.06-0.2 mm, containing 8-10% water. The triglycerides were chromatographed using 5 g silica per gram triglyceride and a gradient of 0 - 8 % (v/v) diethyl ether in light petroleum (b.p. 40-60°C). G. Analyses 1. TLC on silica gel plates Merck Type 60F 254, thickness 0.25 mm was used. Eluents were toluene (A) and toluene/diethyl-ether, 80 : 20, v/v (B). A 10% solution of phosphomolybdic acid in 96% ethanol was used for detection; the plates were heated at 150°C. Alternatively, a 0.2% solution of 2,7-dichlorofluorescein in 96% alcohol and UV light was used. 2. GLC The composition of the products wasdetermined on OV-17, after treatment of the sample with trimethyl silylating agents. Satisfactory base-line separation was

112

A.P.J. Mank et al., 1,3-Di. and triglyceride synthesis

obtained for 1,2- and 1,3-diglycerides. With this method impurities in the crude intermediates could be detected and their removal during subsequent purification stages monitored. For details, see ref. [8]. 3. Lipolysis

The positions of the acyl groups in many but not all of the triglycerides were determined by the lipase method [ 1,9]. There was no evidence of acyl migration in the final acylation of di- to triglyceride. 4. Melting points

Melting points were determined with the micro melting apparatus "Kofler". M.p. data refer usually to the most stable polymorphic form 03) [10]. H. Examples o f diglycerides

1.1,3-Dipentadecanoylglycerol [C15:o - (OH) - C15:o]

An amount of 30.8 g glycidol pentadecanoate (purity 95%; 0.1 mol) with 24.2 g (0.1 mol) pentadecanoic acid and 0.81 g (0.0039 mol) TEAB was heated for 2;5 hr at i00°C. The product was dissolved in chloroform, the catalyst was removed by extraction with water and the organic layer was dried (MgSO4), fdtered and evaporated to dryness. The diglycerides were isomerized for 72 hr in a drying oven at 50°C. After crystallization from acetone (1 : 6 at 0°C) and hexane (1 : 6 at 0°C), 41.5 g 1,3-dipentadecanoylglycerol was obtained (77% of theory); m.p. 70°C (lit. [11 ] 68.5°C), purity 99% (GLC). 2. 1-Palmitoyl-3-stearoylglycerol [C 16:0 - (OH) - C18:0 ]

An amount of 11.4 g glycidol stearate (purity 98%; 0.034 mol) with 8.7 g palmit. ic acid (0.034 mol) and 0.29 g (0.0014 mol) TEAB was heated for 3 hr at 100°C. The catalyst was extracted as above. The diglycerides were isomerized for 100 hr at 50-55°C. After crystallization from acetone (1 : 8 at 5°C) and hexane (1 : 8 at 20°C), 14 g (70% of theory) 1-palmitoyl-3-stearoylglycerol was obtained; m.p. 71 °C (lit. [ 12] 71.5°C), purity 99% (GLC). 3. 1,3-Dioleoylglycerol 1C18:1 - (OH) - C18:1]

An amount of 30 g glycidol oleate (purity 98%; 0.09 mol), 25.4 g (0.09 mol) oleic acid and 0.75 g (0.0036 mol) TEAB was heated for 3 hr at 100°C under nitrogen. The product was dissolved in chloroform, washed with water and after drying the chloroform solution was evaporated. The residue was crystallized twice from acetone directly (1 : 10 at -25°C). The yield was 28.8 g (52% of theory) 1,3-dioleoylglycerol; m.p. 26-28°C (lit. [13] 25°C), purity 99% (GLC). Table 2 shows some other 1,3-diglycerides prepared in the way described.

A.P.J. Mank et aL, 1,3-Di- and triglyceride synthesis

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Table 2

1,3-Diglycerides prepared by the catalysed addition of fatty acid to glycidol esters. Diglyceride

M.p. CC)

Lit. M.p. (°C)

Yield after two crystallizations (%)

C10:0 - (OH) - C10:0 C10:0 - (OH) - C16:0 C13:0 - (OH) - C13:0 C14:0-(OH)-C14:0 C15:0 - (OH) - C15:0 C16:0 - (OH) -? C12:0 C16:0-(OH)-C16:0 C16:0 - (OH) - C18:0 C16:0 - (OH) - C22:0 C18:0-(OH)-C18:0 C18:0 - (OH) - C18:1 C18:1-(OH)-C18:1 C18:0 - (OH) - C20:0

44.0 54.5 62.0 66-67 70.0 58-59 73-74 71.0 76-77 80-81 48.0 26-28 76.0

44.5 [11]

68.5 69.0 67.0 60.0 a 77.0 47.0 a 69.0 70.0 52.0 a 74,0 60.0 52.0 a 68.0

59.5 67 68.5 59.5 74 71.5

[11] [14] [11] [16] [12] [12]

80 [12] 49 [12]; 54 [15] 25 [13]

a Yields obtained without solid phase isomerization.

L Examples of triglycerides 1. 3-Oleoyl-2-palmitoyl-l-stearoylglycerol A mixture of 48.0 g (77 mmol) 3-oleoyl-l-stearoylglycerol, 7.9 g (100 mmol) pyridine, 25.4 g (93 mmol) palmitoyl chloride and 190 ml hexane was heated for 2.5 hr under reflux. After addition of sufficient water to destroy the excess of acid chloride, the organic layer was cooled, washed with water, dried (MgSO4) and the solvent removed, The product was deacidified by the modified Borgstrt~m procedure and the triglyceride crystallized from acetone. A yield of 61.5 g (93% of theory) triglyceride was obtained; m.p. 40°C (lit. [12] 41°C). Lipase analysis in the 2-position showed 95% C16:0, 3% C18:0 and 2% C18:1.

2. 2-Oleoyl-l-palmitoyl-3-stearoylglycerol A mixture of 89.5 g (0.15 mol) 1-palmitoyl-3-stearoylglycerol, 15.8 g (0.2 mol) pyridine, 54 g (0.18 mol) oleoyl chloride and 400 ml hexane was reacted and worked up as above. After further purification by chromatography 108 g (84% of theory) triglyceride was obtained; m.p. 3 2 - 3 3 ° C , (lit. [ 17,18] 3 1 - 3 3 ° C and lit. [12,18] 3 7 - 3 8 ° C ) . Lipase analysis in the 2-position showed 97% C18:1, 2% C16:0, 1% C18:0.

3. 2-Pentadecanoyl-l,3-ditridecanoylglyeerol A mixture of 16.1 g (33 mmol) 1,3-ditridecanoylglycerol, 3.4 g (43 mmol) pyri-

114

A.P.J. Mank et aL, 1,3.Di- and triglyceride synthesis

dine, 10.4 g (40 mmol) pentadecanoyl chloride and 95 ml benzene was reacted as above. After the crude product had been washed and the solvent removed it was purified at once by two crystallisations from benzene/ethanol yielding 20.7 g triglyceride (89% of theory); m.p. 58.5°C, GLC 99%. Besides a large number of triglycerides described elsewhere, the following hitherto undescribed compounds were prepared similarly: 2-heptadecanoyl-1,3-dipentadecanoyl-glycerol (m.p. 66°C), 2~trans-9-hexadecenoyl)-1,3-dipalmitoylglycerol (m.p. 54°C), 2-elaidoyl-3-oleoyl-l-palmitoylglycerol(m.p. 26°C), 3-elaidoyl-2oleoyl-l-palmitoylglycerol (m.p. 2 2 - 2 3 ° C ) , 3-eicosanoyl-2-oleoyl-l-stearoylglycerol (m.p. 4 1 - 4 2 ° C) an d 2-(cis- 13-doc osenoyl)- 1,3-diste aroylglyce rol (m.p. 47 oC).

References [ 1] R.G. Jensen, Synthetic Glycerides, Topics in Lipid Chemistry (Ed. F.D. Gunstone), Vol. 3, Elek Science, London (1972) p. 1 [2] G. Maerker, E.J. Saggese and W.S. Port, J. Amer. Oil Chem. Soc. 38 (1961) 194; G. Maerker, J.F. Carmichael and W.S. Port, J. Org. Chem. 26 (1961) 2681; W.J.M. Rootsaert and J.G.v.d. Vusse, Chem. Eng. Sci. 21 (1966) 1067 [3] A.P.J. Mank, Dutch Patent Application 7.103.013 Chem. Abstr. 76 (1972) 13849; cf. US Pat. 2.523.309 (to E.B. Kester), Chem. Abstr. 45 (1951) 885 [4] A. Crossley, I.P. Freeman, B.J.F. Hudson and J.H. Pierce. J. Chem. Soc. (1959) 760; I.P. Freeman and I.D. Morton, J. Chem. Soc. (C) (1966) 1710 [5] W.Th.M. de Groot, Lipids 7 (1972) 626 [6] C.M. Lok, J.P. Ward and D.A. van Dorp this issue, p. 115 [7] P. Belfrage and M. Vaughan, J. Lipid Res. 10 (1969) 341 [8] J.A.W. Engbersen and F. van Stijn this issue, p. 133 [9] F.H. Mattson and R.A. Volpenhein, J. Lip. Res. 2 (1961) 58 [ 10] D. Chapman, The Structure of Lipids, Methuen and Co, London (1965) [ 11 ] T. Malkin et al., J. Chem. Soc. (1937) 1409 [12] D. Chapman, A. Crossley and A,C. Davies, J. Chem. Soc. (1957) 1502 [13] M.G.R. Carter and T. Malkin, J. Chem. Soc. (1947) 554 [14] F.J. Baur and A.W. Lange, J. Amer. Chem. Soc. 73 (1951) 3926 [15] B.F. Daubert and H.E. Longenecker, J. Amer. Chem. Soc. 66 (1944) 53 [16] S.S. Sidhu and F.B. Daubert, J. Amer. Chem. Soc. 68 (1946) 2603 [17] J.P. Carreau, Bull. Soc. Chim. Fr. (1970) 4111 [18] T.P. Hilditch and P.N. Williams,The Chemical Constitution of Natural Fats, Chapman and Hall, London (1964) p. 664