Chemistry and Physics o f Lipids 16 (1976) 123-132 © North-Holland Publishing Company

DETERMINATION OF ENANTIOMERIC PURITY OF GLYCERIDES WITH A CHIRAL PMR SHIFT REAGENT J. BUS, C.M. LOK and A. GROENEWEGEN Unilever Research, Vlaardingen, The Netherlands

Received July 27, 1975,

accepted November 27, 1975

Tris(3-heptafluorobutyryl-d-camphorato)europium(Ill),Eu(hfbc) 3 was used to determine the optical purities of enantiomeric mixtures of tri-, di- and monoglycerides with various fatty acid chain lengths by proton magnetic resonance (PMR). Synthesized model enantiomers were used to assign PMR signals. Enantiomeric signal separation becomes more difficult if the chain length difference between the fatty acids in the 1- and 3-positions of glycerol becomes smaller. The sign of the enantiomeric shift difference (AA6) of the terminal acyl CH3 group of 1-acyl2,3-distearoyl-sn-glycerolvs its enantiometer remains the same in the series acyl is hexanoyl, butyryl, propionyl, but is reversed for acetyl. The absolute configuration of the main triglyceride of the seed oil of Euonymus alatus was determined to be 3-acetyl-1,2-distearoyl-sn-glyceroland that of a monobutyryl triglyceride fraction from hydrogenated bovine butterfat was confirmed to be mainly 1,2-diacyl-3-butyryl-snglycerol. The enantiotopic behaviour of the glycerol CH2 groups in (nearly) symmetric di- and triglycerides is discussed.

I. Introduction Natural fats are composed o f triglycerides, the esters o f glycerol and a variety of fatty acids. If the primary h y d r o x y l groups o f glycerol are esterified with different fatty acids, the central carbon atom becomes asymmetric and consequently the triglyceride can exist in two enantiomeric forms. Few methods for establishing the chirality o f organic molecules are applicable to chiral triglycerides. The methods for analysing mixtures, based on the stereospecificity o f phospholipase A 2 for synthetic phospholipids derived from unknown triglycerides [1 ], are very time-consuming. Additionally, most physical methods like optical rotatory dispersion (ORD), melting point determination, X-ray diffraction and piezoelectricity are confined to relatively pure triglycerides. Reviews of these methods were published b y Schlenk [2] and Smith [1]. The isolation and syntheses o f optically active glycerides [3] prompted us to investigate the possibility o f measuring the enantiomeric purity * of glyceride samples * The enantiomeric (or optical) purity is defined as [S-enantiomer] - [R-enantiomer]/[S-enantiomer] + [R-enantiomer]. 123

124

J. Bus et al., Enantiomeric purity o f glycerides

with proton magnetic resonance spectrometry (PMR) after addition of chiral shift reagents (CSR). Recent publications have indicated that lanthanide shift reagents are very useful for the analysis of PMR spectra of saturated and unsaturated triglycerides [4-8]. The CSRs form complexes via the free electron pairs of the ester groups of triglycerides and of the ester and hydroxyl groups of di- and mono-glycerides. The hydroxyl groups have a better complexing ability than ester groups [9]. Two enantiomers of a glyceride form diastereoisomeric associates, the PMR spectra of which are basically different. Since the complex formation takes place near the chiral centre (C-2 of the glycerol part), it is expected that only the protons near this centre will behave differently in the two enantiomers. Because of its good Lewis acidity [10,11 ] we used as CSR tris(3-heptafluorobutyryl-d-camphorato)europium(III), Eu(hfbc)3. An experimental difficulty was that the commercially available CSRs were not yet available in a fully deuterated form. This resulted in a number of extra signals in the spectra, the intensities of which were gradually enhanced upon addition of each new portion of CSR, giving overlap in some cases with substrate signals of interest. The molar ratios of CSR/substrate given in table 1 might not be optimal in all cases, but are meant to be indications for some useful regions. As far as possible the glyceride nomenclature following the convention of Hirschmann, now adopted by the IUPAC-IUB [ref. 12], will be used. The application to glycerol is particularly simple: If the secondary hydroxyl group is drawn to the left side of C-2 in a Fischer projection, C-1 is above and C-3 is below C-2. In these cases the prefLx "sn-" (stereospecifically numbered) will be included in the glyceride names. The prefix "rac-" will be used for racemic mixtures. CH2 OH

sn- 1 position

I

t t 0 P. C .q H

sn-2 position

I

CH2 OH

sn-3 position.

Apart from several synthesized mono-, di-, and triglycerides two natural products, the seed oil of E u o n y m u s alatus and a butterfat fraction, were studied and compared with model glycerides.

II. Experimental PMR spectra were measured on Varian A-60, HA-100 FT and HR-220 (TNO, Delft) spectrometers at 35, 27 and 29°C respectively (0.3-0.4 ml CDC13 solutions'). Chemical shifts (6) are given in ppm downfield from internal tetramethylsilane (TMS) and coupling constants (J) in Hz. The lanthanide-induced shifts (A/i) as well as the differential shifts of protons in different enantiomers (AAS) are given in ppm.

J. Bus et al., Enantiorneric purity o f glycerides

125

Eu(hfbc)3 was obtained from Willow Brook Laboratories, Inc. USA (trade name "Eu-optishift II") and was used without further purification. The seeds of Euonymus alatus were purchased from Van Tubergen B.V., Haarlem, The Netherlands. The syntheses of the racemic and optically active glycerides will be described elsewhere [3].

III. Results and discussion

A. Triglycerides The best chance for a successful separation of proton signals of enantiomeric triglycerides occurs if a large difference exists between the fatty acids in the sn-1- and sn-3. positions, because in that case the asymmetry is large. Fig. 1 shows that the acetyl methyl signals of 1,2-diacetyl-3-stearoyl-sn-glycerol and of 2,3-diacetyl-1stearoyl-sn-glycerol are separated well enough, after addition of Eu(hfbc)3, to allow the determination of the optical purity. Addition of more shift reagent as used in fig. 1 does increase the AA6 for the low-field methyl signal, but line broadening prevents a better resolution of these signals. Good resolution can only be effectuated by measuring at 220 MHz. The assignments of the methyl signals to the 1- or 2- sn-glycerol positions in 1,2diacetyl-3-stearoyl-sn-glycerol are considered to be too speculative because of the uncertainty that still exists about the actual structure of the glyceride complex [4-6], but it is nevertheless clear which set of methyl signals belongs to which enantiomer. The upfield signal of the a-CH2 triplet contains a small impurity signal (fig. 1D). The acetyl proton signals of 1-acetyl-2,3-distearoyl-rac-glycerol are also well sep-

A

i

I

I

I

I

I

I

25

2.0

55

5,0

5.5

5,0

6.0

I

5.5 8(ppm)

Fig. 1. PMR spectra of 1,2-diacetyl-3-stearoyiglycerol in CDCI3 at 60 MHz; CH3C=O (singlets) and CH2C=O (triplets) signals only. A. racemic without CSR; B. racemic; C. 1,2-diacetyl-3-

steaxoyl-sn-glycerol, optical purity 45%; D. 2,3-diacetyl-l-stearoyl-sn-glycerol,optical purity 87%; E. idem + racemic. Molar ratios Eu(hfbc)3/substrate: 0 (A), 1.25 (B), 1.02 (C), 1.39 (D and E).

126

J. Bus et al., Enantiomeric purity of glycerides A

B

C

D

7.7

7.8

6.8

0.2 ppm

6.9

8 ( ppm )

Fig. 2. PMR spectra of 1,2-distearoyl-3-acetylglycerolat 60 MHz;CH3C=O and the overlapping upfield CH2C=O signals only. A. racemic; B. l-acetyl-2,3-distearoyl-sn-glycerolplus the racemic mixture; C. isolated and hydrogenated acetyl triglyceridesfrom Euonyrnus alatus seed oil; D. Euonymus alatus seed oil after addition of 1,2-distearoyl-3-acetyl-rac-glycerolin CDCI3. Molar ratios Eu(hfbc)3/substrate about 3.3.

arated, even at 60 MHz (fig. 2A, table 1), but at all CSR/substrate ratios where the enantiomer signals are separated the upfield absorbing a-CH 2 signal overlaps with the CH3C=O signals. This prevents an accurate determination of the optical purity. The acetyl signals were assigned by addition of synthesized 1-acetyl-2,3-distearoyl-snglycerol to the solution of the racemic mixture (fig. 2B). The seed oil of E u o n y m u s alatus contains about 98% 3-acetyl-1,2-diacylglycerol [13]. Chromatographic separation and hydrogenation yield mainly triglycerides with acetic, palmitic, and stearic acids. Only one acetyl signal is present in the spectrum of the glyceride mixture (fig. 2C) but a second signal appears upfield from the first one upon addition of 1-acetyl-2,3-distearoyl-rac-glycerol(fig. 2D) indicating that the natural triglyceride is the 3-acetyl-sn-glycerol enantiomer. This in in agreement with the results of Kleiman et al. [13], who found a similar result for Euonymus verrucosis seed oil by ORD measurements. The lower-field u-CH2 triplet was never observed to be resolved into two triplets. The higher-field one in some cases, but this is not always visible due to the overlap problems mentioned before. An extra difficulty is formed by some minor impurities of unknown origin, often found in the region of the relevant signals. The next example, 1-propionyl-2,3-distearoyl-rac-glycerol shows that the only useful signal is the propionyl methyl triplet. Fig. 3A shows this triplet of a mixture of 3-propionyl-1,2-distearoyl-sn-glycerol and of the racemic glyceride at 220 MHz. Even at 60 MHz the sepraration is clearly visible. The stronger high-field triplet comes from the 3-propionyl-sn-glycerol derivative.

127

J. Bus et al., Enantiomeric purity o f glycerides

Table 1 Enantiomeric shift differences (AA6 ppm) of proton signals from various glyceridesin CDC13. -rac-Glycerol

Substrate Molar strate ratio 0zmol) CSR/ Substrate

MHz

Resonance observed

AA6/A6

1,2-diacetyl3-stearoyl

32.8

1.56

60 220

sn-I-CH3C=O~ sn'2"CH3C=O/

0.10 /3.82 0.04 /4.08

1-acetyl-2,3-distearoyl-

26.1

3.36 2.32

60 60

CH3C=O CH2C=O

0.04 /5.93 0.08 /4.48 /4.93

1-propionyl-2,3-distearoyl-

66.6

1.84

60

CH3CH2C=O

0.04 /2.57

1-butyryl-2,3-distearoyl-

26.2

2.80

220

CH2C=O CH2C=O CR3(CH2)2C=O

0.005/1.68 0.01 /1.78 0.01 /0.50

1-hexanoyl-2,3-dipalmitoyl-

35.3

4.1

220

CH3(CH2)4C=O

0.006/0.13

1-benzoyl-3-stearoyl-

48

1.20

60

CH2C=O

0.04 /2.93

1,2-dipalmitoyl

38.7

0.45 1.36

100 100

CH2C=O CH2C=O

0.04 /1.07 0.075/2.95

1-stearoyl-

65.4

1.30

60

CH2C=O

0.18 /2.27

-

The 1-butyryl-2,3-distearoyl-rac-glycerol can be studied by the CH2 C=O signals, but even better by the butyryl methyl signal. The line separations become smaller, however (table 1). In practice this means that the separation can only be studied at 220 MHz, or with higher-field spectrometers. Two of the CH2C=O signals at 220 MHz (fig. 3C) are partly resolved but the upfield one is not. Too much speculation is required to assign these CH2 signals to the 1-, 2- or 3-positions in the glyceride. The assignment of the butyryl methyl signals (fig. 3D) was performed by mixing 3butyryl-1,2-distearoyl-sn-glycerol with the racemic mixture, which proved that the upfield absorbing triplet belongs to the 3-butyryl-1,2-distearoyl-sn-glycerol. This result can be used for the determination of the absolute configuration of a natural triglyceride. A bovine butterfat fraction containing a relatively large amount of monobutyryl glycerides was obtained by repeated crystallization of hydrogenated bovine butterfat from acetone. After addition of CSR the butyryl methyl triplet (fig. 3E) suggests the presence of one enantiomer. After addition of 1-butyryl-2,3-distearoyl-rac-glycerol the spectrum of fig. 3F was obtained. The upfield triplet has the higher intensity.

J. Bus et aL, Enantiomeric purity of glyceride~

128 A

C

I

&0

I

I

100

1.45

/,.00 '

'

|

250

I

1.90 8(pprn)

Fig. 3. PMR spectra of various triglycerides in CDC13 at 220 MHz. A. Proplonyl CH 3 signal of a mixture of 1-propionyl-2,3-distearoyl-rac-glyceroland 3-propionyl-1,2-distearoyl-sn-glycerol (~1.5 : 1); B. Hexanoyl CH 3 signal of 1-hexanoyl-2,3-palmitoyl-rac-glycerol;C, D. 1-butyryl2,3-distearoyl-rac-glycerol,CH2C=O (C) and butyryl CH 3 (D) protons. The upfield part of D is overlapped by a spinning side band. E. 3-butyryl-1,2-diacyl-sn-glycerolfraction fro m bovine butterfat (25.8 mg) and 146.3 ~tmol/1 CSR; F. The butterfat fraction E, after addition of 1butyryl-2,3-distearoyl-rac-glycerol. Molar ratios Eu(hfbc)3/substrate: 1.53 (A), 4.1 (B), 2.80 (C and D), not determined (F).

This confirms that the b u t y r y l group in butterfat triglycerides is mainly in the sn-3 position, a result that was also obtained via an enzymatic procedure [14,15]. Finally a very small AA8 value was found for the hexanoyl methyl group o f 1hexanoyl-2,3-dipalmitoyl-rac-glycerol at 220 MHz (AA~ = 0.006 at a molar ratio o f not less than 4.1). Fig. 3B shows that only if the enantiomer ratio is near 1 : 1 b o t h isomers can be detected but if the ratio deviates too much from this value the minor component can hardly be detected. The spectrum o f a mixture o f 3-hexanoyl-1,2distearoyl-rac-glycerol with 3-hexanoyl-1,2-distearoyl-sn-glycerol reveals also an enhanced intensity o f the upfield triplet. All other proton signals give too much overlap, thus preventing their use for the determination o f enantiomeric purity. It is logical to expect that this compound is about the last homologue in this series o f triglycerides for w h i c h signals o f enantiomers can be separated. In 2-acetyl-l-palmitoyl-3-stearoyl-rac-glycerolno line separation due to the two enantiomers could be observed because the molecule has a pseudoplane o f symmetry. However, a chemical shift difference was found between the sn-I and sn-3 CH 2 groups in the glycerol part o f the molecule after addition o f CSR. These CH 2 groups

J. Bus et al., Enantiomeric purity of glycerides

129

C

A

! 9.0

I 8.5

I 4.9

! 4.5 ~(ppm)

Fig. 4. PMR spectra in CDCI 3 with l~u(hfbc) 3 at 60 MHz. A. glycerol CH 2 protons o f 2-acetyl-

1-palmitoyl-3-stearoyl-rac-glycerol,molar ratio Eu(hfbc)3/substrate 1.19; B. CH2C=O protons of 1-stearoyl-sn-glycerol;C. as B with 1-stearoyl-rac-glyceroladded. Molar ratio Eu(hfbc)3/substrate for B and C about 1.33. can be dealt with as "AB" parts of "ABX" systems which in an achiral medium lead to two coinciding sets of two doublets of doublets, but in a chiral medium these CH2 groups are, in principle, non-equivalent, they are "enantiotopic by internal comparison" (ref. [16]), an effect which has also been demonstrated in the NMR signals of the methyl groups of 2-propanol [17]. In fig. 4A the complete signal of the two glycerol CH2-groups is reproduced after addition of CSR. Only the up field protons of each CH2 group exhibit a measureable chemical shift difference. At a CSR/substrate ratio of 1.19 we found AA~5 = 0.04 (A~i = 4.6), at a radio of 2.01, AA~ = 0.07 (A6 not calibrated). The other glycerol CH2 proton broadens a littele in this case. A further discussion of this phenomenon is given in the 1,3-diglyceride paragraph. The sn-1- and sn-3-CH2C=O protons developed no measurable chemical shift difference. B. Diglycerides

The study of diglycerides might be complicated by the fact that shift reagents are Lewis acids capable of causing isomerizations. Pfeffer [18] found that both pure 1,3and 1,2-isomers equilibrate at room temperature to a mixture of 72% 1,3- and 28% 1,2-diglycerides in a solution of tris(1,1,1,2,2,3,3,-heptafhioro-7,7-dimethyl-4,6octanedionato) europium(Ill) d30 [Eu(fod)3 ] in CDC13 within 24 hr. We found, however, that Eu(hfbc)3 does not cause a measurable isomerization within 24 hr under the same conditions, but that Eu(fod)3 does indeed (thin-layer chromatography; toluene/diethyl ether 85:15). This result enabled us to study also the diglycerides with Eu(hfbc)3. 1. 1,3-diglycerides The strongly asymmetric 1-benzoyl-3-stearoyl-rac-glycerol shows a chemical shift difference for the CH2C---Oprotons of the two enantiomers (table 1) after addition of CSR. The 1-palmitoyl-3-stearoyl-rac-glyceroldoes not. A pseudoplane of sym-

130

J. Bus et al., Enantiomeric purity of glycerides

metry prevents this, as could be expected. However, in 1-palmitoyl-3-stearoyl-racglycerol, 1,3-dipalmitoyl-glycerol and 1,3-distearoylglycerol the sn-1- and sn-3CH2 C=O protons are enantiotopic by internal comparison [16,17]. The triplets show a differential chemical shift (AA5) after CSR addition. That the effect in 1-palmitoyl3-stearoyl-rac-glycerol is not caused by the presence of two enantiomers could be proven by measuring 3-palmitoyl-I -stearoyl-sn-glycerol, where the same phenomenon was found. At a molar ratio CSR/substrate of about 2, AA5 is about 0.15 with only small variations (AS ~ 4.2-4.4). The sn-1- and sn-3-CH2 groups of the glycerol moiety of 1,3-dipalmitoylglycerol show a similar behaviour to that described above for 2-acetyl-l-palmitoyl-3-stearoyl-rac-glycerol but now only the downfield doublet of doublets of the CH 2 signal shows a chemical shift difference. At a molar ratio CSR/1,3-dipalmitoylglycerol = 1.17, A5 = 3.8 and AA5 = 0.18. The much stronger effect must be due to the much greater complex-forming ability of the hydroxyl group [9]. The signals from the glycerol protons are often very much broadened after addition of large amounts of shift reagent. We have attempted to explain why in this case as well as in the 2-acetyl-l-palmitoyl-3-stearoyl-rac-glycerol spectrum only one sn-l-CH 2 proton of the glycerol moiety gives a chemical shift difference with one sn-3-CH2 proton and the other one does not (or is very small). If this behaviour has a more general character, one would expect that e.g. 1,3-dichloro-2-propanol and 1,3-dimethoxy-2-propanol behave similarly. However, this was apparently not true. In the dichloro compound, AA5 for the faster shifting methylene C - H proton is twice as large as that for the other one and in the dimethoxy compound both AA5 values are nearly equal.

2. 1,2-diglycerides We investigated 1,2-dipalmitoyl-rac-glycerol, 2,3-dipalmitoyl-sn-glycerol and 1,2distearoyl-rac-glycerol as members of the class of 1,2-diglycerides. In these compounds protons of both CH 2 C=O groups show enantiomeric splitting. Table 1 only shows data of the less shifted, high-field CH2C=O triplet at two CSR/substrate ratios (2,3-dipalmitoyl-sn-glycerol at lower field than the sn-1,2 isomer; at molar ratios