- Email: [email protected]
Acyl migration in glycerides. I. A bimolecular resonant ion complex as intermediate in acyl migration of monoglycerides
Chem. Phys. Lipids 1 (1967) 113-127 © North-Holland Publ. Co., Amsterdam
ACYL MIGRATION I. A B I M O L E C U L A R
IN GLYCERIDES.
RESONANT
AS INTERMEDIATE ACYL MIGRATION
ION COMPLEX IN
OF MONOGLYCERIDES
DMYTRO BUCHNEA* Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada Received 30 May 1966 Acyl migration and the rate of racemization of D- and L-monostearin and the rate of conversion of 2-monostearin into DL-isomer catalyzed by hydrogen chloride under anhydrous conditions have been studied. It was found that the rate of racemization and the rate of conversion differ considerably under identical conditions. The rate of racemization and the rate of conversion of these three isomers into DL-monostearin increases as follows: D-monostearin, L-monostearin and 2-monostearin. 2-Monostearin is one of the most unstable isomers and is completely convertible into DL-isomer (100%). The rate of the racemization of D- and L-monostearin to DL-isomer coincides perfectly with the rate of the conversion of these two isomers into bismonostearin ethers. D-monostearin is inherently more than twice as stable as L-isomer. All four isomers were converted into bismonostearin ethers. The synthesis of the bismonostearin ethers is described. The formation of a bimolecular resonant ion complex as an intermediate in acyl migration is suggested.
Introduction D u r i n g the synthetic w o r k o n optically active, m i x e d fatty acid diglycerides 1) it was f o u n d t h a t D - l - t r i p h e n y l m e t h y l - 2 - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether, on r e m o v a l o f its protective t r i p h e n y l m e t h y l g r o u p with h y d r o g e n chloride in ice-cold a n h y d r o u s p e t r o l e u m ether solution, forms an equilibriu m m i x t u r e o f f o u r c o m p o u n d s . These f o u r c o m p o u n d s were s e p a r a t e d b y c h r o m a t o g r a p h y on a silicic acid c o l u m n , a n d were c h a r a c t e r i z e d as: D-2stearoylglyceryl-3-benzyl ether; a p p r o x . 5%, D - l - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether; a p p r o x . 55% to 65%, b i s ( D - 2 - s t e a r o y l g l y c e r y l - 3 - b e n z y l ) - l - e t h e r , a p prox. 20%; and D - l - c h l o r o - 2 - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether, a p p r o x . 8% to 20%. T h e v a r i a t i o n o f D - l - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether a n d D - l - c h l o r o 2-stearoylglyceryl-3-benzyl ether in the e q u i l i b r i u m m i x t u r e is due to the stearoyl g r o u p m i g r a t i o n f r o m C-1 b a c k to C-2, resulting in D - l - c h l o r o - 2 stearoylglyceryl-3-benzyl ether, which is caused by longer action o f h y d r o g e n c h l o r i d e u p o n D - l - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether. The presence o f benzyl g r o u p assured t h a t the a s y m m e t r y o f glyceryl m o i e t y a n d therewith the * Address: Banting Institute, 100 College Street, Toronto 5, Canada.
114
DMYTRO
BUCHNEA
optical property is fully maintained throughout these conversions. On removal of their protective benzyl groups by catalytic hydrogenolysis with hydrogen in presence of palladium black in glacial acetic acid, these compounds were converted into 2-stearoylglycerol, D-l-stearoylglycerol, bis(D2-stearoylglyceryl)-l-ether and D-l-chloro-2-stearoylglycerol respectively. Upon the introduction of a second stearoyl group in the position-3 of bis(D-2-stearoylglyceryl)-l-ether and D-l-chloro-2-stearoylglycerol, an optically active bis(D-2,3glyceryldistearate)-l-ether with a specific rotation of + 1 °, in chloroform, and D-l-chloro-2, 3-glyceryldistearate, with a specific rotation of + 3 °, in chloroform, were obtained. These results prove that the ether linkage and chlorine atom are in the position-1 ; otherwise symmetrical compounds would have resulted. The fact that the removal of triphenylmethyl group with hydrogen chloride is connected with the acyl migration and also with the formation of bis(D-2stearoylglyceryl-3-benzyl)-l-ether led to the idea that this bisglyceride ether might be the intermediate in acyl group migration 2). However, the kinetic study of the reactions between monostearins and hydrogen chloride revealed that the formation of bis(stearoylglyceryl) ethers takes place independently of stearoyl group migration. This fact was established by treatment of 2-stearoylglycerol (1) with hydrogen chloride in ice-cold anhydrous ether, by taking aliquots at various intervals, and titrating with periodic acid for glycol determination (fig. 1), and also analysing on thinlayer chromatography. % 100 ~
•
o
~o
2
._..
L-
-.
o,-.
-
.... zx D '~-
i
[
•~
f%.. 0 °A
t
• /',
/
\
~0
-Stearoylglycerol
% -
o_
",.,
\
\ '.,J(
0
~
!
20
.'x. I
I
~
L
40
i
o
60 Minutes
!
80
,
I
I00
~
!
120
Fig. l. The rates of conversion of 2-stearoylglycerol,L-3-stearoylglycerol,D-l-stearoylglycerol and DL-l-stearoylglycerol into bisglyceride ethers in presence of dry, gaseous hydrogen chloride in anhydrous ether at 0°C.
ACYL MIGRATION IN GLYCERIDES
+
I
115
I 0 I 0
1 0
I 0
~,
I ''~,.o 0"" I
0 I 0 I 0 L
0 ÷
/ r 0
I 0
+
+~
I 0 [
0 I
~-u-~
--
L 0 I,~ e.l
L~ -F
I
o @ I ©
©
~ -u-~
I 0 I 0 I
.-~
L
J
I 0 0
•
[
I
o I 0
0 o I ~ I
0
L 0 I
0 I
I
o
I o I
IT I 0 I
116
DMYTRO BUCHNEA
The conversion of 2-stearoylglycerol to DL-l-stearoylglycerol proceeds at a constant rate as indicated by the rate of consumption of periodic acid (fig. 1). Within 20 min stearoyl groups migrated from positions C-2(a) and C-2(b) to the positions C-l(a) and C-3(b) (fig. 2, n) almost completely (98~o). Then the amount of periodic acid added slowly decreases for each successive sample and within 50 rain reaches 0-value again. After 70 min 2-stearoylglycerol (1) was completely converted by way of DL-l-stearoylglycerol (nI) to a mixture of bis(DL-l-stearoylglyceryl)-3-ether (v), bis(DL-2-stearoylglyceryl)-3-ether (v0 and bis(DL-l-chloro-2-stearoylglyceryl)-3-ether (vi0, (fig. 2). This study was extended to the optically active D-l-stearoylglycerol and L-3-stearoylglycerol as well as to the racemic DL-l-stearoylglycerol. It was found that there is a considerable difference in the stability of D- and L-, and DL-l-stearoylglycerol in presence of hydrogen chloride under identical conditions. L-3-Stearoylglycerol was converted to the mixture of compounds v, Vl, vii, within 30 min, D-l-stearoylglycerol needs 70 min to be converted to the same mixture, whereas DL-l-stearoylglycerol took 120 min to be converted to the mixture of bis(stearoylglyceryl) ethers. This observation indicates that D-l-stearoylglycerol is more than twice as stable as L-isomer. Both D- and L-isomers racemise to DE-form which is the most stable of isomers. Contrary to this, 2-monoglyceride is one of the most unstable of isomers, and it is also completely convertible to DL-isomer. Therefore it is impossible that 2-monoglyceride could be a component of the equilibrium mixture reported by Martina), van Lohuizen and Verkade 4) and others5). The observation that the isomers of monostearin differ in their stability was entirely confirmed by the results obtained from the kinetic study of the rate of racemization of D-l- and L-3-stearoylglycerol and the rate of conversion of 2-stearoylglycerol in presence of known concentration of hydrogen chloride in anhydrous ether solution. The racemization rate of these isomers was studied in presence of a concentration equal to 4 N hydrogen chloride in anhydrous ether solution at 0 °C. It was found that 2-stearoylglycerol converts 100~ to DL-isomer within 10 to 15 min under these conditions. L-3stearoylglycerol needs 80 min to be completely racemized to DL-isomer under identical conditions, whereas D-l-stearoylglycerol under the same conditions was only 40~ racemized (fig. 3). Furthermore, similar results were also obtained from the study of the racemization of D-l-stearoylglycerol and L-3-stearoylglycerol and conversion of 2-stearoylglycerol into DL-isomer in presence of various concentrations of hydrogen chloride in anhydrous ether solution at constant time and temperature. 2-Stearoylglycerol needs a concentration equal to 2-3 N of hydrogen chloride in anhydrous ether solution to undergo a complete conversion to DL-
ACYL MIGRATION
117
IN GLYCERIDES
I00
8C
"% i
so Sl~ sS
0
,/
.~ 60
/''*
Steoroylglycerol
--O#SS
t
,'" ,-"
,A.
,
o 2[] L . . . . -
,,*,
-
zx D ......
z%
~b s
4.0
ooo-
•
20
o. A o"
,~. , , - "
.o...o../k
• •#
0,
°°-ZX
so./k.-
0 0
'~"
~
L
t
20
40
60
I
I
80
!00
Minutes
Fig. 3. The rates of racemization of D-l-stearoylglycerol and L-3-stearoylglycerol, and the rate of conversion of 2-stearoylglycerol into DL-isomer in presence of 4 N hydrogen chloride in anhydrous ether solution at 0 °C. isomer within 50 min at 0°C. L-3-stearoylglycerol requires a concentration over 4 N of hydrogen chloride to undergo 100% racemization, in the same time, whereas D-l-stearoylglycerol at a concentration of over 5 N hydrogen chloride in anhydrous ether solution, under identical conditions, was only 30% racemized. These results prove that the D-isomer is inherently far more stable than the L-isomer, and this might be the reason why D-isomers are predominant in nature. The difference in the stability of D- and L-isomers of monostearins has been completely confirmed by their difference in energy. The determination of the heat of combustion revealed that there exists a difference in energy between D-l-stearoylglycerol and L-3-stearoylglycerol. It was found for example that L-3-stearoylglycerol has about 170 calories per gram more energy than D-l-stearoylglycerol. A preliminary investigation of D- and L-glyceraldehyde indicated that there exists an even greater difference of energy between D- and L-isomers; about 700 calories per gram more energy in favour of L-glyceraldehyde (see following table). This difference of energy between D- and L-isomer might be a tension energy, originating in the distance between 0-3 and 0-2 of L-isomer, which seems to be greater than the distance between 0-1 and 0-2 of D-isomer*. * The experimental details of this investigation will be reported later.
118
DMYTRO BUCHNEA TABLE 1 The heat of combustion of following isomers:
D-l-stearoylglycerol L-3-stearoylglycerol D-glyceraldehyde L-glyceraldehyde
Cal./g
Difference
8417 8590 3992 4698
173
703
Procedures and data MATERIALS
D-l-triphenylmethyl-2-stearoylglyceryl-3-benzyl ether was prepared by the method of Buchnea and Baerl). 2-Stearoylglycerol was prepared either by catalytic hydrogenolysis of D-l-triphenylmethyl-2-stearoylglyceryl-3benzyl ether or from 2-stearoylglyceryl-l-3-benzylidine as described by Bergmann and Carter 6) and Simmel and King7). D-l-stearoylglycerol was obtained by catalytic hydrogenolysis of D-l-stearoylglyceryl-3-benzyletherl). L-3-stearoylglycerol and DL-l-stearoylglycerol were prepared from D-l, 2-isopropylideneglycerol and DL-1, 2-isopropylideneglycerol, as described by Baer and Fischer s) and E. Fischer, et al.°). Analar petroleum ether (b.p. 35-60 °C) and analytical reagent ether were dried over sodium wire. Chloroform analar for use in polarimetric and infrared work was freed of ethanol by distillation over phosphorus pentoxide. Silicic acid Mallinckrodt, 100 mesh (powder), Analytical Reagent, with 12~o weight loss on ignition was used. Palladium catalyst was prepared as described by Tausz and Putnokyl0), except that the palladium after having been washed thoroughly with water, was then stored under the water until used. Glacial acetic acid (C.P.Reagent) as provided by the manufacturer was used as solvent for catalytic hydrogenolysis. Hydrogen chloride was generated from a mixture of concentrated hydrochloric acid and sodium chloride with concentrated sulfuric acid, and dried by passage through concentrated sulfuric acid to the reaction mixture. SYNTHESIS OF BIs(DL-STEAROYLGLYCERYL)-3-ETHER
Bis(DL-l-triphenylmethylglyceryl)-3-ether To a solution of 17.0 g (0.06 M) of triphenylmethyl chloride dissolved in 100 ml of dry benzene was quickly added at 0°C to a solution of 5.0 g (0.03 M) of bis(DL-glycerol)-3-ether,*) dissolved in 50 ml of pyridine. The reaction * The preparation of bis (DL-glycerol)-3-ether is reported in Part III of this series, to be published in Chem. Phys. Lipids. Vol. 1, no. 2.
ACYL M I G R A T I O N IN GLYCERIDES
119
mixture was shaken at room temperature for 24 hr. At the end of this time the reaction mixture was freed from most of benzene and pyridine by distillation under reduced pressure. The residue was then triturated with 200 ml of ice-cold water, centrifuged and the supernatant decanted. The heavy syrup was dissolved in 500 ml of benzene, and then washed successively with two 250 ml portions of ice-cold 2 N sulfuric acid, two 250 ml portions of saturated sodium bicarbonate solution, and finally with 250 ml of distilled water. The solution was dried with 100 g of anhydrous sodium sulfate and after filtration the solution was concentrated by distillation under reduced pressure at 30-35°C. The remaining material was dried at 0.02 mm Hg to its constant weight. Recrystallization of this material from ether at - 6 °C gave 16.0 g (80Y/o of theory) of a white crystalline material, which was highly soluble in benzene, chloroform, sparingly soluble in ether, and insoluble in petroleum ether or water. Analysis. Calculated for C4~H4205(651): C, 81.20; H, 6.51 Found C, 81.14; H, 6.62.
Bis( D L-l-triphenylmethyl-2-stearoylglyceryl )-3-ether To a solution of 13.2 g (0.02 M) of freshly prepared bis(DL-l-triphenylmethylglyceryl)-3-ether and 8.0 g (0.1 M) of anhydrous pryidine in 150 ml of dry benzene was added a solution of freshly distilled 12.1 g (0.04 M) stearoyl chloride in 50 ml of dry benzene. The reaction mixture was kept at 40°C for 20 hr. At the end of this period the reaction mixture was diluted with 500 ml of ether and then washed successively with three 500 ml portions of ice-cold 2 N sulfuric acid, two 500 ml portions of saturated sodium bicarbonate solution and finally with two 500 ml portions of water. The solution was dried over anhydrous sodium sulfate, and the solvents were removed by distillation under reduced pressure. The residue was redissolved in low boiling petroleum ether, cleared by centrifugation and the supernatant was concentrated under reduced pressure. The residue (weight 22.5 g) was chromatographed on silicic acid column with benzene as solvent. The benzene eluate was concentrated under reduced pressure and the residue was recrystallized twice from petroleum ether at - 6 °C. Bis(DL-l-triphenylmethyl2-stearoylglyceryl)-3-ether melted at 50-51 °C and was highly soluble in chloroform, ether, benzene or petroleum ether and insoluble in water. Analysis. Calculated for C8oHlloOv(l184): C, 81.17; H, 9.37 Found C, 80.90; H, 9.39.
Bis ( D L-2-stearo ylglycer yl )-3-ether Bis(DL-2-stearoylglyceryl)-3-ether was obtained on removal of triphenylmethyl groups by catalytic hydrogenolysis as described1). On two recrystalli-
120
DMYTRO BUCHNEA
zations from ether it gave pure bis(DL-2-stearoylglyceryl)-3-ether; m.p. 74.5-75.5 °C. Analysis. Calculated for C42H8207(699): C, 72.16; H, 11.82 Found C, 72.21; H, 11.90.
Bis( DL-l-stearoylglyceryl)-3-ether This ether was obtained on removal of triphenylmethyl groups with hydrogen chloride in ice-cold, anhydrous ether solution as described1). On chromatography on a silicic acid column using benzene and ether as solvents, and on two recrystallizations from ether it gave pure bis(DL-l-stearoylglyceryl)-3-ether; m.p. 80-81 °C. Analysis. Calculated for C42H8207(699): C, 72.16; H, 11.82 Found C, 72.16; H, 11.41. R E M O V A L OF P R O T E C T I V E T R I P H E N Y L M E T H Y L G R O U P W I T H H Y D R O G E N C H L O R I D E
D-1-Triphenylmethyl-2-stearoylglyceryl-3-benzyl ether, m.p. 30-31 °C, specific rotation - 9 . 5 °, c, 10 chloroform was used as a starting material. Thirty g of this material was freed of its protective triphenylmethyl group as described by Buchnea and Baert), except that the hydrogen chloride stream was passed through the ice-cold reaction mixture only until triphenylmethyl chloride precipitated, which was approximately 20 min. After preliminary purification, 20.5 g of crude material was obtained, consisting, according to TLC analysis, of D-2-stearoylglyceryl-3-benzyl ether, D-l-stearoylglyceryl3-benzyl ether, bis(D-2-stearoylglyceryl-3-benzyl)-l-ether, D-l-chloro-2stearoylglyceryl-3-benzyl ether and some triphenylcarbinol. The crude reaction mixture (20.0 g) was dissolved in 200 ml of benzene and the benzene solution was passed through a column of 400 g of silicic acid, approximately 60 cm long and 4.5 cm wide. The effluent was collected with a fraction collector. First D-l-chloro-2-stearoylglyceryl-3-benzyl ether, then bis(D-2-stearoylglyceryl-3-benzyl)-l-ether were recovered from silicic acid column with benzene as eluate. After the effluent was free of solute, the benzene was replaced by a mixture of benzene/ether (3:1, v/v). This mixture eluted first D-l-stearoylglyceryl-3-benzyl ether, and then D-2-stearoylglyceryl-3-benzyl ether.
D-l-chloro-2-stearoylglyceryl-3-benzyl ether The first benzene eluate was checked on TLC and the combined fractions were concentrated under reduced pressure, and the residue was freed of the rest of the solvent by keeping it in a vacuum of 0.02 mm until its weight was constant. D-l-chloro-2-stearoylglyceryl-3-benzyl ether, weighed 4.0 g~(20% of theory based on 20 g mixture put through the silicic acid column). Two
ACYL MIGRATION IN GLYCERIDES
] 21
recrystallizations from dry acetone at -80°C, gave chromatographically pure D-l-chloro-2-stearoylglyceryl-3-benzylether that melted at 18.5-19.5 °C, and has a specific rotation of + 5.5 °,c, 10 in chloroform. Analysis. Calculated for C28H4703C1(467): C, 71.97; H, 10.14; C1, 7.54 Found C, 72.03; H, 10.18; C1, 7.46.
Bis( D-2-stearoylglyceryl-3-benzyl)-l-ether The detection of this compound was made also on TLC, and then recovered from the solvent by concentration under reduced pressure, 3.8 g (19~o of theory based on 20 g mixture) were obtained. On recrystallization from petroleum ether chromatographically pure bis(D-2-stearoylglyceryl-3benzyl)-l-ether, m.p. 53-54°C and a specific rotation + 6.0 °, c, l0 in chloroform, was obtained. Analysis. Calculated for C56H9407(879): C, 76.48; H, 10.77 Found (882) C, 76.51; H, 10.80.
D-l-stearoylglyceryl-3-benzyl ether The recovery of D-l-stearoylglyceryl-3-benzyl ether from the column was performed as described with the mixture of benzene/ether (3 : 1, v/v) and the compound was detected on TCL. Eleven g (55~o of theory based on 20 g) were obtained after concentration of solvents. Two recrystallizations from petroleum ether gave pure D-l-stearoylglyceryl-3-benzyl ether that melted at 43-44°C, and had a specific rotation of +2.0 °, c, 10 in chloroform. Analysis. Calculated for C28H4804(449): C, 74.95; H, 10.78 Found C, 75.05; H, 10.88.
D-2-stearoylglyceryl-3-benzyl ether From the last fractions was recovered 1.0 g (5~o of theory) of D-2-stearoylglyceryl-3-benzyl ether, which on recrystallization from petroleum ether gave a chromatographically pure compound that melted at 34-35 °C, and had a specific rotation of + 3.0 °, c, 10 in chloroform. Analysis. Calculated for C28H4804(449): C, 74.95; H, 10.78 Found C, 75.03; H, 10.78. REMOVAL
OF P R O T E C T I V E
BENZYL GROUPS
BY C A T A L Y T I C H Y D R O G E N O L Y S I S
All four compounds: D-l-chloro-2-stearoylglyceryl-3-benzyl ether, bis(D-2-stearoylglyceryl-3-benzyl)-l-ether, D-l-stearoylglyceryl-3-benzyl ether and D-2-stearoylglyceryl-3-benzyl ether were freed of their protective benzyl groups by catalytic hydrogenolysis with hydrogen in presence of palladium black in acetic acid, as described1), and after removal of acetic acid by distillation under reduced pressure, the compounds were purified individually as follows:
122
DMYTRO BUCHNEA
D-l-chloro-2-stearoylglyeerol Was obtained in a yield of 95~. Two recrystallizations from ether at - 6 °C gave chromatographically pure D-l-chloro-2-stearoylglycerol that melted at 51-52°C, and had a specific rotation of - 2 . 5 °, c, 10 in chloroform. Analysis. Calculated for C21H4103C1(377): C, 66.90; H, 10.96; CI, 9.14 Found C, 67.00; H, 11.13; C1, 9.14. On introduction of the second stearoyl group there resulted D-l-chloro2,3-distearin, with a specific rotation of + 3.0 °, c, 10 in chloroform.
Bis(D-2-stearoylglycerol)-l-ether This compound was obtained in a yield of 98~ by hydrogenolysis, and on recrystallization from ether at + 6 °C, gave chromatographically pure bis(D-2-stearoylglycerol)-l-ether that melted at 74.5-75.5 °C, and had a specific rotation of - 2 . 6 °, c, 10 in chloroform. Analysis. Calculated for C42H8207(699); C, 72.16; H, 11.82 Found (700) C, 72.30; H, 12.01. This compound on acylation with stearoyl chloride gave an asymmetric bis(D-2,3-distearoylglyceryl)-l-ether with a specific rotation of + 1.0 °, c, 10 in chloroform.
D-l-stearoylglycerol The catalytic hydrogenolysis of D-l-stearoylglyceryl-3-benzyl ether resulted in a 98~ yield of D-l-stearoylglycerol. Two recrystallizations from ether gave chromatographically pure D-l-stearoylglycerol that melted at 76-77°C, and had a specific rotation of +4.0 °, c, 10 in pyridine. Baer and Fischer 10) reported for L-isomer a m.p. 76-77 °C and a specific rotation of -3.58 °, c, 12.3 in pyridine. On titration with periodic acid it was proved that only 1-isomer was present. Analysis. Calculated for C11H4204(358.5): C, 70.35; H, 11.81 Found C, 70.51 ; H, 11.90.
2-Stearoylglycerol The reduction of D-2-stearoylglyceryl-3-benzyl ether afforded 95~o of 2-stearoylglycerol, which on recrystallization from ether gave chromatographically pure 2-stearoylglycerol with a m.p. of 73-74°C. Analysis. Calculated for Cz~H4204(358.5): C, 70.35; H, 11.81 Found C, 70.51 ; H, 11.75. On acylation of D-l-stearoylglycerol and 2-stearoylglycerol with stearoyl chloride 11) a symmetrical tristearin was obtained in both cases.
ACYL MIGRATION IN GLYCERIDES
123
CONVERSION OF 2-STEAROYLGLYCEROL VIA DL-STEAROYLGLYCEROL TO BIS(STEAROYLGLYCERYL) ETHERS
DL-l-stearoylglycerol (III)from 2-stearoylglycerol (1) Six g of 2-stearoylglycerol were dissolved in 120 ml of anhydrous ether and the container was immersed in an ice bath. Then a stream of dry hydrogen chloride was passed through the precipitated suspension of 2-stearoylglycerol for 20 min. Every ten minutes aliquots were taken and titrated with periodic acid as described by Handschumaker and Lintersl2). According to the data obtained from periodic acid addition, 2-stearoylglycerol was almost completely (98%) converted to 1-isomer within 20 min. After removal of ether by distillation under reduced pressure the remaining material was recrystallized twice from ether. A yield of 88% of chromatographically pure DL-l-stearoylglycerol, which melted at 82-83 °C, and was identical in every respect with authentic DL-l-stearoylglycerol, was obtained.
Bis(DL-l-stearoylglyceryl)-3-ether
(v)
This compound was prepared by the action of dry hydrogen chloride in ice-cold, anhydrous ether, on 2-stearoylglycerol, D-l-stearoylglycerol, L-3stearoylglycerol and DL-l-stearoylglycerol, respectively. All four isomers were treated in the same manner, viz., 3.0 g of each isomer were dissglved in 60 ml of anhydrous ether and cooled in an ice bath (at this temperature the monostearin precipitates again). Through the suspension was passed a stream of hydrogen chloride, and every ten minutes aliquots were taken and titrated with periodic acid. According to the titration results the four isomers required the following time to be converted to bis(DL-lstearoylglyceryl)-3-ether: L-3-stearoylglycerol 30 rain, D-l-stearoylglycerol and 2-stearoylglycerol 70 min and DL-l-stearoylglycerol 120 rain. At the end of these periods the solvent was removed by distillation under reduced pressure and after two recrystallizations from ether gave chromatographically pure bis(DL-l-stearoylglyceryl)-3-ether(v) that melted at 80-81°C. The melting points, analysis on TLC and infra red spectra of all four compounds were identical. Analysis. Calculated for C42H8207(699): C, 72.16; H, 11.82 Found (700): C,72.20; H, 11.75.
Bis(DL-2-stearoylglyceryl)-3-ether
(v0
This bisglyceride ether was isolated from mother liquor after recrystallization of compound v. It was not possible to obtain this bisglyceride ether chromatographically pure, but TLC analysis and infrared spectrum were identical with that of synthetic bis (DL-2-stearoylglyceryl)-3-ether.
124
DMYTRO BUCHNEA
Bis ( D L-l-chloro-2-stearoylglyceryl)-3-ether
(vii)
Three g of each bis(DL-l-stearoylglyceryl)-3-ether(v) or bis (DL-2-stearoylglyceryl)-3-ether(vl) were dissolved in anhydrous ether and a stream of hydrogen chloride was passed through the solution at 25 °C for 3 hr. Then the ether was removed under reduced pressure, and the residue was recrystallized from ether at - 6 °C resulting in a chromatographically pure bis(DL-l-chloro-2-stearoylglyceryl)-3-ether(vII), m.p. 59-60°C. Analysis. Calculated for C42H8005C12(736 ) : C, 68.53; H, 10.95; C1, 9.63. Found (740); C, 68.60; H, 11.02; CI, 9.03. R A C E M I Z A T I O N OF MONOSTEARINS IN PRESENCE OF H Y D R O G E N CHLORIDE IN A N H Y D R O U S ETHER SOLUTION
The hydrogen chloride in anhydrous ether solution was prepared by passing a stream of dry hydrogen chloride through anhydrous ether at 0 °C for 8 hr. The concentration of hydrogen chloride dissolved in anhydrous ether at 0°C was determined by titration with 1 N methanolic potassium hydroxide solution at 0 ° using phenolphthalein as indicator. It was found that anhydrous ether at 0°C is able to dissolve in 8 hr a quantity equal to 7 N to 7.5 N of hydrogen chloride. For the study of the rate of racemization in presence of different concentrations of hydrogen chloride, the above concentration was diluted with anhydrous ether.
The rate of raeemization of D-1 stearoylglycerol The racemization was carried out at 0 °C in 4 N dry hydrogen chloride in anhydrous ether solution. One and a half grams of D-1-stearoylglyceryl were added at once to the stirred 100 ml hydrogen chloride solution. The first sample was analysed at zero time, and then, every ten min, aliquots were taken and after removal of the ether and hydrogen chloride by evaporation under reduced pressure at a temperature not exceeding 20 °C the remaining material was dissolved in anhydrous pyridine and adjusted to a concentration of 10~. F r o m these solutions the rotations were taken individually. At zero time treatment, the specific rotation was + 3.7 °, which is equal to zero per cent of racemization. Then the rotation decreased for each successive sample and within 80 min of treatment reached a specific rotation of + 2.3 °, which is equal to 40~o racemization. After 100 min of treatment, D-l-stearoylglycerol was converted 50~ to DL-l-stearoylglycerol. The per cent of racemization was plotted as ordinate and the time as abscissa (fig. 3). At the end of 100 min of treatment the rest of material was recovered from ether and hydrogen chloride and recrystallized from ether, m.p. 76-81 °C, and on
ACYL MIGRATION IN GLYCERIDES
125
titration with periodic acid proved to be 100~o 1-isomer. According to the specific rotation ( + 1.9 °) and to the melting point the material is a mixture of D-l-stearoylglycerol and DL-l-stearoylglycerol.
The rate of racemization of L-3-stearoylglycerol One and a half grams amount of L-3-stearoylglycerol with a specific rotation of - 3 . 6 ° was treated with 4 N hydrogen chloride in anhydrous ether solution under identical conditions as the D-isomer. It was found that the specific rotation of L-3-stearoylglycerol decreased from - 3 . 6 ° at zero time treatment to zero value within 80 rain of treatment, which is equal to 100~ racemization. On removal of the ether and hydrogen chloride under reduced pressure; and, recrystallization from ether this material melted at 82-83 °C and on titration with periodic acid proved to be 100~ DL-isomer.
The rate of conversion of 2-stearoylglycerol into DL-isomer To a stirred 100 ml, 0°C cold, 4 N hydrogen chloride in anhydrous ether solution 1.5 g of 2-stearoylglycerol was added at once. Then the aliquots were taken as follows: Five, ten, fifteen and twenty minutes. After removal of the ether and hydrogen chloride under reduced pressure at a temperature not exceeding 20 °C the residues were titrated with periodic acid as described by Handschumaker and Linters 1~) According to the values obtained from periodic acid addition, 2-stearoylglycerol was completely (100~) converted to the DL-isomer within 10 to 15 min (fig. 3). The rest of material was recovered from reaction medium and after recrystallization from ether, melted at 82-83°C, and was identical with the authentic DL-l-stearoylglycerol.
Racemization of D-l- and L-3-stearoylglycerol and conversion of 2-stearoylglycerol to DL-isomer in presence of different concentrations of hydrogen chloride in anhydrous ether solution One and a half gram amounts of each D-l-, L-3- and 2-stearoylglycerol were separately t~eated at 0 °C for 50 min in the following concentrations of dry hydrogen chloride in anhydrous ether solution: 1 N, 2 N, 3 N, 4 N, 5 N, 6 N and 7 N. All three isomers were not affected by 1 N to 2 N hydrogen chloride at 0 °C within 50 min. The concentration of 2 N to 3 N hydrogen chloride in ether solution converted 2-stearoylglycerol to DL-isomer at 0 °C in about 50 min. The L-3-stearoylglycerol racemized at a concentration of over 4 N hydrogen chloride in anhydrous ether solution at 0 °C within 50 min, whereas D-l-stearoylglycerol at a concentration of over 5 N hydrogen chloride in anhydrous ether solution at 0 °C within 50 min was racemized only 30~o.
126
DMYTRO BUCHNEA
The first two products, after recrystallization from ether melted at 82-83 °C and were identical with the authentic DL-l-stearoylglycerol. The last product, according to the specific rotation (+ 2.6 °) and to the melting point 76-80 ° was a mixture of D-l- and DL-l-stearoylglycerol. Discussion
The results of this investigation show that the acyl group migration in partially acylated glycerides catalysed by hydrogen chloride is accompanied by the formation of bisglyceride ethers. Therefore it is assumed that the mechanism of these reactions might proceed in two steps: First hydrogen chloride chlorinates 2-stearoylglycerol (0 and forms a bimolecular resonant ion complex intermediate (II), regeneration of hydrogen chloride with the simultaneous exchange of stearoyl groups between radicals (a) and (b) from positions C-2(a) and C-2(b) to the positions C-l(b) and C-3(a) resulting in DL-l-stearoylglycerol (In), fig. 2. In the second step, reaction of DL-l-stearoylglycerol (Ill) with hydrogen chloride results in the formation of bimolecular resonant ion complex intermediate (IV), followed by the regeneration of hydrogen chloride with the formation of bis(DL-l-stearoylglyceryl)-3-ether (v), and the migration of the stearoyl groups from positions C-1 (a) and C-1 (b) to the positions C-2(a) and C-2(b) resulting in bis (DL-2-stearoylglyceryl)-3-ether (Vl), and finally the reaction of the primary hydroxyl groups of compound vl with hydrogen chloride to give bis(DL-l-chloro-2-stearoylglyceryl)-3-ether (vii) as the final product in these reactions. The presence of the secondary hydroxyl groups in the second step requires more energy to facilitate the migration of the stearoyl groups from positions C-l(a) and C-l(b) to the positions C-2(a) and C-2(b), (vI) fig. 2. This takes place only by longer treatment of compound v (fig. 2) with gaseous hydrogen chloride in anhydrous ether solution at 0 °C. Therefore, when hydrogen chloride is first dissolved in anhydrous ether and then this solution is used for treatment of D-, L- and 2-stearoylglycerol, in this case, only racemization and conversion to DL-isomer takes place. It seems that the formation of the bimolecular resonance ion complex takes place only between two identical glyceride molecules. This could be the reason why, in Doerschuk's 13) experimental results, the glycerol-C14 was not incorporated in the resulting DL-l-palmitoylglycerol. The hypothesis of a bimolecular resonant ion complex intermediate (II and IV) formation during the acyl migration is also validated by the fact that during the migration of the acyl groups the formation of bisglyceride ethers takes place. Winstein and his co-workers 14) were the first to visualize the formation of similar ion pairs in the interpretation of rearrangement, ex-
ACYL MIGRATION IN GLYCERIDES
127
change, r a c e m i z a t i o n a n d solvolysis reactions o f n e u t r a l o r g a n i c substrates. W i n s t e i n s studies represent a p r e c e d e n t for the i n t e r p r e t a t i o n o f the f o r m a tion o f the b i m o l e c u l a r r e s o n a n t ion c o m p l e x i n t e r m e d i a t e s in the reactions described here. Bevan et al. 15) have p o s t u l a t e d the f o r m a t i o n o f a n i n t e r m e d i a t e r e s o n a n t ion for certain similar reactions, b u t this p o s t u l a t i o n implies only the form a t i o n o f a m o n o m o l e c u l a r r e s o n a n t ion. V a n L o h u i z e n a n d V e r k a d e 4) c o n s i d e r e d an i n t r a m o l e c u l a r r e a c t i o n m e c h a n i s m for acyl g r o u p m i g r a t i o n , which is b a s e d essentially o n the form a t i o n o f the o r t h o o e s t e r i n t e r m e d i a t e as p u t f o r w a r d by Emil Fischer18).
Acknowledgement The a u t h o r wishes to t h a n k Mr. F r e d e r i c k L a m m e r i c h , M e d i c a l A r t Dep a r t m e n t , U n i v e r s i t y o f T o r o n t o , for the d r a w i n g o f the figures.
References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14)
D. Buchnea and E. Baer, J. Lipid Res. I (1960) 405 D. Buchnea, Fette, Seifen, Anstrichmittel 64 (1962) 887 J. B. Martin, J. Am. Chem. Soc. 75 (1953) 5483 O. E. Van Lohuizen and P. E. Verkade, Rec. Trav. Chim. 79 (1960) 133 A. Crossley, I. P. Freeman, B. J. F. Hudson and J. H. Pierce, J. Chem. Soc. 760 (1959) M. Bergmann and N. M. Carter, Z. Physiol. Chem. 191 (1930) 211 B. F. Simmel and C. G. King, J. Am. Chem. Soc. 56 (1934) 1724 E. Baer and H. O. L. Fischer, J. Am. Chem. Soc. 67 (1945) 2031 Emil Fischer, M. Bergmann and E. Barwind, Chem. Ber. 53 (1920) 1589 J. Tausz and N. von Putnoky, Chem. Ber. 52 (1919) 1573 H. E. Fierz-David and W. Kusters, Helv. Acta 22 (1939) 82 E. Handschumaker and L. Linters, J. Am. Oil Chemists' Soc. 24 (1947) 143 A. P. Doerschuk, J. Am. Chem. Soc. 74 (1952) 4202 S. Winstein, E. Clippinger, A. H. Fainber and G. C. Robinson, J. Am. Chem. Soc. 76 (1954) 2597 15) T. H. Bevan, D. A. Brown, G. I. Gregory and T. Malkin, J. Chem. Soc. 24 (1953)127 16) Emil Fischer, Chem. Ber. 53 (1920) 1623
ACYL MIGRATION I. A B I M O L E C U L A R
IN GLYCERIDES.
RESONANT
AS INTERMEDIATE ACYL MIGRATION
ION COMPLEX IN
OF MONOGLYCERIDES
DMYTRO BUCHNEA* Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada Received 30 May 1966 Acyl migration and the rate of racemization of D- and L-monostearin and the rate of conversion of 2-monostearin into DL-isomer catalyzed by hydrogen chloride under anhydrous conditions have been studied. It was found that the rate of racemization and the rate of conversion differ considerably under identical conditions. The rate of racemization and the rate of conversion of these three isomers into DL-monostearin increases as follows: D-monostearin, L-monostearin and 2-monostearin. 2-Monostearin is one of the most unstable isomers and is completely convertible into DL-isomer (100%). The rate of the racemization of D- and L-monostearin to DL-isomer coincides perfectly with the rate of the conversion of these two isomers into bismonostearin ethers. D-monostearin is inherently more than twice as stable as L-isomer. All four isomers were converted into bismonostearin ethers. The synthesis of the bismonostearin ethers is described. The formation of a bimolecular resonant ion complex as an intermediate in acyl migration is suggested.
Introduction D u r i n g the synthetic w o r k o n optically active, m i x e d fatty acid diglycerides 1) it was f o u n d t h a t D - l - t r i p h e n y l m e t h y l - 2 - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether, on r e m o v a l o f its protective t r i p h e n y l m e t h y l g r o u p with h y d r o g e n chloride in ice-cold a n h y d r o u s p e t r o l e u m ether solution, forms an equilibriu m m i x t u r e o f f o u r c o m p o u n d s . These f o u r c o m p o u n d s were s e p a r a t e d b y c h r o m a t o g r a p h y on a silicic acid c o l u m n , a n d were c h a r a c t e r i z e d as: D-2stearoylglyceryl-3-benzyl ether; a p p r o x . 5%, D - l - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether; a p p r o x . 55% to 65%, b i s ( D - 2 - s t e a r o y l g l y c e r y l - 3 - b e n z y l ) - l - e t h e r , a p prox. 20%; and D - l - c h l o r o - 2 - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether, a p p r o x . 8% to 20%. T h e v a r i a t i o n o f D - l - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether a n d D - l - c h l o r o 2-stearoylglyceryl-3-benzyl ether in the e q u i l i b r i u m m i x t u r e is due to the stearoyl g r o u p m i g r a t i o n f r o m C-1 b a c k to C-2, resulting in D - l - c h l o r o - 2 stearoylglyceryl-3-benzyl ether, which is caused by longer action o f h y d r o g e n c h l o r i d e u p o n D - l - s t e a r o y l g l y c e r y l - 3 - b e n z y l ether. The presence o f benzyl g r o u p assured t h a t the a s y m m e t r y o f glyceryl m o i e t y a n d therewith the * Address: Banting Institute, 100 College Street, Toronto 5, Canada.
114
DMYTRO
BUCHNEA
optical property is fully maintained throughout these conversions. On removal of their protective benzyl groups by catalytic hydrogenolysis with hydrogen in presence of palladium black in glacial acetic acid, these compounds were converted into 2-stearoylglycerol, D-l-stearoylglycerol, bis(D2-stearoylglyceryl)-l-ether and D-l-chloro-2-stearoylglycerol respectively. Upon the introduction of a second stearoyl group in the position-3 of bis(D-2-stearoylglyceryl)-l-ether and D-l-chloro-2-stearoylglycerol, an optically active bis(D-2,3glyceryldistearate)-l-ether with a specific rotation of + 1 °, in chloroform, and D-l-chloro-2, 3-glyceryldistearate, with a specific rotation of + 3 °, in chloroform, were obtained. These results prove that the ether linkage and chlorine atom are in the position-1 ; otherwise symmetrical compounds would have resulted. The fact that the removal of triphenylmethyl group with hydrogen chloride is connected with the acyl migration and also with the formation of bis(D-2stearoylglyceryl-3-benzyl)-l-ether led to the idea that this bisglyceride ether might be the intermediate in acyl group migration 2). However, the kinetic study of the reactions between monostearins and hydrogen chloride revealed that the formation of bis(stearoylglyceryl) ethers takes place independently of stearoyl group migration. This fact was established by treatment of 2-stearoylglycerol (1) with hydrogen chloride in ice-cold anhydrous ether, by taking aliquots at various intervals, and titrating with periodic acid for glycol determination (fig. 1), and also analysing on thinlayer chromatography. % 100 ~
•
o
~o
2
._..
L-
-.
o,-.
-
.... zx D '~-
i
[
•~
f%.. 0 °A
t
• /',
/
\
~0
-Stearoylglycerol
% -
o_
",.,
\
\ '.,J(
0
~
!
20
.'x. I
I
~
L
40
i
o
60 Minutes
!
80
,
I
I00
~
!
120
Fig. l. The rates of conversion of 2-stearoylglycerol,L-3-stearoylglycerol,D-l-stearoylglycerol and DL-l-stearoylglycerol into bisglyceride ethers in presence of dry, gaseous hydrogen chloride in anhydrous ether at 0°C.
ACYL MIGRATION IN GLYCERIDES
+
I
115
I 0 I 0
1 0
I 0
~,
I ''~,.o 0"" I
0 I 0 I 0 L
0 ÷
/ r 0
I 0
+
+~
I 0 [
0 I
~-u-~
--
L 0 I,~ e.l
L~ -F
I
o @ I ©
©
~ -u-~
I 0 I 0 I
.-~
L
J
I 0 0
•
[
I
o I 0
0 o I ~ I
0
L 0 I
0 I
I
o
I o I
IT I 0 I
116
DMYTRO BUCHNEA
The conversion of 2-stearoylglycerol to DL-l-stearoylglycerol proceeds at a constant rate as indicated by the rate of consumption of periodic acid (fig. 1). Within 20 min stearoyl groups migrated from positions C-2(a) and C-2(b) to the positions C-l(a) and C-3(b) (fig. 2, n) almost completely (98~o). Then the amount of periodic acid added slowly decreases for each successive sample and within 50 rain reaches 0-value again. After 70 min 2-stearoylglycerol (1) was completely converted by way of DL-l-stearoylglycerol (nI) to a mixture of bis(DL-l-stearoylglyceryl)-3-ether (v), bis(DL-2-stearoylglyceryl)-3-ether (v0 and bis(DL-l-chloro-2-stearoylglyceryl)-3-ether (vi0, (fig. 2). This study was extended to the optically active D-l-stearoylglycerol and L-3-stearoylglycerol as well as to the racemic DL-l-stearoylglycerol. It was found that there is a considerable difference in the stability of D- and L-, and DL-l-stearoylglycerol in presence of hydrogen chloride under identical conditions. L-3-Stearoylglycerol was converted to the mixture of compounds v, Vl, vii, within 30 min, D-l-stearoylglycerol needs 70 min to be converted to the same mixture, whereas DL-l-stearoylglycerol took 120 min to be converted to the mixture of bis(stearoylglyceryl) ethers. This observation indicates that D-l-stearoylglycerol is more than twice as stable as L-isomer. Both D- and L-isomers racemise to DE-form which is the most stable of isomers. Contrary to this, 2-monoglyceride is one of the most unstable of isomers, and it is also completely convertible to DL-isomer. Therefore it is impossible that 2-monoglyceride could be a component of the equilibrium mixture reported by Martina), van Lohuizen and Verkade 4) and others5). The observation that the isomers of monostearin differ in their stability was entirely confirmed by the results obtained from the kinetic study of the rate of racemization of D-l- and L-3-stearoylglycerol and the rate of conversion of 2-stearoylglycerol in presence of known concentration of hydrogen chloride in anhydrous ether solution. The racemization rate of these isomers was studied in presence of a concentration equal to 4 N hydrogen chloride in anhydrous ether solution at 0 °C. It was found that 2-stearoylglycerol converts 100~ to DL-isomer within 10 to 15 min under these conditions. L-3stearoylglycerol needs 80 min to be completely racemized to DL-isomer under identical conditions, whereas D-l-stearoylglycerol under the same conditions was only 40~ racemized (fig. 3). Furthermore, similar results were also obtained from the study of the racemization of D-l-stearoylglycerol and L-3-stearoylglycerol and conversion of 2-stearoylglycerol into DL-isomer in presence of various concentrations of hydrogen chloride in anhydrous ether solution at constant time and temperature. 2-Stearoylglycerol needs a concentration equal to 2-3 N of hydrogen chloride in anhydrous ether solution to undergo a complete conversion to DL-
ACYL MIGRATION
117
IN GLYCERIDES
I00
8C
"% i
so Sl~ sS
0
,/
.~ 60
/''*
Steoroylglycerol
--O#SS
t
,'" ,-"
,A.
,
o 2[] L . . . . -
,,*,
-
zx D ......
z%
~b s
4.0
ooo-
•
20
o. A o"
,~. , , - "
.o...o../k
• •#
0,
°°-ZX
so./k.-
0 0
'~"
~
L
t
20
40
60
I
I
80
!00
Minutes
Fig. 3. The rates of racemization of D-l-stearoylglycerol and L-3-stearoylglycerol, and the rate of conversion of 2-stearoylglycerol into DL-isomer in presence of 4 N hydrogen chloride in anhydrous ether solution at 0 °C. isomer within 50 min at 0°C. L-3-stearoylglycerol requires a concentration over 4 N of hydrogen chloride to undergo 100% racemization, in the same time, whereas D-l-stearoylglycerol at a concentration of over 5 N hydrogen chloride in anhydrous ether solution, under identical conditions, was only 30% racemized. These results prove that the D-isomer is inherently far more stable than the L-isomer, and this might be the reason why D-isomers are predominant in nature. The difference in the stability of D- and L-isomers of monostearins has been completely confirmed by their difference in energy. The determination of the heat of combustion revealed that there exists a difference in energy between D-l-stearoylglycerol and L-3-stearoylglycerol. It was found for example that L-3-stearoylglycerol has about 170 calories per gram more energy than D-l-stearoylglycerol. A preliminary investigation of D- and L-glyceraldehyde indicated that there exists an even greater difference of energy between D- and L-isomers; about 700 calories per gram more energy in favour of L-glyceraldehyde (see following table). This difference of energy between D- and L-isomer might be a tension energy, originating in the distance between 0-3 and 0-2 of L-isomer, which seems to be greater than the distance between 0-1 and 0-2 of D-isomer*. * The experimental details of this investigation will be reported later.
118
DMYTRO BUCHNEA TABLE 1 The heat of combustion of following isomers:
D-l-stearoylglycerol L-3-stearoylglycerol D-glyceraldehyde L-glyceraldehyde
Cal./g
Difference
8417 8590 3992 4698
173
703
Procedures and data MATERIALS
D-l-triphenylmethyl-2-stearoylglyceryl-3-benzyl ether was prepared by the method of Buchnea and Baerl). 2-Stearoylglycerol was prepared either by catalytic hydrogenolysis of D-l-triphenylmethyl-2-stearoylglyceryl-3benzyl ether or from 2-stearoylglyceryl-l-3-benzylidine as described by Bergmann and Carter 6) and Simmel and King7). D-l-stearoylglycerol was obtained by catalytic hydrogenolysis of D-l-stearoylglyceryl-3-benzyletherl). L-3-stearoylglycerol and DL-l-stearoylglycerol were prepared from D-l, 2-isopropylideneglycerol and DL-1, 2-isopropylideneglycerol, as described by Baer and Fischer s) and E. Fischer, et al.°). Analar petroleum ether (b.p. 35-60 °C) and analytical reagent ether were dried over sodium wire. Chloroform analar for use in polarimetric and infrared work was freed of ethanol by distillation over phosphorus pentoxide. Silicic acid Mallinckrodt, 100 mesh (powder), Analytical Reagent, with 12~o weight loss on ignition was used. Palladium catalyst was prepared as described by Tausz and Putnokyl0), except that the palladium after having been washed thoroughly with water, was then stored under the water until used. Glacial acetic acid (C.P.Reagent) as provided by the manufacturer was used as solvent for catalytic hydrogenolysis. Hydrogen chloride was generated from a mixture of concentrated hydrochloric acid and sodium chloride with concentrated sulfuric acid, and dried by passage through concentrated sulfuric acid to the reaction mixture. SYNTHESIS OF BIs(DL-STEAROYLGLYCERYL)-3-ETHER
Bis(DL-l-triphenylmethylglyceryl)-3-ether To a solution of 17.0 g (0.06 M) of triphenylmethyl chloride dissolved in 100 ml of dry benzene was quickly added at 0°C to a solution of 5.0 g (0.03 M) of bis(DL-glycerol)-3-ether,*) dissolved in 50 ml of pyridine. The reaction * The preparation of bis (DL-glycerol)-3-ether is reported in Part III of this series, to be published in Chem. Phys. Lipids. Vol. 1, no. 2.
ACYL M I G R A T I O N IN GLYCERIDES
119
mixture was shaken at room temperature for 24 hr. At the end of this time the reaction mixture was freed from most of benzene and pyridine by distillation under reduced pressure. The residue was then triturated with 200 ml of ice-cold water, centrifuged and the supernatant decanted. The heavy syrup was dissolved in 500 ml of benzene, and then washed successively with two 250 ml portions of ice-cold 2 N sulfuric acid, two 250 ml portions of saturated sodium bicarbonate solution, and finally with 250 ml of distilled water. The solution was dried with 100 g of anhydrous sodium sulfate and after filtration the solution was concentrated by distillation under reduced pressure at 30-35°C. The remaining material was dried at 0.02 mm Hg to its constant weight. Recrystallization of this material from ether at - 6 °C gave 16.0 g (80Y/o of theory) of a white crystalline material, which was highly soluble in benzene, chloroform, sparingly soluble in ether, and insoluble in petroleum ether or water. Analysis. Calculated for C4~H4205(651): C, 81.20; H, 6.51 Found C, 81.14; H, 6.62.
Bis( D L-l-triphenylmethyl-2-stearoylglyceryl )-3-ether To a solution of 13.2 g (0.02 M) of freshly prepared bis(DL-l-triphenylmethylglyceryl)-3-ether and 8.0 g (0.1 M) of anhydrous pryidine in 150 ml of dry benzene was added a solution of freshly distilled 12.1 g (0.04 M) stearoyl chloride in 50 ml of dry benzene. The reaction mixture was kept at 40°C for 20 hr. At the end of this period the reaction mixture was diluted with 500 ml of ether and then washed successively with three 500 ml portions of ice-cold 2 N sulfuric acid, two 500 ml portions of saturated sodium bicarbonate solution and finally with two 500 ml portions of water. The solution was dried over anhydrous sodium sulfate, and the solvents were removed by distillation under reduced pressure. The residue was redissolved in low boiling petroleum ether, cleared by centrifugation and the supernatant was concentrated under reduced pressure. The residue (weight 22.5 g) was chromatographed on silicic acid column with benzene as solvent. The benzene eluate was concentrated under reduced pressure and the residue was recrystallized twice from petroleum ether at - 6 °C. Bis(DL-l-triphenylmethyl2-stearoylglyceryl)-3-ether melted at 50-51 °C and was highly soluble in chloroform, ether, benzene or petroleum ether and insoluble in water. Analysis. Calculated for C8oHlloOv(l184): C, 81.17; H, 9.37 Found C, 80.90; H, 9.39.
Bis ( D L-2-stearo ylglycer yl )-3-ether Bis(DL-2-stearoylglyceryl)-3-ether was obtained on removal of triphenylmethyl groups by catalytic hydrogenolysis as described1). On two recrystalli-
120
DMYTRO BUCHNEA
zations from ether it gave pure bis(DL-2-stearoylglyceryl)-3-ether; m.p. 74.5-75.5 °C. Analysis. Calculated for C42H8207(699): C, 72.16; H, 11.82 Found C, 72.21; H, 11.90.
Bis( DL-l-stearoylglyceryl)-3-ether This ether was obtained on removal of triphenylmethyl groups with hydrogen chloride in ice-cold, anhydrous ether solution as described1). On chromatography on a silicic acid column using benzene and ether as solvents, and on two recrystallizations from ether it gave pure bis(DL-l-stearoylglyceryl)-3-ether; m.p. 80-81 °C. Analysis. Calculated for C42H8207(699): C, 72.16; H, 11.82 Found C, 72.16; H, 11.41. R E M O V A L OF P R O T E C T I V E T R I P H E N Y L M E T H Y L G R O U P W I T H H Y D R O G E N C H L O R I D E
D-1-Triphenylmethyl-2-stearoylglyceryl-3-benzyl ether, m.p. 30-31 °C, specific rotation - 9 . 5 °, c, 10 chloroform was used as a starting material. Thirty g of this material was freed of its protective triphenylmethyl group as described by Buchnea and Baert), except that the hydrogen chloride stream was passed through the ice-cold reaction mixture only until triphenylmethyl chloride precipitated, which was approximately 20 min. After preliminary purification, 20.5 g of crude material was obtained, consisting, according to TLC analysis, of D-2-stearoylglyceryl-3-benzyl ether, D-l-stearoylglyceryl3-benzyl ether, bis(D-2-stearoylglyceryl-3-benzyl)-l-ether, D-l-chloro-2stearoylglyceryl-3-benzyl ether and some triphenylcarbinol. The crude reaction mixture (20.0 g) was dissolved in 200 ml of benzene and the benzene solution was passed through a column of 400 g of silicic acid, approximately 60 cm long and 4.5 cm wide. The effluent was collected with a fraction collector. First D-l-chloro-2-stearoylglyceryl-3-benzyl ether, then bis(D-2-stearoylglyceryl-3-benzyl)-l-ether were recovered from silicic acid column with benzene as eluate. After the effluent was free of solute, the benzene was replaced by a mixture of benzene/ether (3:1, v/v). This mixture eluted first D-l-stearoylglyceryl-3-benzyl ether, and then D-2-stearoylglyceryl-3-benzyl ether.
D-l-chloro-2-stearoylglyceryl-3-benzyl ether The first benzene eluate was checked on TLC and the combined fractions were concentrated under reduced pressure, and the residue was freed of the rest of the solvent by keeping it in a vacuum of 0.02 mm until its weight was constant. D-l-chloro-2-stearoylglyceryl-3-benzyl ether, weighed 4.0 g~(20% of theory based on 20 g mixture put through the silicic acid column). Two
ACYL MIGRATION IN GLYCERIDES
] 21
recrystallizations from dry acetone at -80°C, gave chromatographically pure D-l-chloro-2-stearoylglyceryl-3-benzylether that melted at 18.5-19.5 °C, and has a specific rotation of + 5.5 °,c, 10 in chloroform. Analysis. Calculated for C28H4703C1(467): C, 71.97; H, 10.14; C1, 7.54 Found C, 72.03; H, 10.18; C1, 7.46.
Bis( D-2-stearoylglyceryl-3-benzyl)-l-ether The detection of this compound was made also on TLC, and then recovered from the solvent by concentration under reduced pressure, 3.8 g (19~o of theory based on 20 g mixture) were obtained. On recrystallization from petroleum ether chromatographically pure bis(D-2-stearoylglyceryl-3benzyl)-l-ether, m.p. 53-54°C and a specific rotation + 6.0 °, c, l0 in chloroform, was obtained. Analysis. Calculated for C56H9407(879): C, 76.48; H, 10.77 Found (882) C, 76.51; H, 10.80.
D-l-stearoylglyceryl-3-benzyl ether The recovery of D-l-stearoylglyceryl-3-benzyl ether from the column was performed as described with the mixture of benzene/ether (3 : 1, v/v) and the compound was detected on TCL. Eleven g (55~o of theory based on 20 g) were obtained after concentration of solvents. Two recrystallizations from petroleum ether gave pure D-l-stearoylglyceryl-3-benzyl ether that melted at 43-44°C, and had a specific rotation of +2.0 °, c, 10 in chloroform. Analysis. Calculated for C28H4804(449): C, 74.95; H, 10.78 Found C, 75.05; H, 10.88.
D-2-stearoylglyceryl-3-benzyl ether From the last fractions was recovered 1.0 g (5~o of theory) of D-2-stearoylglyceryl-3-benzyl ether, which on recrystallization from petroleum ether gave a chromatographically pure compound that melted at 34-35 °C, and had a specific rotation of + 3.0 °, c, 10 in chloroform. Analysis. Calculated for C28H4804(449): C, 74.95; H, 10.78 Found C, 75.03; H, 10.78. REMOVAL
OF P R O T E C T I V E
BENZYL GROUPS
BY C A T A L Y T I C H Y D R O G E N O L Y S I S
All four compounds: D-l-chloro-2-stearoylglyceryl-3-benzyl ether, bis(D-2-stearoylglyceryl-3-benzyl)-l-ether, D-l-stearoylglyceryl-3-benzyl ether and D-2-stearoylglyceryl-3-benzyl ether were freed of their protective benzyl groups by catalytic hydrogenolysis with hydrogen in presence of palladium black in acetic acid, as described1), and after removal of acetic acid by distillation under reduced pressure, the compounds were purified individually as follows:
122
DMYTRO BUCHNEA
D-l-chloro-2-stearoylglyeerol Was obtained in a yield of 95~. Two recrystallizations from ether at - 6 °C gave chromatographically pure D-l-chloro-2-stearoylglycerol that melted at 51-52°C, and had a specific rotation of - 2 . 5 °, c, 10 in chloroform. Analysis. Calculated for C21H4103C1(377): C, 66.90; H, 10.96; CI, 9.14 Found C, 67.00; H, 11.13; C1, 9.14. On introduction of the second stearoyl group there resulted D-l-chloro2,3-distearin, with a specific rotation of + 3.0 °, c, 10 in chloroform.
Bis(D-2-stearoylglycerol)-l-ether This compound was obtained in a yield of 98~ by hydrogenolysis, and on recrystallization from ether at + 6 °C, gave chromatographically pure bis(D-2-stearoylglycerol)-l-ether that melted at 74.5-75.5 °C, and had a specific rotation of - 2 . 6 °, c, 10 in chloroform. Analysis. Calculated for C42H8207(699); C, 72.16; H, 11.82 Found (700) C, 72.30; H, 12.01. This compound on acylation with stearoyl chloride gave an asymmetric bis(D-2,3-distearoylglyceryl)-l-ether with a specific rotation of + 1.0 °, c, 10 in chloroform.
D-l-stearoylglycerol The catalytic hydrogenolysis of D-l-stearoylglyceryl-3-benzyl ether resulted in a 98~ yield of D-l-stearoylglycerol. Two recrystallizations from ether gave chromatographically pure D-l-stearoylglycerol that melted at 76-77°C, and had a specific rotation of +4.0 °, c, 10 in pyridine. Baer and Fischer 10) reported for L-isomer a m.p. 76-77 °C and a specific rotation of -3.58 °, c, 12.3 in pyridine. On titration with periodic acid it was proved that only 1-isomer was present. Analysis. Calculated for C11H4204(358.5): C, 70.35; H, 11.81 Found C, 70.51 ; H, 11.90.
2-Stearoylglycerol The reduction of D-2-stearoylglyceryl-3-benzyl ether afforded 95~o of 2-stearoylglycerol, which on recrystallization from ether gave chromatographically pure 2-stearoylglycerol with a m.p. of 73-74°C. Analysis. Calculated for Cz~H4204(358.5): C, 70.35; H, 11.81 Found C, 70.51 ; H, 11.75. On acylation of D-l-stearoylglycerol and 2-stearoylglycerol with stearoyl chloride 11) a symmetrical tristearin was obtained in both cases.
ACYL MIGRATION IN GLYCERIDES
123
CONVERSION OF 2-STEAROYLGLYCEROL VIA DL-STEAROYLGLYCEROL TO BIS(STEAROYLGLYCERYL) ETHERS
DL-l-stearoylglycerol (III)from 2-stearoylglycerol (1) Six g of 2-stearoylglycerol were dissolved in 120 ml of anhydrous ether and the container was immersed in an ice bath. Then a stream of dry hydrogen chloride was passed through the precipitated suspension of 2-stearoylglycerol for 20 min. Every ten minutes aliquots were taken and titrated with periodic acid as described by Handschumaker and Lintersl2). According to the data obtained from periodic acid addition, 2-stearoylglycerol was almost completely (98%) converted to 1-isomer within 20 min. After removal of ether by distillation under reduced pressure the remaining material was recrystallized twice from ether. A yield of 88% of chromatographically pure DL-l-stearoylglycerol, which melted at 82-83 °C, and was identical in every respect with authentic DL-l-stearoylglycerol, was obtained.
Bis(DL-l-stearoylglyceryl)-3-ether
(v)
This compound was prepared by the action of dry hydrogen chloride in ice-cold, anhydrous ether, on 2-stearoylglycerol, D-l-stearoylglycerol, L-3stearoylglycerol and DL-l-stearoylglycerol, respectively. All four isomers were treated in the same manner, viz., 3.0 g of each isomer were dissglved in 60 ml of anhydrous ether and cooled in an ice bath (at this temperature the monostearin precipitates again). Through the suspension was passed a stream of hydrogen chloride, and every ten minutes aliquots were taken and titrated with periodic acid. According to the titration results the four isomers required the following time to be converted to bis(DL-lstearoylglyceryl)-3-ether: L-3-stearoylglycerol 30 rain, D-l-stearoylglycerol and 2-stearoylglycerol 70 min and DL-l-stearoylglycerol 120 rain. At the end of these periods the solvent was removed by distillation under reduced pressure and after two recrystallizations from ether gave chromatographically pure bis(DL-l-stearoylglyceryl)-3-ether(v) that melted at 80-81°C. The melting points, analysis on TLC and infra red spectra of all four compounds were identical. Analysis. Calculated for C42H8207(699): C, 72.16; H, 11.82 Found (700): C,72.20; H, 11.75.
Bis(DL-2-stearoylglyceryl)-3-ether
(v0
This bisglyceride ether was isolated from mother liquor after recrystallization of compound v. It was not possible to obtain this bisglyceride ether chromatographically pure, but TLC analysis and infrared spectrum were identical with that of synthetic bis (DL-2-stearoylglyceryl)-3-ether.
124
DMYTRO BUCHNEA
Bis ( D L-l-chloro-2-stearoylglyceryl)-3-ether
(vii)
Three g of each bis(DL-l-stearoylglyceryl)-3-ether(v) or bis (DL-2-stearoylglyceryl)-3-ether(vl) were dissolved in anhydrous ether and a stream of hydrogen chloride was passed through the solution at 25 °C for 3 hr. Then the ether was removed under reduced pressure, and the residue was recrystallized from ether at - 6 °C resulting in a chromatographically pure bis(DL-l-chloro-2-stearoylglyceryl)-3-ether(vII), m.p. 59-60°C. Analysis. Calculated for C42H8005C12(736 ) : C, 68.53; H, 10.95; C1, 9.63. Found (740); C, 68.60; H, 11.02; CI, 9.03. R A C E M I Z A T I O N OF MONOSTEARINS IN PRESENCE OF H Y D R O G E N CHLORIDE IN A N H Y D R O U S ETHER SOLUTION
The hydrogen chloride in anhydrous ether solution was prepared by passing a stream of dry hydrogen chloride through anhydrous ether at 0 °C for 8 hr. The concentration of hydrogen chloride dissolved in anhydrous ether at 0°C was determined by titration with 1 N methanolic potassium hydroxide solution at 0 ° using phenolphthalein as indicator. It was found that anhydrous ether at 0°C is able to dissolve in 8 hr a quantity equal to 7 N to 7.5 N of hydrogen chloride. For the study of the rate of racemization in presence of different concentrations of hydrogen chloride, the above concentration was diluted with anhydrous ether.
The rate of raeemization of D-1 stearoylglycerol The racemization was carried out at 0 °C in 4 N dry hydrogen chloride in anhydrous ether solution. One and a half grams of D-1-stearoylglyceryl were added at once to the stirred 100 ml hydrogen chloride solution. The first sample was analysed at zero time, and then, every ten min, aliquots were taken and after removal of the ether and hydrogen chloride by evaporation under reduced pressure at a temperature not exceeding 20 °C the remaining material was dissolved in anhydrous pyridine and adjusted to a concentration of 10~. F r o m these solutions the rotations were taken individually. At zero time treatment, the specific rotation was + 3.7 °, which is equal to zero per cent of racemization. Then the rotation decreased for each successive sample and within 80 min of treatment reached a specific rotation of + 2.3 °, which is equal to 40~o racemization. After 100 min of treatment, D-l-stearoylglycerol was converted 50~ to DL-l-stearoylglycerol. The per cent of racemization was plotted as ordinate and the time as abscissa (fig. 3). At the end of 100 min of treatment the rest of material was recovered from ether and hydrogen chloride and recrystallized from ether, m.p. 76-81 °C, and on
ACYL MIGRATION IN GLYCERIDES
125
titration with periodic acid proved to be 100~o 1-isomer. According to the specific rotation ( + 1.9 °) and to the melting point the material is a mixture of D-l-stearoylglycerol and DL-l-stearoylglycerol.
The rate of racemization of L-3-stearoylglycerol One and a half grams amount of L-3-stearoylglycerol with a specific rotation of - 3 . 6 ° was treated with 4 N hydrogen chloride in anhydrous ether solution under identical conditions as the D-isomer. It was found that the specific rotation of L-3-stearoylglycerol decreased from - 3 . 6 ° at zero time treatment to zero value within 80 rain of treatment, which is equal to 100~ racemization. On removal of the ether and hydrogen chloride under reduced pressure; and, recrystallization from ether this material melted at 82-83 °C and on titration with periodic acid proved to be 100~ DL-isomer.
The rate of conversion of 2-stearoylglycerol into DL-isomer To a stirred 100 ml, 0°C cold, 4 N hydrogen chloride in anhydrous ether solution 1.5 g of 2-stearoylglycerol was added at once. Then the aliquots were taken as follows: Five, ten, fifteen and twenty minutes. After removal of the ether and hydrogen chloride under reduced pressure at a temperature not exceeding 20 °C the residues were titrated with periodic acid as described by Handschumaker and Linters 1~) According to the values obtained from periodic acid addition, 2-stearoylglycerol was completely (100~) converted to the DL-isomer within 10 to 15 min (fig. 3). The rest of material was recovered from reaction medium and after recrystallization from ether, melted at 82-83°C, and was identical with the authentic DL-l-stearoylglycerol.
Racemization of D-l- and L-3-stearoylglycerol and conversion of 2-stearoylglycerol to DL-isomer in presence of different concentrations of hydrogen chloride in anhydrous ether solution One and a half gram amounts of each D-l-, L-3- and 2-stearoylglycerol were separately t~eated at 0 °C for 50 min in the following concentrations of dry hydrogen chloride in anhydrous ether solution: 1 N, 2 N, 3 N, 4 N, 5 N, 6 N and 7 N. All three isomers were not affected by 1 N to 2 N hydrogen chloride at 0 °C within 50 min. The concentration of 2 N to 3 N hydrogen chloride in ether solution converted 2-stearoylglycerol to DL-isomer at 0 °C in about 50 min. The L-3-stearoylglycerol racemized at a concentration of over 4 N hydrogen chloride in anhydrous ether solution at 0 °C within 50 min, whereas D-l-stearoylglycerol at a concentration of over 5 N hydrogen chloride in anhydrous ether solution at 0 °C within 50 min was racemized only 30~o.
126
DMYTRO BUCHNEA
The first two products, after recrystallization from ether melted at 82-83 °C and were identical with the authentic DL-l-stearoylglycerol. The last product, according to the specific rotation (+ 2.6 °) and to the melting point 76-80 ° was a mixture of D-l- and DL-l-stearoylglycerol. Discussion
The results of this investigation show that the acyl group migration in partially acylated glycerides catalysed by hydrogen chloride is accompanied by the formation of bisglyceride ethers. Therefore it is assumed that the mechanism of these reactions might proceed in two steps: First hydrogen chloride chlorinates 2-stearoylglycerol (0 and forms a bimolecular resonant ion complex intermediate (II), regeneration of hydrogen chloride with the simultaneous exchange of stearoyl groups between radicals (a) and (b) from positions C-2(a) and C-2(b) to the positions C-l(b) and C-3(a) resulting in DL-l-stearoylglycerol (In), fig. 2. In the second step, reaction of DL-l-stearoylglycerol (Ill) with hydrogen chloride results in the formation of bimolecular resonant ion complex intermediate (IV), followed by the regeneration of hydrogen chloride with the formation of bis(DL-l-stearoylglyceryl)-3-ether (v), and the migration of the stearoyl groups from positions C-1 (a) and C-1 (b) to the positions C-2(a) and C-2(b) resulting in bis (DL-2-stearoylglyceryl)-3-ether (Vl), and finally the reaction of the primary hydroxyl groups of compound vl with hydrogen chloride to give bis(DL-l-chloro-2-stearoylglyceryl)-3-ether (vii) as the final product in these reactions. The presence of the secondary hydroxyl groups in the second step requires more energy to facilitate the migration of the stearoyl groups from positions C-l(a) and C-l(b) to the positions C-2(a) and C-2(b), (vI) fig. 2. This takes place only by longer treatment of compound v (fig. 2) with gaseous hydrogen chloride in anhydrous ether solution at 0 °C. Therefore, when hydrogen chloride is first dissolved in anhydrous ether and then this solution is used for treatment of D-, L- and 2-stearoylglycerol, in this case, only racemization and conversion to DL-isomer takes place. It seems that the formation of the bimolecular resonance ion complex takes place only between two identical glyceride molecules. This could be the reason why, in Doerschuk's 13) experimental results, the glycerol-C14 was not incorporated in the resulting DL-l-palmitoylglycerol. The hypothesis of a bimolecular resonant ion complex intermediate (II and IV) formation during the acyl migration is also validated by the fact that during the migration of the acyl groups the formation of bisglyceride ethers takes place. Winstein and his co-workers 14) were the first to visualize the formation of similar ion pairs in the interpretation of rearrangement, ex-
ACYL MIGRATION IN GLYCERIDES
127
change, r a c e m i z a t i o n a n d solvolysis reactions o f n e u t r a l o r g a n i c substrates. W i n s t e i n s studies represent a p r e c e d e n t for the i n t e r p r e t a t i o n o f the f o r m a tion o f the b i m o l e c u l a r r e s o n a n t ion c o m p l e x i n t e r m e d i a t e s in the reactions described here. Bevan et al. 15) have p o s t u l a t e d the f o r m a t i o n o f a n i n t e r m e d i a t e r e s o n a n t ion for certain similar reactions, b u t this p o s t u l a t i o n implies only the form a t i o n o f a m o n o m o l e c u l a r r e s o n a n t ion. V a n L o h u i z e n a n d V e r k a d e 4) c o n s i d e r e d an i n t r a m o l e c u l a r r e a c t i o n m e c h a n i s m for acyl g r o u p m i g r a t i o n , which is b a s e d essentially o n the form a t i o n o f the o r t h o o e s t e r i n t e r m e d i a t e as p u t f o r w a r d by Emil Fischer18).
Acknowledgement The a u t h o r wishes to t h a n k Mr. F r e d e r i c k L a m m e r i c h , M e d i c a l A r t Dep a r t m e n t , U n i v e r s i t y o f T o r o n t o , for the d r a w i n g o f the figures.
References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14)
D. Buchnea and E. Baer, J. Lipid Res. I (1960) 405 D. Buchnea, Fette, Seifen, Anstrichmittel 64 (1962) 887 J. B. Martin, J. Am. Chem. Soc. 75 (1953) 5483 O. E. Van Lohuizen and P. E. Verkade, Rec. Trav. Chim. 79 (1960) 133 A. Crossley, I. P. Freeman, B. J. F. Hudson and J. H. Pierce, J. Chem. Soc. 760 (1959) M. Bergmann and N. M. Carter, Z. Physiol. Chem. 191 (1930) 211 B. F. Simmel and C. G. King, J. Am. Chem. Soc. 56 (1934) 1724 E. Baer and H. O. L. Fischer, J. Am. Chem. Soc. 67 (1945) 2031 Emil Fischer, M. Bergmann and E. Barwind, Chem. Ber. 53 (1920) 1589 J. Tausz and N. von Putnoky, Chem. Ber. 52 (1919) 1573 H. E. Fierz-David and W. Kusters, Helv. Acta 22 (1939) 82 E. Handschumaker and L. Linters, J. Am. Oil Chemists' Soc. 24 (1947) 143 A. P. Doerschuk, J. Am. Chem. Soc. 74 (1952) 4202 S. Winstein, E. Clippinger, A. H. Fainber and G. C. Robinson, J. Am. Chem. Soc. 76 (1954) 2597 15) T. H. Bevan, D. A. Brown, G. I. Gregory and T. Malkin, J. Chem. Soc. 24 (1953)127 16) Emil Fischer, Chem. Ber. 53 (1920) 1623