Chemistry and Physics of Lipids, 60 (1991) 189-199 Elsevier Scientific Publishers Ireland Ltd.
189
Regio- and stereoselective enzymatic esterification of glycerol and its derivatives Adam W. Mazur, George D. Hiler II, Susannie S.C. Lee, Molly P. Armstrong and Jack D. Wendel The Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 398707. Cincinnati, OH 45239-8707 (U.S.A.)
(Received July 25th, 1991; revision received October 29th, 1991; accepted October 29th, 1991)
A methodology for regio- and stereoselectivepreparation of acyl glycerol derivativesis presented. It offers easy access to specific 1,2-, 1,3-diglyceridesand triglyceridesas well as alkyl glycerolesters, phospholipids and glycolipids. These compounds are prepared by esterification of the corresponding glycerol derivatives such as 2-monoglycerides, alkyl glycerols, glyceryl glycosides, glyceryl phosphate esters, or unsubstituted glycerol. The regio- and stereoselectivity in the esterification is achieved by using fatty acid anhydrides and an enzymaticcatalyst, 1,3-specificlipase. NMR methods for determining the regio- and stereoselectivityof esterification are discussed. Key words: enzymaticesterification; glycerylesters; lipases; lipid synthesis; regioselectiveesterification; stereoselectiveesterification
Introduction Efficient preparation of pure isomers of glycerol esters, phospholipids and glycolipids in multigram quantities is frequently a difficult task [1]. A typical synthesis of the regio- and stereospecific acyl glycerol derivatives involves rather complex, multi step procedures requiring extensive use of protecting groups [2-5]. This is because one has to differentiate the hydroxyl groups for selective derivatization and at the same time prevent isomerization of the acyl substituents. This acyl migration is often difficult to control [6] since it can be facilitated by polar solvents and catalyzed by minute quantities of ions or free radicals generated in the reactions. Enzymic esterification of glycerol and its derivatives in the presence of 1,3-specific lipases [7,8] potentially offers a simple alternative to an Correspondence to: Adam W. Mazur, The Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 398707. Cincinnati, OH 45239-8707 (U.S.A.)
elaborate chemical methodology. These lipases are generally selective in the esterification of the primary hydroxyls in glycerol, the reaction can be done in low polarity solvents, and formation o f charged species can be avoided. However, if fatty acids or esters are used as substrates, the degree of conversion is unsatisfactory unless the water or alcohol formed in the process are eliminated from the reaction mixture. Although the yields o f glycerides can be improved by using large excesses of acid or ester substrates, these conditions may lead to the loss of regiospecificity through the enzyme-catalyzed exchange of the ester groups. An alternative method which avoids large excesses of the acylating reagents is a lipase-catalyzed esterification with active esters [9]. This is an irreversible process minimizing the possibility of acyl migration, but the high cost of active esters limits the application of this approach. Our strategy comprises the use of readily available fatty acid anhydrides for lipase catalyzed regio- a n d stereoselective esterifications. Acid anhydrides are common reagents for non-specific
0009-3084/91/$03.50 © 199t Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
190 obtained by one of two routes based on the enzymatic regiospecific hydrolysis of triglycerides. A selection of the specific route depends upon the required length of acyl group in the 2-position. Medium chain 2-monoglycerides (up to tetradecanoyl Ci4:0), can be obtained through the hydrolysis of simple commercially available triglycerides Ill] (e.g. tridecanoate, Fig. l). In contrast, preparation of 2-monoglycerides with acyl groups longer than Cl4:0 is not practical by this route because the rate of regiospecific hydrolysis of triglycerides decreases with the increase in acyl chain length. This is a result of decreasing solubility of longer chain substrates in the reaction mixture and particularly in the interface where the hydrolysis takes place. On the other hand, mixed triesters containing long chain acyl group in the 2-position but short or medium groups in l- and 3-positions, e.g. 1,3-decanoyl-2-docosanoyl glycerol, are hydrolyzed selectively to long.chain 2-monoglycerides (Fig. 2).
chemical acylations. On the other hand, in enzymatic reactions, these reagents can display stereoselectivity. Bianchi et al. [10] have reported resolution of racemic monohydroxyl alcohols by selective esterification of one enantiomer with acetic anhydride in the presence of Pseudomonas lipase. We have found that lipase catalysis allows regio- and stereoselective acylation of the hydroxyl groups in glycerols with fatty acid anhydrides. This method can be easily applied to large scale preparation of various glycerol esters. Lipozyme, a commercial 1,3-specific lipase, is a very effective catalyst for this purpose. Results and Discussion
The presented method offers easy access to specific 1,2-, 1,3-diglycerides and triglycerides as well as to alkyl glycerol esters, phospholipids and glycolipids. 2-Monoglycerides, the key substrates for the regiospecific synthesis of triglycerides, are
OCOR
OH
1 Lipose
R lC00 ~
b,,=._
Rl C 0 0 ~
Buffer/hexane OCOR 1
OH
65 - 70
R I = CgH19 (3) R I = C7H15 (4.)
R 1 - C9H19 (1) RI == C7H15 (2)
65 - 8 0 + Z
OCOR
OH
DMAP 3
(" 3C0)20
RICO0~
Llpozyme (Rp'O)20 CH2Cl2
R1 C 0 0 ~
80+ Z
O00R 2
OCOR2
e 1= %H19 R2= C2 ,3 e3= c-p15 (7)
R 1= %H19 R 2 = ~2 11443 (5)
R 1"= C7H15 R2 '= %11"14"3 R 3= C9H19 (8)
R 1= C7H15
Fig. I. Strategy for preparation of triglycerides with medium chain acyl group in the 2-position.
R 2 = C'21P~3 (6)
191
OH
OCOR
Ll~=y~ (R1C0)20 .---
HO OH
75-
1
DMAP
OCOR
140
R2COO--
80Z
OCOR 1
(9)
1
80+ Z
OCOR 1
R 1 ,, C9H19 (10)
R 1"= C9H19
R 2 " C21H43 (12)
R 1"
R 1"= C7H15
R 2 " C211"!4.3 (15)
C7H15 (11)
45 - 5 0 Z
Lipo=e Buffer/he=cane
DMAP
2(:°°'--
OCOR4
OH
(R4C0)20
R
Lipo~Jme
(.3coho
OH
R2CO0 OCOR3
R2" %1 3
70 - 75 g
%H19 R4= C?H15 (10
OCOR 3
80+ Z
I~2 == C21H43 R3== C¢jH19 (15)
OH
R 2 " C211"143 (14)
Fig. 2. Strategy for preparation of triglycerides with long chain acyl group in the 2-position.
2-Monoglycerides can be converted to 1,2-diglycerides by a lipase catalyzed reaction in methylene chloride (Fig. 1). Under similar conditions, 1,3-diglycerides are conveniently prepared from glycerol and the appropriate fatty acid anhydrides (Fig. 2). In methylene chloride, both reactions are very selective and only small amounts (below 1%) of triglycerides are formed. In contrast, substantial amounts of triglycerides (up to 10%) are produced in hexane which may be the result of higher lipase activity in this solvent than in methylene chloride [12]. Further Lipozyme catalyzed esterification of 1,2-diglycerides to the corresponding triglycerides occurs with substantial loss of regioselectivity. Consequently, the esterification of diglycerides to specific triglycerides is accomplished with a chemical catalyst, N,N-dimethylaminopyridine (DMAP) instead of Lipozyme. The regioselective enzymatic acylation method leading to 1,3-diacylglycerols can easily be extended to the preparation of other glycerol derivatives.
Using analogous procedures, a broad range of important compounds used in lipid research can be obtained in good yield. Examples of these syntheses, shown in Table I include glycolipid (18), phospholipids (20), (22) and esters of alkyl glycerol (24). Although extremely low solubility of some substrates such as dibenzylglycerolphosphate (25) and monobenzylglycerolphosphate [13] (26) (Fig. 3) prevents esterification under these conditions, other poorly but sufficiently soluble compounds, e.g. (19), are esterified when suspended in the reaction mixture. Regioselectivity of' the esterifications has been investigated using C-13 NMR spectroscopy. Positional isomers of mono- and diglycerides are easy to detect because each isomer gives a distinctive resonance pattern of the glycerol backbone carbon atoms, as shown in Tables II and III. The spectra indicate that mono- and diglycerides as well as other lipids listed in Table I are single positional isomers. Isomeric composition of triglycerides, on the other hand, has been determined from chemi-
192 TABLE 1 Lipozymecatalyzed acylation of various glycerol derivatives using acid anhydrides (RICO)20.
Substrote
HO
Yield
(g)
56
Product
140
e ~- (%h4c.3
OH
OH
(17)
(18)
OH HO
71
(m)
(20) -~OCOR 1 35
HO
" 1" (c~12c" 3
CH
(r.=..W=)
_ / °t ~- '(, ) 2
"'~(0-
189
Regio- and stereoselective enzymatic esterification of glycerol and its derivatives Adam W. Mazur, George D. Hiler II, Susannie S.C. Lee, Molly P. Armstrong and Jack D. Wendel The Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 398707. Cincinnati, OH 45239-8707 (U.S.A.)
(Received July 25th, 1991; revision received October 29th, 1991; accepted October 29th, 1991)
A methodology for regio- and stereoselectivepreparation of acyl glycerol derivativesis presented. It offers easy access to specific 1,2-, 1,3-diglyceridesand triglyceridesas well as alkyl glycerolesters, phospholipids and glycolipids. These compounds are prepared by esterification of the corresponding glycerol derivatives such as 2-monoglycerides, alkyl glycerols, glyceryl glycosides, glyceryl phosphate esters, or unsubstituted glycerol. The regio- and stereoselectivity in the esterification is achieved by using fatty acid anhydrides and an enzymaticcatalyst, 1,3-specificlipase. NMR methods for determining the regio- and stereoselectivityof esterification are discussed. Key words: enzymaticesterification; glycerylesters; lipases; lipid synthesis; regioselectiveesterification; stereoselectiveesterification
Introduction Efficient preparation of pure isomers of glycerol esters, phospholipids and glycolipids in multigram quantities is frequently a difficult task [1]. A typical synthesis of the regio- and stereospecific acyl glycerol derivatives involves rather complex, multi step procedures requiring extensive use of protecting groups [2-5]. This is because one has to differentiate the hydroxyl groups for selective derivatization and at the same time prevent isomerization of the acyl substituents. This acyl migration is often difficult to control [6] since it can be facilitated by polar solvents and catalyzed by minute quantities of ions or free radicals generated in the reactions. Enzymic esterification of glycerol and its derivatives in the presence of 1,3-specific lipases [7,8] potentially offers a simple alternative to an Correspondence to: Adam W. Mazur, The Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 398707. Cincinnati, OH 45239-8707 (U.S.A.)
elaborate chemical methodology. These lipases are generally selective in the esterification of the primary hydroxyls in glycerol, the reaction can be done in low polarity solvents, and formation o f charged species can be avoided. However, if fatty acids or esters are used as substrates, the degree of conversion is unsatisfactory unless the water or alcohol formed in the process are eliminated from the reaction mixture. Although the yields o f glycerides can be improved by using large excesses of acid or ester substrates, these conditions may lead to the loss of regiospecificity through the enzyme-catalyzed exchange of the ester groups. An alternative method which avoids large excesses of the acylating reagents is a lipase-catalyzed esterification with active esters [9]. This is an irreversible process minimizing the possibility of acyl migration, but the high cost of active esters limits the application of this approach. Our strategy comprises the use of readily available fatty acid anhydrides for lipase catalyzed regio- a n d stereoselective esterifications. Acid anhydrides are common reagents for non-specific
0009-3084/91/$03.50 © 199t Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
190 obtained by one of two routes based on the enzymatic regiospecific hydrolysis of triglycerides. A selection of the specific route depends upon the required length of acyl group in the 2-position. Medium chain 2-monoglycerides (up to tetradecanoyl Ci4:0), can be obtained through the hydrolysis of simple commercially available triglycerides Ill] (e.g. tridecanoate, Fig. l). In contrast, preparation of 2-monoglycerides with acyl groups longer than Cl4:0 is not practical by this route because the rate of regiospecific hydrolysis of triglycerides decreases with the increase in acyl chain length. This is a result of decreasing solubility of longer chain substrates in the reaction mixture and particularly in the interface where the hydrolysis takes place. On the other hand, mixed triesters containing long chain acyl group in the 2-position but short or medium groups in l- and 3-positions, e.g. 1,3-decanoyl-2-docosanoyl glycerol, are hydrolyzed selectively to long.chain 2-monoglycerides (Fig. 2).
chemical acylations. On the other hand, in enzymatic reactions, these reagents can display stereoselectivity. Bianchi et al. [10] have reported resolution of racemic monohydroxyl alcohols by selective esterification of one enantiomer with acetic anhydride in the presence of Pseudomonas lipase. We have found that lipase catalysis allows regio- and stereoselective acylation of the hydroxyl groups in glycerols with fatty acid anhydrides. This method can be easily applied to large scale preparation of various glycerol esters. Lipozyme, a commercial 1,3-specific lipase, is a very effective catalyst for this purpose. Results and Discussion
The presented method offers easy access to specific 1,2-, 1,3-diglycerides and triglycerides as well as to alkyl glycerol esters, phospholipids and glycolipids. 2-Monoglycerides, the key substrates for the regiospecific synthesis of triglycerides, are
OCOR
OH
1 Lipose
R lC00 ~
b,,=._
Rl C 0 0 ~
Buffer/hexane OCOR 1
OH
65 - 70
R I = CgH19 (3) R I = C7H15 (4.)
R 1 - C9H19 (1) RI == C7H15 (2)
65 - 8 0 + Z
OCOR
OH
DMAP 3
(" 3C0)20
RICO0~
Llpozyme (Rp'O)20 CH2Cl2
R1 C 0 0 ~
80+ Z
O00R 2
OCOR2
e 1= %H19 R2= C2 ,3 e3= c-p15 (7)
R 1= %H19 R 2 = ~2 11443 (5)
R 1"= C7H15 R2 '= %11"14"3 R 3= C9H19 (8)
R 1= C7H15
Fig. I. Strategy for preparation of triglycerides with medium chain acyl group in the 2-position.
R 2 = C'21P~3 (6)
191
OH
OCOR
Ll~=y~ (R1C0)20 .---
HO OH
75-
1
DMAP
OCOR
140
R2COO--
80Z
OCOR 1
(9)
1
80+ Z
OCOR 1
R 1 ,, C9H19 (10)
R 1"= C9H19
R 2 " C21H43 (12)
R 1"
R 1"= C7H15
R 2 " C211"!4.3 (15)
C7H15 (11)
45 - 5 0 Z
Lipo=e Buffer/he=cane
DMAP
2(:°°'--
OCOR4
OH
(R4C0)20
R
Lipo~Jme
(.3coho
OH
R2CO0 OCOR3
R2" %1 3
70 - 75 g
%H19 R4= C?H15 (10
OCOR 3
80+ Z
I~2 == C21H43 R3== C¢jH19 (15)
OH
R 2 " C211"143 (14)
Fig. 2. Strategy for preparation of triglycerides with long chain acyl group in the 2-position.
2-Monoglycerides can be converted to 1,2-diglycerides by a lipase catalyzed reaction in methylene chloride (Fig. 1). Under similar conditions, 1,3-diglycerides are conveniently prepared from glycerol and the appropriate fatty acid anhydrides (Fig. 2). In methylene chloride, both reactions are very selective and only small amounts (below 1%) of triglycerides are formed. In contrast, substantial amounts of triglycerides (up to 10%) are produced in hexane which may be the result of higher lipase activity in this solvent than in methylene chloride [12]. Further Lipozyme catalyzed esterification of 1,2-diglycerides to the corresponding triglycerides occurs with substantial loss of regioselectivity. Consequently, the esterification of diglycerides to specific triglycerides is accomplished with a chemical catalyst, N,N-dimethylaminopyridine (DMAP) instead of Lipozyme. The regioselective enzymatic acylation method leading to 1,3-diacylglycerols can easily be extended to the preparation of other glycerol derivatives.
Using analogous procedures, a broad range of important compounds used in lipid research can be obtained in good yield. Examples of these syntheses, shown in Table I include glycolipid (18), phospholipids (20), (22) and esters of alkyl glycerol (24). Although extremely low solubility of some substrates such as dibenzylglycerolphosphate (25) and monobenzylglycerolphosphate [13] (26) (Fig. 3) prevents esterification under these conditions, other poorly but sufficiently soluble compounds, e.g. (19), are esterified when suspended in the reaction mixture. Regioselectivity of' the esterifications has been investigated using C-13 NMR spectroscopy. Positional isomers of mono- and diglycerides are easy to detect because each isomer gives a distinctive resonance pattern of the glycerol backbone carbon atoms, as shown in Tables II and III. The spectra indicate that mono- and diglycerides as well as other lipids listed in Table I are single positional isomers. Isomeric composition of triglycerides, on the other hand, has been determined from chemi-
192 TABLE 1 Lipozymecatalyzed acylation of various glycerol derivatives using acid anhydrides (RICO)20.
Substrote
HO
Yield
(g)
56
Product
140
e ~- (%h4c.3
OH
OH
(17)
(18)
OH HO
71
(m)
(20) -~OCOR 1 35
HO
" 1" (c~12c" 3
CH
(r.=..W=)
_ / °t ~- '(, ) 2
"'~(0-
