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Zinc(II)-facilitated hydrolysis of 2-hydroxyacid esters. A model for carboxypeptidase A
J. inorg, nucl. Chem. Vol. 42, pp. 1 5 5 - 1 5 6 (~)Pergamon Press Ltd., 1981). Printed in G r e a t Britain
0022-1902/80/0101~)155/$02.00/0
ZINC(II)-FACILITATED HYDROLYSIS OF 2-HYDROXYACID ESTERS. A MODEL FOR CARBOXYPEPTIDASE A RODNEY F. BOYER Department of Chemistry, Hope College, Holland, MI 49423, U.S.A.
(Received 9 April 1979) Abstract--Zn(II) complexes of naturally-occurring 2-hydroxyacids have been prepared by two separate methods: (1) Reaction between 2-hydroxyacid and Zn(II); and (2) Zn(II)-facilitated hydrolysis of esters of 2-hydroxyacids. For example, reaction of 0-acetyl 2-hydroxyoctadecanoic acid with Zn(II) in warm 95% ethanol led to formation of Zn(2-hydroxyoctadecanoate) 2. This general reaction should provide insight into the importance of metal-ion activation of the leaving group in carboxypeptidasecatalyzed hydrolysis of esters.
INTRODUCTION
EXPERIMENTAL
The metal coordinating abilities of complex lipids and lipid components have been the subject of few investigations [1]. 2-Hydroxycarboxylic acids [2HA] are an exception as several authors have reported isolation and purification methods for these compounds based on their ability to act as bidendate ligands to copper. These methods have been applied to the isolation of long-chain 2-HA [C18-C26] from brain cerebrosides [2], various rat tissues [3], wool wax [4] and mushrooms [5]. Although copper(II) has most often been utilized for complex formation with 2-HA [6], these naturally-occurring lipid components should also coordinate to other physiologically important metals. Complex formation between short-chain 2-HA and Zn(II) [7-9], Mn(II) [7, 10] and Fe(II) [9] has been reported. The discovery that carboxypeptidase A catalyzes the hydrolysis of the ester bond in certain 0-acyl 2-hydroxyacids [11] prompted us to investigate the Zn(II)-mediated hydrolysis of various ester derivatives of 2-HA. Here we report the preparation of zinc complexes of several biochemically important 2-HA (Table 1) by the direct reaction of Zn(II) with the free 2-HA. Of special interest is our observation of Zn(II)-promoted hydrolysis of methyl esters and 0-acetyl derivatives of 2-HA as a method for the synthesis of Zn(2-HA)2. Zn(II)-mediated reactions of this type should provide a useful model for the study of the catalytic role of Zn(II) in carboxypeptidase.
Reagents and instrumentation The 2-HA were obtained from Sigma Chemical Company and ICN-K and K. The esters were prepared as previously described [6]. IR measurements, using KBr pellets, were made on a Perkin Elmer Model 621. Quantitative microanalyses were performed by Galbraith Laboratories. Preparation of complexes Zn(2-HA)2 complexes were prepared in ethanol solution by reaction between the 2-HA (Immole) and Zn(NO3)2 (0.5 mmole). In every case the yields were greater than 95%. Zn(II)-promoted hydrolysis was carried out on methylmandelate(I), methyl 2-hydroxyoctadecanoate(II) and 0acetyl 2-hydroxyoctadecanoic acid(III). The ester (1 mmole), dissolved in 95% ethanol, was heated to 60°C with Zn(NO3)2 (1 mmole). The time required for 75% hydrolysis of each ester was I (10hr), II (8 hr) and III (4 hr). All complexes were identified by IR, microanalysis (Table 1) and by recovery of the free 2-HA by EDTA treatment [6]. Important IR bands of the complexes included 3100--3300cm-I (O--H), 1560-1570 cm i (C=O) and 1060-1080 cm-1 (C--OH). RESULTS A N D DISCUSSION
Zinc complexes of mandelic acid [9, 12, 13] and short-chain 2-HA [7, 8, 10] have previously been reported, however, ours is the first report on longchain, naturally occurring 2-HA as Zn ligands. 2-HA of chain length C18--C26 are found in both plant and animal tissue [2-5]. The complexes reported here are white, crystalline solids that can be
Table 1. Elemental analysis of Zn(2-HA)2 CalcuIated
2-Hydroxyacid
(2-HA)
C
(%)
Found (%)
H
Zn
C
H
Zn
2-hydroxydodecanoate
58.14
9.29
13.20
58.23
9.55
13.07
2-hydroxyoctadecanoate
65.12
10.55
9.85
65.31
10.28
9.70
2-hydroxytetracosanoate
69.28
I1.31
7.86
69.35
I1.20
7.90
2-hydroxyhexacosanoate
70.32
11.50
7.37
70.60
11.20
7,08
mandelate
52.23
3.81
17.79
52.10
3.90
17.70
155
R. F. BOYER
156
described by the formula Zn(2-HA)2 as is supported by the IR data and microanalyses (Table 1). Examination of the IR data confirms that the v(COO-), v(O---H) and v ( C - - O H ) are shifted toward lower frequencies, relative to the free acid, indicating that both the carboxylate and hydroxyl functional groups are coordinated to the metal. A n X-ray crystallographic investigation of bis(glycino)-zinc(II) monohydrate has established that the metal coordination is octahedral and the glycine ligands are arranged in a trans position [14], however, when the complex was dehydrated, IR analysis indicated tetrahedral coordination [15]. A recent crystal structure analysis of bis(/3-alanino) zinc nitrate tetrahydrate established an octahedral coordination [16]. In amino acid and peptide complexes of zinc the coordination of the metal atom may be tetrahedral, octahedral or intermediate (irregular coordination) [17]. The Zn(2HA)2 complexes described here are anhydrous and may best be characterized as tetrahedral, however, our data do not allow differentiation. Model reactions to elucidate the action of carboxypeptidase A have provided insight into substrate binding [17], geometrical alignment of Z n 2+ [18], anhydride intermediates [19] and metal ion activation of the leaving group [20]. We believe that the Zn(II)-mediated hydrolysis of compound ItI represents a viable model for a study of the catalytic role of Zn(II) in the esterase action of carboxypeptidase.
OCH3 -O
O~c/O ~
/ RCH O ," x
I
H A R = phenyl (I) or hexadecyl (II)
I
z/
RCH O / ' "
X
1 C-------O I
CH3
B R = hexadecyl (III)
Structures A and B depict the two types of ester derivatives used here and their most likely coordination with Zn(II). Structure A represents a possible intermediate for hydrolysis of compounds I and II, whereas B represents a possible intermediate for compound III. We propose that the metal ion acts as a template to polarize the carbonyl bond and activate it toward hydrolytic attack. The Zn(II)mediated hydrolysis of ester III may involve a seven-membered ring intermediate (coordination through the acetyl carbonyl) rather than a five membered ring intermediate (Structure B), however, our earlier investigation of Cu(II)-facilitated hydrolysis supports the importance of metal ion stabilization (complexation) of the leaving a hydroxyacid [6]. We intend to study the kinetics of the Zn(II)-mediated hydrolysis of several esters including I, II, III, 0-acetylmandelate and O-(trans-
cinnamoyl)-/3-phenyllactate, the latter two being known substrates of carboxypeptidase [11]. From these data we should be able to quantitate the importance of a metal ion: leaving group interaction in Zn(II)-facilitated hydrolysis and in ¢arboxypeptidase action. Since most 2-HA occur in nature as amides in cerebrosides and as esters in triacylglycerols [3], it is intriguing to speculate that metalloenzymes may be involved in the in vitro hydrolysis of these complex lipids. Acknowledgement--This work was supported by Grant NS-13081 from the National Institutes of Health. The assistance of B. Smith is gratefully acknowledged.
REFERENCES 1. A. Lindenbaum, in Metal Ions in Biological Systems (Edited by S. K. Dhar). (Advances in Experimental Medicine and Biology, Vol. 40), p. 67. Plenum, New York (1973). 2. N. Radin, J. Brown and F. Lavin, J. Biol. Chem. 219, 977 (1956). 3. Y. Kishimoto and N. Radin, J. Lipid Res. 4, 139 (1963). 4. A. Milburn and E. Truter, Z Appl. Chem. 12, 156 (1962). 5. M. Prostenik, I. Burcar, A. Castek, C. Cosovic, J. Golem, Z. Jandric, K. Kljaic and V. Ondrusek, Chem. Phys. Lipids 22, 97 (1978). 6. R. Boyer, M. Wernette, S. Van Wylen, E. Faustman and R. Titus, Inorg. Biochem. in press (1979). 7. H. Sigel, R. Griesser, B. Prijs, D. McCormick and M. Joiner, Arch. Biochem. Biophys. 130, 514 (1969). 8. I. Filipovic, I. Piljae, B. Bach-Dragufinovic, I. Kruhak, and B. Grabarie, Croat. Chem. Acta 45, 447 (1973). 9. P. Khadikar, R. Ameria, M. Kekre and S. Chauhan, J. Inorg. Nucl. Chem. 35, 4301 (1973). 10. H. Thun, W. Guns and F. Verbeek, Anal. Chim. Acta 37, 332 (1967). 11. E. Kaiser and B. Kaiser, Ace. Chem. Res. $, 219 (1972). 12. B. Folkesson and R. Larsson, Acta Chem. Scand. 22, 16 (1968). 13. A. Fischinger, A. Sarapu and A. Companion, Can. Y. Chem. 47, 2629 (1969). 14. B. Low, F. Hirsideld and F. Richards, J. Am. Chem. Soc. 81, 4412 (1959). 15. A. Saraceno, I. Nakagawa, S. Mizushima, C. Curran and J. Quagliano, J. Am. Chem. Soc. 80, 5018 (1958). 16. F. Dejehet, R. De Buyst, B. Ledieu, J. Deelercq, G. Germain and M. Van Meerssche, Inorg. Chim. Acta 30, 197 (1978). 17. H. Freeman in Inorganic Biochemistry (Edited by G. Eichhorn), p. 121. Elsevier, Amsterdam (1973). 18. J. Groves and R. Dias, Z Am. Chem. Soc. 101, 1033 (1979). 19. R. Breslow, D. McClure, R. Brown and J. Eisenach, J. Am. Chem. Soc. 97, 194 (1975). 20. R. Hay and C. Clark, J. Chem. Soc. Dalton 1993 (1977).
0022-1902/80/0101~)155/$02.00/0
ZINC(II)-FACILITATED HYDROLYSIS OF 2-HYDROXYACID ESTERS. A MODEL FOR CARBOXYPEPTIDASE A RODNEY F. BOYER Department of Chemistry, Hope College, Holland, MI 49423, U.S.A.
(Received 9 April 1979) Abstract--Zn(II) complexes of naturally-occurring 2-hydroxyacids have been prepared by two separate methods: (1) Reaction between 2-hydroxyacid and Zn(II); and (2) Zn(II)-facilitated hydrolysis of esters of 2-hydroxyacids. For example, reaction of 0-acetyl 2-hydroxyoctadecanoic acid with Zn(II) in warm 95% ethanol led to formation of Zn(2-hydroxyoctadecanoate) 2. This general reaction should provide insight into the importance of metal-ion activation of the leaving group in carboxypeptidasecatalyzed hydrolysis of esters.
INTRODUCTION
EXPERIMENTAL
The metal coordinating abilities of complex lipids and lipid components have been the subject of few investigations [1]. 2-Hydroxycarboxylic acids [2HA] are an exception as several authors have reported isolation and purification methods for these compounds based on their ability to act as bidendate ligands to copper. These methods have been applied to the isolation of long-chain 2-HA [C18-C26] from brain cerebrosides [2], various rat tissues [3], wool wax [4] and mushrooms [5]. Although copper(II) has most often been utilized for complex formation with 2-HA [6], these naturally-occurring lipid components should also coordinate to other physiologically important metals. Complex formation between short-chain 2-HA and Zn(II) [7-9], Mn(II) [7, 10] and Fe(II) [9] has been reported. The discovery that carboxypeptidase A catalyzes the hydrolysis of the ester bond in certain 0-acyl 2-hydroxyacids [11] prompted us to investigate the Zn(II)-mediated hydrolysis of various ester derivatives of 2-HA. Here we report the preparation of zinc complexes of several biochemically important 2-HA (Table 1) by the direct reaction of Zn(II) with the free 2-HA. Of special interest is our observation of Zn(II)-promoted hydrolysis of methyl esters and 0-acetyl derivatives of 2-HA as a method for the synthesis of Zn(2-HA)2. Zn(II)-mediated reactions of this type should provide a useful model for the study of the catalytic role of Zn(II) in carboxypeptidase.
Reagents and instrumentation The 2-HA were obtained from Sigma Chemical Company and ICN-K and K. The esters were prepared as previously described [6]. IR measurements, using KBr pellets, were made on a Perkin Elmer Model 621. Quantitative microanalyses were performed by Galbraith Laboratories. Preparation of complexes Zn(2-HA)2 complexes were prepared in ethanol solution by reaction between the 2-HA (Immole) and Zn(NO3)2 (0.5 mmole). In every case the yields were greater than 95%. Zn(II)-promoted hydrolysis was carried out on methylmandelate(I), methyl 2-hydroxyoctadecanoate(II) and 0acetyl 2-hydroxyoctadecanoic acid(III). The ester (1 mmole), dissolved in 95% ethanol, was heated to 60°C with Zn(NO3)2 (1 mmole). The time required for 75% hydrolysis of each ester was I (10hr), II (8 hr) and III (4 hr). All complexes were identified by IR, microanalysis (Table 1) and by recovery of the free 2-HA by EDTA treatment [6]. Important IR bands of the complexes included 3100--3300cm-I (O--H), 1560-1570 cm i (C=O) and 1060-1080 cm-1 (C--OH). RESULTS A N D DISCUSSION
Zinc complexes of mandelic acid [9, 12, 13] and short-chain 2-HA [7, 8, 10] have previously been reported, however, ours is the first report on longchain, naturally occurring 2-HA as Zn ligands. 2-HA of chain length C18--C26 are found in both plant and animal tissue [2-5]. The complexes reported here are white, crystalline solids that can be
Table 1. Elemental analysis of Zn(2-HA)2 CalcuIated
2-Hydroxyacid
(2-HA)
C
(%)
Found (%)
H
Zn
C
H
Zn
2-hydroxydodecanoate
58.14
9.29
13.20
58.23
9.55
13.07
2-hydroxyoctadecanoate
65.12
10.55
9.85
65.31
10.28
9.70
2-hydroxytetracosanoate
69.28
I1.31
7.86
69.35
I1.20
7.90
2-hydroxyhexacosanoate
70.32
11.50
7.37
70.60
11.20
7,08
mandelate
52.23
3.81
17.79
52.10
3.90
17.70
155
R. F. BOYER
156
described by the formula Zn(2-HA)2 as is supported by the IR data and microanalyses (Table 1). Examination of the IR data confirms that the v(COO-), v(O---H) and v ( C - - O H ) are shifted toward lower frequencies, relative to the free acid, indicating that both the carboxylate and hydroxyl functional groups are coordinated to the metal. A n X-ray crystallographic investigation of bis(glycino)-zinc(II) monohydrate has established that the metal coordination is octahedral and the glycine ligands are arranged in a trans position [14], however, when the complex was dehydrated, IR analysis indicated tetrahedral coordination [15]. A recent crystal structure analysis of bis(/3-alanino) zinc nitrate tetrahydrate established an octahedral coordination [16]. In amino acid and peptide complexes of zinc the coordination of the metal atom may be tetrahedral, octahedral or intermediate (irregular coordination) [17]. The Zn(2HA)2 complexes described here are anhydrous and may best be characterized as tetrahedral, however, our data do not allow differentiation. Model reactions to elucidate the action of carboxypeptidase A have provided insight into substrate binding [17], geometrical alignment of Z n 2+ [18], anhydride intermediates [19] and metal ion activation of the leaving group [20]. We believe that the Zn(II)-mediated hydrolysis of compound ItI represents a viable model for a study of the catalytic role of Zn(II) in the esterase action of carboxypeptidase.
OCH3 -O
O~c/O ~
/ RCH O ," x
I
H A R = phenyl (I) or hexadecyl (II)
I
z/
RCH O / ' "
X
1 C-------O I
CH3
B R = hexadecyl (III)
Structures A and B depict the two types of ester derivatives used here and their most likely coordination with Zn(II). Structure A represents a possible intermediate for hydrolysis of compounds I and II, whereas B represents a possible intermediate for compound III. We propose that the metal ion acts as a template to polarize the carbonyl bond and activate it toward hydrolytic attack. The Zn(II)mediated hydrolysis of ester III may involve a seven-membered ring intermediate (coordination through the acetyl carbonyl) rather than a five membered ring intermediate (Structure B), however, our earlier investigation of Cu(II)-facilitated hydrolysis supports the importance of metal ion stabilization (complexation) of the leaving a hydroxyacid [6]. We intend to study the kinetics of the Zn(II)-mediated hydrolysis of several esters including I, II, III, 0-acetylmandelate and O-(trans-
cinnamoyl)-/3-phenyllactate, the latter two being known substrates of carboxypeptidase [11]. From these data we should be able to quantitate the importance of a metal ion: leaving group interaction in Zn(II)-facilitated hydrolysis and in ¢arboxypeptidase action. Since most 2-HA occur in nature as amides in cerebrosides and as esters in triacylglycerols [3], it is intriguing to speculate that metalloenzymes may be involved in the in vitro hydrolysis of these complex lipids. Acknowledgement--This work was supported by Grant NS-13081 from the National Institutes of Health. The assistance of B. Smith is gratefully acknowledged.
REFERENCES 1. A. Lindenbaum, in Metal Ions in Biological Systems (Edited by S. K. Dhar). (Advances in Experimental Medicine and Biology, Vol. 40), p. 67. Plenum, New York (1973). 2. N. Radin, J. Brown and F. Lavin, J. Biol. Chem. 219, 977 (1956). 3. Y. Kishimoto and N. Radin, J. Lipid Res. 4, 139 (1963). 4. A. Milburn and E. Truter, Z Appl. Chem. 12, 156 (1962). 5. M. Prostenik, I. Burcar, A. Castek, C. Cosovic, J. Golem, Z. Jandric, K. Kljaic and V. Ondrusek, Chem. Phys. Lipids 22, 97 (1978). 6. R. Boyer, M. Wernette, S. Van Wylen, E. Faustman and R. Titus, Inorg. Biochem. in press (1979). 7. H. Sigel, R. Griesser, B. Prijs, D. McCormick and M. Joiner, Arch. Biochem. Biophys. 130, 514 (1969). 8. I. Filipovic, I. Piljae, B. Bach-Dragufinovic, I. Kruhak, and B. Grabarie, Croat. Chem. Acta 45, 447 (1973). 9. P. Khadikar, R. Ameria, M. Kekre and S. Chauhan, J. Inorg. Nucl. Chem. 35, 4301 (1973). 10. H. Thun, W. Guns and F. Verbeek, Anal. Chim. Acta 37, 332 (1967). 11. E. Kaiser and B. Kaiser, Ace. Chem. Res. $, 219 (1972). 12. B. Folkesson and R. Larsson, Acta Chem. Scand. 22, 16 (1968). 13. A. Fischinger, A. Sarapu and A. Companion, Can. Y. Chem. 47, 2629 (1969). 14. B. Low, F. Hirsideld and F. Richards, J. Am. Chem. Soc. 81, 4412 (1959). 15. A. Saraceno, I. Nakagawa, S. Mizushima, C. Curran and J. Quagliano, J. Am. Chem. Soc. 80, 5018 (1958). 16. F. Dejehet, R. De Buyst, B. Ledieu, J. Deelercq, G. Germain and M. Van Meerssche, Inorg. Chim. Acta 30, 197 (1978). 17. H. Freeman in Inorganic Biochemistry (Edited by G. Eichhorn), p. 121. Elsevier, Amsterdam (1973). 18. J. Groves and R. Dias, Z Am. Chem. Soc. 101, 1033 (1979). 19. R. Breslow, D. McClure, R. Brown and J. Eisenach, J. Am. Chem. Soc. 97, 194 (1975). 20. R. Hay and C. Clark, J. Chem. Soc. Dalton 1993 (1977).