- Email: [email protected]
Biotransformation of citronellol by means of horseradish peroxidase
Biotransformation of citroneHol by means of horseradish peroxidase Janina Kaminska, Lidia Markowicz, Jolanta Stoi~owska and Jozef Gora Institute o f General F o o d Chemistry, Technical University o f 4zOd~., L~)d~, Poland
Oxidation of citronellol by means of a horseradish peroxiduse-hydrogen peroxide system in the presence of ascorbic acid has been studied. Incubations have been performed in two-phase medium (water-ethyl acetate or water-citronellol) and in homogeneous solution (water-methanol). The influence of pH, incubation time, hydrogen peroxide excess, and enzyme concentration on citronellol conversion and composition of the reaction products has been determined. The main oxidation products were isolated and identified. They were d~[ferent from compounds Jormed during the sensitized photooxidation of citronellol. This seems to exclude the participation of singlet oxygen in reactions mediated by horseradish peroxidase.
Keywords: Citronellol; horseradish peroxidase; oxidation
Introduction Citronellol (I), in Figure I, is a known p e r f u m e r y and flavoring c o m p o u n d as well as a substrate for the synthesis of other o d o r chemicals, such as rose oxide (IV), an important minor sensory c o m p o n e n t of Bulgarian rose oil. The first step of rose oxide synthesis is based on sensitized photooxidation of citronellol to a mixture o f allyl h y d r o p e r o x i d e s , which are subsequently reduced to the corresponding diols II and III. ~ In the p r e s e n c e of acid, diol II is dehydrated to oxide IV. 2 The biosynthesis of IV probably also proceeds from citronellol via ailyl diol II. In p h o t o o x y g e n a t i o n of alkenes, singlet oxygen is a reactive intermediate yielding allyl hydroperoxides. 3 It is n o t e w o r t h y that a serious disadvantage of the photooxidation process (especially in large scale) is the very high energy consumption. The same kind of allyl h y d r o p e r o x i d e s were found to arise from some steroids (containing C-C double bond) after tre~/tment with a p e r o x i d a s e hydrogen peroxide system. 4-6 M o r e o v e r , spectral evidence for the existence of singlet oxygen in the e n z y m e - h y d r o g e n peroxide mixture was reported. 7 For these reasons, we have undertaken an investigation of the e n z y m a t i c hydroxylation of citronellol as an alternative route to preparation of diol II. We have
Address reprint requests to Dr. Kamifiska at the Institute of General Food Chemistry, Technical University of &6d~', ul. Zwirki 36, 90-924 ~6d~', Poland Received 12 April 1988; revised 1 July 1988
438
Enzyme Microb. Technol., 1989, vol. 11, July
chosen horseradish peroxidase (EC I. 11.1.7.) as biocatalyst, taking into account its availability and stability during storage and operation.
Materials and methods Horseradish peroxidase was prepared from fresh roots according to a procedure described in ref. 8. The e n z y m e activity was determined by the guaiacol method according to ref. 9. All reagents and solvents were of analytical grade. Phosphate buffer (pH 5-8) or borate buffer (pH 8-10) were used for making the e n z y m e solutions. H y d r o g e n peroxide was applied as a 3% (w/v) solution in water. G a s - l i q u i d c h r o m a t o g r a phy analyses were performed on a 2 m × 4 m m column packed with 3% OV-61. ~H-NMR spectra were recorded on a Bruker 300M H z apparatus in d e u t e r o c h l o r o f o r m solution with tetramethylsilane as internal standard. Optical rotation was measured on a Perkin-Elmer 241 MC polarimeter in chloroform solution. Biotransformation procedure
The mixture of buffer solution (10 cc), methanol (20 cc), ascorbic acid (1 raM), citronellol (1 mM), e n z y m e (7 U), and hydrogen peroxide (10-50 mM) was kept at a t e m p e r a t u r e of 303°K on a water bath shaker for 3 days. The mixture was then treated with potassium sulfite (10-50 mM) for 24 h and subjected to continuous extraction with 70 cc of dichloromethane for 3-3.5 h. The extract was dried o v e r magnesium sulfate and the
© 1989 But t er wor t h Publishers
Biotransformation of citronellol: J. Kamihska et al.
1. O21SENS. 2REDUCTION
H
Figure 1 Synthesis of rose oxide from citronellol
solvent was removed by evaporation under reduced pressure. Products V-VII and VIII-X were only partially resolved. Pure samples for identification were isolated by preparative t.l.c, on commercial plates precoated with silica gel 60, using a chloroformmethanol mixture (92 : 8) as the developing system. The ~H-NMR data of products obtained are as follows: 6-Methoxy-3,7-dimethyl-l,7-octanediol (V) and 7methoxy-3,7-dimethyl-l,6-octanediol (V1); 0.84 ppm, d, 3H, CH3 at C-3; 1.02 and 1.05 ppm, 2s, 6H, 2 x CH3 at C-7; 3.15 ppm, s, 3H, OCH3; 3.34 ppm, t, IH at C-6; 3.61 ppm, m, 2H at C-I.
In the hydroxylation of the aromatic ring of L-tyrosine or D-p-hydroxyphenyiglycine, dihydroxyfumaric acid has been used as a proton donor.11 We have chosen ascorbic acid for this purpose. After preliminary experiments on the enzymatic oxidation of citronellol, we have found that a reasonable reaction rate can be achieved in homogeneous solution only (methanolwater). In a two-phase medium like water-citronellol or water-ethyl acetate, conversion of substrate did not exceed 5% after 14 days of reaction. Blank experiments (without enzyme) have shown that hydrogen peroxide in the presence of ascorbic acid has led to nonenzymatic substrate oxidation. However, the enzymatic process is 1.5 to 2.5 times faster. A similar phenomenon has already been noticed for an o x y g e n dihydroxyfumaric acid system. ~ On the other hand, ferrous ions at a concentration equivalent to the quantity of iron present in the enzyme do not catalyse oxidation of citronellol. Horseradish peroxidase is stable over a broad pH range from 3.5 to 12. The optimum pH of enzyme activity for citronellol transformation was found to be at pH values between 8 and 9. Therefore, further transformations were conducted in borate buffer solution at pH 9. Contrary to our expectations, oxidation of citronellol by means of the horseradish peroxidasehydrogen peroxide system did not yield allyl diols II and III. The main transformation products are shown in Figure 2. The major products were compounds V-VII, which probably arise via epoxidation of substrate and then solvolysis of epoxide by methanol or water. An accompanying side reaction is the addition of methanol or water to the double bond of citronellol, yielding compounds VIll, IX, and X. The products of nonenzymatic oxidation are qualitatively the same; the dif-
~
3,7-Dimethyl-l,6,7-octanetriol (VII); 0.91 ppm, d, 3H, CH3 at C-3; 1.15 and !.21 ppm, 2s, 6H, 2 x CH3 at C-7; 3.34 ppm, t, IH at C-6; 3.68 ppm, m, 2H at C-I.
OH
PEROXlDASE
H202
7-Methoxy-3,7-dimethyl-l-octanoi (VIII); 0.91 ppm, d, 3H, CH3 at C-3; 1.14 ppm, s, 6H, 2 x CH3 at C-7; 3.18 ppm, s, 3H, OCH3; 3.67 ppm, m, 2H at C-I. 6-Methoxy-3,7-dimethyl-l-octanol (IX); 0.87 ppm, d, 3H, CH3 at C-3; 1.09 ppm, d, 6H, 2 × CH3 at C-7; 3.18 ppm, s, 3H, OCH3; 3.67 ppm, m, 2H at C-I.
o. 3 V
24%
VI
24%
"/~OH VII
18%
X
11%
3,7-Dimethyl-l,7-octanediol (X); 0.91 ppm, d, 3H, CH3 at C-3; 1.21 ppm, s, 6H, 2 x CH3 at C-7; 3.67 ppm, m, 2H at C-1. 3
Results and discussion For the proposed mechanism of horseradish peroxidase action, as well as hydrogen peroxide or molecular oxygen, the presence of a proton donor is necessary. 10
H3 VIII 11%
IX
3%
Figure 2 Products of citronellol transformation by means of horseradish peroxidase-hydrogen peroxide system
Enzyme Microb. Technol., 1989, vol. 11, July
437
Papers % 6"
70 (~ Z
Z
O o ~_ 60 o Q O
0 6O o I--
50 ~ 0
50.
ne
40"
30"
20
20-~
10
:
2
4
I
6
,
~
108
10
12
1~1
TIME (DAYS) Figure 3 Appearance of the main products of citronellol transf o r m a t i o n as a function of time. Conditions: 10 cc of borate buffer (pH 9), 20 cc of methanol, 1 mM of citronellol, 1 mM of ascorbic acid, 7 U of peroxidase, 20 mM of h y d r o g e n peroxide [24 cc of 3% (w/v) solution] every 3 days; t e m p e r a t u r e 303°K; (f~) products V-VII; (e) products VIII-X
ferences were found in quantitative composition only. However, the product obtained in the presence of enzyme has shown slight optical activity, whereas the product from the blank test was optically inactive. The product ratio V-VII/VIII-X was always 30-40% higher for the enzymatic than for the nonenzymatic process. The formation of products V-VII and VIII-X with increasing reaction time is shown in Figure 3. Compounds V-VII were found to be rather stable in the reaction conditions when a low enzyme concentration was employed. The quantity of products V-VII rapidly decreases with increasing peroxidase concentration. Unidentified new products with longer retention time in GLC appear (see Figure 4). At the same time, the quantity of addition products V|I1-X increases only slightly with increasing enzyme concentration. Work on the isolation and identification of products formed at high enzyme concentration is now in progress.
Conclusions The absence of diols 1I and III among the products of transformation of citronellol by a horseradish peroxidase-hydrogen peroxide system seems to exclude the participation of singlet oxygen as an active intermediate, j2 The predominant reaction in this system is epoxidation of the C-C double bond followed by
438 EnzymeMicrob.Technol.,1989,vol.11,July
2'o
4'0
e'o
e~o
1~o
1~o
PEROXIDAS E [U] Figure 4 Product distribution as a function of enzyme concentration. Conditions: 10 cc of borate buffer (pH 9), 20 cc of methanol, 1 mM of citronellol, 1 mM of ascorbic acid, 40 mM of h y d r o g e n peroxide [48 cc of 3% (w/v) solution]; t e m p e r a t u r e 303°K; time 4 days; (c~) products V-VII; (v) products VIII-X; (e) unidentified products
epoxide solvolysis. An accompanying side reaction is solvent addition to the double bond.
Acknowledgement This research was supported by the Institute of Technical Biochemistry, Technical University of L6d~,, grant C.P.B.P. 04.11/2.39.
References I 2 3 4 5 6 7 8 9
10 II 12
Ohloff, G., Klein, E. and Schenck, O. Angew. C/tent. 1961, 73, 578 Ohloff, G. and Lienhard, B. Heir. Chim. Acta, 1965, 48, 182 Denny, R. W. and Nickon, A. Org. Reactions, 1973, 20, 133 Teng, J. I. and Smith, L. L. J. Am. Chem. Sot'. 1973, 95, 4060 Teng, J. I. and Smith, L. L. Bioorg. Chem. 1976, 5, 99 Yu, P. H. and Tan, L. J. Steroid Biochem. 1977, 8, 825 Khan, A. U. J. Am. Chem. Soc. 1983, 105, 7195 Kenten, R. H. and Mann, P. J. G. Biochem. J. 1954, 57, 347 Bergmeyer, H. U., G a w e h n , K. and Grassl, M. Methods t~/' Enzymatic Analysis. Verlag Chemie Weinheim, Academic Press, 1974, 1 , 4 9 4 Saunders, B. C. Inorganic BiochemisoT Elsevier, Amsterdam, 1973, p. 991 Klibanov, A. M., Berman, Z. and Alberti, B. N. J. Am. Chem. Soc. 1981, 103, 6263 Metelica, D. 1. Aktivacija kisloroda fi, rmentnimi sistemami N a u k a , Moscow, 1982, p. 61
Oxidation of citronellol by means of a horseradish peroxiduse-hydrogen peroxide system in the presence of ascorbic acid has been studied. Incubations have been performed in two-phase medium (water-ethyl acetate or water-citronellol) and in homogeneous solution (water-methanol). The influence of pH, incubation time, hydrogen peroxide excess, and enzyme concentration on citronellol conversion and composition of the reaction products has been determined. The main oxidation products were isolated and identified. They were d~[ferent from compounds Jormed during the sensitized photooxidation of citronellol. This seems to exclude the participation of singlet oxygen in reactions mediated by horseradish peroxidase.
Keywords: Citronellol; horseradish peroxidase; oxidation
Introduction Citronellol (I), in Figure I, is a known p e r f u m e r y and flavoring c o m p o u n d as well as a substrate for the synthesis of other o d o r chemicals, such as rose oxide (IV), an important minor sensory c o m p o n e n t of Bulgarian rose oil. The first step of rose oxide synthesis is based on sensitized photooxidation of citronellol to a mixture o f allyl h y d r o p e r o x i d e s , which are subsequently reduced to the corresponding diols II and III. ~ In the p r e s e n c e of acid, diol II is dehydrated to oxide IV. 2 The biosynthesis of IV probably also proceeds from citronellol via ailyl diol II. In p h o t o o x y g e n a t i o n of alkenes, singlet oxygen is a reactive intermediate yielding allyl hydroperoxides. 3 It is n o t e w o r t h y that a serious disadvantage of the photooxidation process (especially in large scale) is the very high energy consumption. The same kind of allyl h y d r o p e r o x i d e s were found to arise from some steroids (containing C-C double bond) after tre~/tment with a p e r o x i d a s e hydrogen peroxide system. 4-6 M o r e o v e r , spectral evidence for the existence of singlet oxygen in the e n z y m e - h y d r o g e n peroxide mixture was reported. 7 For these reasons, we have undertaken an investigation of the e n z y m a t i c hydroxylation of citronellol as an alternative route to preparation of diol II. We have
Address reprint requests to Dr. Kamifiska at the Institute of General Food Chemistry, Technical University of &6d~', ul. Zwirki 36, 90-924 ~6d~', Poland Received 12 April 1988; revised 1 July 1988
438
Enzyme Microb. Technol., 1989, vol. 11, July
chosen horseradish peroxidase (EC I. 11.1.7.) as biocatalyst, taking into account its availability and stability during storage and operation.
Materials and methods Horseradish peroxidase was prepared from fresh roots according to a procedure described in ref. 8. The e n z y m e activity was determined by the guaiacol method according to ref. 9. All reagents and solvents were of analytical grade. Phosphate buffer (pH 5-8) or borate buffer (pH 8-10) were used for making the e n z y m e solutions. H y d r o g e n peroxide was applied as a 3% (w/v) solution in water. G a s - l i q u i d c h r o m a t o g r a phy analyses were performed on a 2 m × 4 m m column packed with 3% OV-61. ~H-NMR spectra were recorded on a Bruker 300M H z apparatus in d e u t e r o c h l o r o f o r m solution with tetramethylsilane as internal standard. Optical rotation was measured on a Perkin-Elmer 241 MC polarimeter in chloroform solution. Biotransformation procedure
The mixture of buffer solution (10 cc), methanol (20 cc), ascorbic acid (1 raM), citronellol (1 mM), e n z y m e (7 U), and hydrogen peroxide (10-50 mM) was kept at a t e m p e r a t u r e of 303°K on a water bath shaker for 3 days. The mixture was then treated with potassium sulfite (10-50 mM) for 24 h and subjected to continuous extraction with 70 cc of dichloromethane for 3-3.5 h. The extract was dried o v e r magnesium sulfate and the
© 1989 But t er wor t h Publishers
Biotransformation of citronellol: J. Kamihska et al.
1. O21SENS. 2REDUCTION
H
Figure 1 Synthesis of rose oxide from citronellol
solvent was removed by evaporation under reduced pressure. Products V-VII and VIII-X were only partially resolved. Pure samples for identification were isolated by preparative t.l.c, on commercial plates precoated with silica gel 60, using a chloroformmethanol mixture (92 : 8) as the developing system. The ~H-NMR data of products obtained are as follows: 6-Methoxy-3,7-dimethyl-l,7-octanediol (V) and 7methoxy-3,7-dimethyl-l,6-octanediol (V1); 0.84 ppm, d, 3H, CH3 at C-3; 1.02 and 1.05 ppm, 2s, 6H, 2 x CH3 at C-7; 3.15 ppm, s, 3H, OCH3; 3.34 ppm, t, IH at C-6; 3.61 ppm, m, 2H at C-I.
In the hydroxylation of the aromatic ring of L-tyrosine or D-p-hydroxyphenyiglycine, dihydroxyfumaric acid has been used as a proton donor.11 We have chosen ascorbic acid for this purpose. After preliminary experiments on the enzymatic oxidation of citronellol, we have found that a reasonable reaction rate can be achieved in homogeneous solution only (methanolwater). In a two-phase medium like water-citronellol or water-ethyl acetate, conversion of substrate did not exceed 5% after 14 days of reaction. Blank experiments (without enzyme) have shown that hydrogen peroxide in the presence of ascorbic acid has led to nonenzymatic substrate oxidation. However, the enzymatic process is 1.5 to 2.5 times faster. A similar phenomenon has already been noticed for an o x y g e n dihydroxyfumaric acid system. ~ On the other hand, ferrous ions at a concentration equivalent to the quantity of iron present in the enzyme do not catalyse oxidation of citronellol. Horseradish peroxidase is stable over a broad pH range from 3.5 to 12. The optimum pH of enzyme activity for citronellol transformation was found to be at pH values between 8 and 9. Therefore, further transformations were conducted in borate buffer solution at pH 9. Contrary to our expectations, oxidation of citronellol by means of the horseradish peroxidasehydrogen peroxide system did not yield allyl diols II and III. The main transformation products are shown in Figure 2. The major products were compounds V-VII, which probably arise via epoxidation of substrate and then solvolysis of epoxide by methanol or water. An accompanying side reaction is the addition of methanol or water to the double bond of citronellol, yielding compounds VIll, IX, and X. The products of nonenzymatic oxidation are qualitatively the same; the dif-
~
3,7-Dimethyl-l,6,7-octanetriol (VII); 0.91 ppm, d, 3H, CH3 at C-3; 1.15 and !.21 ppm, 2s, 6H, 2 x CH3 at C-7; 3.34 ppm, t, IH at C-6; 3.68 ppm, m, 2H at C-I.
OH
PEROXlDASE
H202
7-Methoxy-3,7-dimethyl-l-octanoi (VIII); 0.91 ppm, d, 3H, CH3 at C-3; 1.14 ppm, s, 6H, 2 x CH3 at C-7; 3.18 ppm, s, 3H, OCH3; 3.67 ppm, m, 2H at C-I. 6-Methoxy-3,7-dimethyl-l-octanol (IX); 0.87 ppm, d, 3H, CH3 at C-3; 1.09 ppm, d, 6H, 2 × CH3 at C-7; 3.18 ppm, s, 3H, OCH3; 3.67 ppm, m, 2H at C-I.
o. 3 V
24%
VI
24%
"/~OH VII
18%
X
11%
3,7-Dimethyl-l,7-octanediol (X); 0.91 ppm, d, 3H, CH3 at C-3; 1.21 ppm, s, 6H, 2 x CH3 at C-7; 3.67 ppm, m, 2H at C-1. 3
Results and discussion For the proposed mechanism of horseradish peroxidase action, as well as hydrogen peroxide or molecular oxygen, the presence of a proton donor is necessary. 10
H3 VIII 11%
IX
3%
Figure 2 Products of citronellol transformation by means of horseradish peroxidase-hydrogen peroxide system
Enzyme Microb. Technol., 1989, vol. 11, July
437
Papers % 6"
70 (~ Z
Z
O o ~_ 60 o Q O
0 6O o I--
50 ~ 0
50.
ne
40"
30"
20
20-~
10
:
2
4
I
6
,
~
108
10
12
1~1
TIME (DAYS) Figure 3 Appearance of the main products of citronellol transf o r m a t i o n as a function of time. Conditions: 10 cc of borate buffer (pH 9), 20 cc of methanol, 1 mM of citronellol, 1 mM of ascorbic acid, 7 U of peroxidase, 20 mM of h y d r o g e n peroxide [24 cc of 3% (w/v) solution] every 3 days; t e m p e r a t u r e 303°K; (f~) products V-VII; (e) products VIII-X
ferences were found in quantitative composition only. However, the product obtained in the presence of enzyme has shown slight optical activity, whereas the product from the blank test was optically inactive. The product ratio V-VII/VIII-X was always 30-40% higher for the enzymatic than for the nonenzymatic process. The formation of products V-VII and VIII-X with increasing reaction time is shown in Figure 3. Compounds V-VII were found to be rather stable in the reaction conditions when a low enzyme concentration was employed. The quantity of products V-VII rapidly decreases with increasing peroxidase concentration. Unidentified new products with longer retention time in GLC appear (see Figure 4). At the same time, the quantity of addition products V|I1-X increases only slightly with increasing enzyme concentration. Work on the isolation and identification of products formed at high enzyme concentration is now in progress.
Conclusions The absence of diols 1I and III among the products of transformation of citronellol by a horseradish peroxidase-hydrogen peroxide system seems to exclude the participation of singlet oxygen as an active intermediate, j2 The predominant reaction in this system is epoxidation of the C-C double bond followed by
438 EnzymeMicrob.Technol.,1989,vol.11,July
2'o
4'0
e'o
e~o
1~o
1~o
PEROXIDAS E [U] Figure 4 Product distribution as a function of enzyme concentration. Conditions: 10 cc of borate buffer (pH 9), 20 cc of methanol, 1 mM of citronellol, 1 mM of ascorbic acid, 40 mM of h y d r o g e n peroxide [48 cc of 3% (w/v) solution]; t e m p e r a t u r e 303°K; time 4 days; (c~) products V-VII; (v) products VIII-X; (e) unidentified products
epoxide solvolysis. An accompanying side reaction is solvent addition to the double bond.
Acknowledgement This research was supported by the Institute of Technical Biochemistry, Technical University of L6d~,, grant C.P.B.P. 04.11/2.39.
References I 2 3 4 5 6 7 8 9
10 II 12
Ohloff, G., Klein, E. and Schenck, O. Angew. C/tent. 1961, 73, 578 Ohloff, G. and Lienhard, B. Heir. Chim. Acta, 1965, 48, 182 Denny, R. W. and Nickon, A. Org. Reactions, 1973, 20, 133 Teng, J. I. and Smith, L. L. J. Am. Chem. Sot'. 1973, 95, 4060 Teng, J. I. and Smith, L. L. Bioorg. Chem. 1976, 5, 99 Yu, P. H. and Tan, L. J. Steroid Biochem. 1977, 8, 825 Khan, A. U. J. Am. Chem. Soc. 1983, 105, 7195 Kenten, R. H. and Mann, P. J. G. Biochem. J. 1954, 57, 347 Bergmeyer, H. U., G a w e h n , K. and Grassl, M. Methods t~/' Enzymatic Analysis. Verlag Chemie Weinheim, Academic Press, 1974, 1 , 4 9 4 Saunders, B. C. Inorganic BiochemisoT Elsevier, Amsterdam, 1973, p. 991 Klibanov, A. M., Berman, Z. and Alberti, B. N. J. Am. Chem. Soc. 1981, 103, 6263 Metelica, D. 1. Aktivacija kisloroda fi, rmentnimi sistemami N a u k a , Moscow, 1982, p. 61