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Lipase catalysed synthesis of sebacic and phthalic esters
Enzyme and Microbial Technology 33 (2003) 952–957
Lipase catalysed synthesis of sebacic and phthalic esters Stanisław Gryglewicz∗ Department of Chemistry, Wrocław University of Technology, ul. Gdañska7/9, 50-344 Wrocław, Poland Received 4 December 2002; received in revised form 7 July 2003; accepted 7 July 2003
Abstract The enzymatic synthesis and hydrolysis of alkyl sebacates and o-, m-, p-phthalates were studied. Biosyntheses were conducted through alcoholysis of dimethyl phthalates and dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol in a solvent-free medium, using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM) and Porcine pancreas (PPL). It was found that the synthesis and hydrolysis of sebacic acid esters were characterised by a satisfactory rate, however, by low enantioselectivity. The yield of synthesis of di-3,5,5-trimethylhexyl sebacate catalysed by Novozym 435 at 50 ◦ C was 84%, after 20 h of reaction. The degree of conversion, 62.9% after 350 h, was obtained for alcoholysis reaction of dimethyl m-phthalate with 3,5,5-trimethylhexanol. For the enzymes used, no activity was detected at all on both the synthesis and hydrolysis of di-2-ethylhexyl o-phthalate and di-3,5,5-trimethylhexyl o-phthalate. © 2003 Elsevier Inc. All rights reserved. Keywords: Alcoholysis; Candida antarctica; Hydrolysis; Phthalate; Porcine pancreas; Rhizomucor miehei; Sebacate
1. Introduction The use of biocatalysts has found widespread application in organic chemistry over the last decade [1]. Triacylglycerol hydrolases [2] known as lipases are of special interest. They are enzymes capable of catalysing cleavage ester bonds in water as well as in non-conventional medium as organic solvents, supercritical fluids and compressed gasses [3]. Lipases catalyse the hydrolysis reaction of synthetic esters as well as their synthesis and transesterification [4]. Many reactions catalysed by lipases are characterised by high stereospecificity, enantioselectivity and substrate specificity [5]. This allows to produce pure stereoisomers of definite compounds on a large scale [6] which is of great importance in the production of food [7,8], flavour materials [9,11] and modern insecticides [10] and in the pharmaceutical industry [12,13]. This is particularly significant considering the fact that different stereoisomers exhibit different physiological influences, some of which are undesirable. In many cases, lipases show high catalytic efficiency and require milder reaction conditions which reduces energy requirements and damage of reaction products. Usually, synthesis or transesterification of esters catalysed by enzymes are carried out in organic solvents. The type of solvent determines the rate and selectivity of the reaction ∗
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[14–16]. Physicochemical properties of substrates and products of the reaction often make it possible to take place in a solvent-free system [17]. This way the rate of reaction can be increased by increasing the concentration of substrates. Moreover, the product separation becomes easier. The selection of the acyl donor is also very important. It is not always possible to carry out a direct biosynthesis of esters starting from alcohol and carboxylic acid. Aromatic acids and short chain carboxylic acids for an example, can lead to the deactivation of enzymes [18,19]. Preparation of esters through the alcoholysis of carboxylic acid methyl esters by respective alcohols permits to overcome this drawback. Since no water is formed, the rate of the side reaction of hydrolysis is reduced. The volatile methanol leaving the reaction environment makes the process irreversible [20,21]. Transesterification of vinyl and oxime esters [22] as acyl donors gives a similar result. The aim of this work was to evaluate the suitability of lipases (EC 3.1.1.3) as catalysts in the synthesis and hydrolysis of di-2-ethylhexyl and 3,5,5-trimethylhexyl phthalates and sebacates. Esters of these chiral alcohols are produced on a large scale in the industry and are widely used as plasticizers of polymers [23]. The use of racemic compounds in particular, raises justified fears. R-(−)-2-Ethylhexanoic acid which is a natural metabolite of R-(−)-2-ethylhexanol exhibits a strong teratogenic activity in the organism of mammals. This implies a much interest in the asymmetric synthesis of S-(+)-2-ethylhexanol.
S. Gryglewicz / Enzyme and Microbial Technology 33 (2003) 952–957
2. Materials and methods
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2 ml of toluene. The obtained extracts were subjected to gas chromatographic analysis.
2.1. Materials 2.4. GC analysis Novozym 435 (Candida antarctica lipase) and Lipozyme IM (Rhizomucor miehei lipase) were a gift from Novo Nordisk. Type II crude lipase from Porcine pancreas (PPL) was obtained from Sigma. Dimethyl phthalates, dimethyl sebacate, isooctane, 2-ethylhexanol, 3,5,5-trimethylhexanol were purchased from Aldrich. The chemicals used were of analytical grade. Calcium methoxide was obtained by a direct reaction of metallic calcium with methanol. Di-2-ethylhexyl and di-3,5,5-trimethylhexyl sebacates and phthalates were synthesised by the transesterification of appropriate dimethyl esters with alcohols. The calcium methoxide was used as the catalyst in the alcoholysis reaction. The synthesis of these esters was conducted in a glass reactor stopped with a Dean-Stark cup. The formed methanol in the course of reaction, was continuously removed by azeotropic distillation with isooctane, in order to obtain a high yield. When the process was over, the solvent and unreacted substrates were separated by distillation (180 ◦ C, 5 mmHg). 2.2. Biosynthesis of esters The synthesis of esters catalysed by enzymes was carried out as follows: 0.05 mole of 2-ethylhexanol or 3,5,5-trimethylhexanol, 0.025 mole of appropriate dimethyl ester of dicarboxylic acid and 5 wt.% of a biocatalyst in relation to the mass of substrates were placed in an Erlenmeyer flask. The synthesis was conducted in a thermostatic shaker in open glass flask which enables the forming methanol to be removed from the reaction environment effectively. The reaction temperature was 50 ◦ C. In alcoholysis reactions of dimethyl phthalates conducted in toluene, a substrate ratio of 1:9 was maintained. In the course of the reaction, the samples were taken for gas chromatographic analysis at different time intervals to evaluate the progress of the reaction. The process was ended by removing the enzyme through filtration. Before reaction, the organic reagents and lipases were subjected to equilibration procedure of water activity to an aw = 0.9. For this purpose, the system was kept for 72 h in a closed vessel above a saturated solution of zinc sulphate of known water activity [24].
GC analysis was performed using a HP 5890II gas chromatograph with a capillary column (HP-5, 30 m × 0.32 mm) and flame ionisation detector (FID). Nitrogen was used as the carrier gas. The column temperature was programmed from 70 to 280 ◦ C at 20 ◦ C/min after an initial 1 min isothermal period. The split ratio was 5:1. The inlet and detector were set at 250 and 280 ◦ C, respectively. Each analysis was repeated three times. 2.5. Enantioselectivity The enantiomeric excess (ee) was determined by gas chromatography and expressed as the ratio of A − B/A + B, where A and B are peaks of individual enantiomers. A HP Chiral-20 capillary column (25 m × 0.25 mm) was used with nitrogen as the carrier gas. The column temperature was programmed from 50 to 150 ◦ C at 5 ◦ C/min after an initial 3 min isothermal period. Sample injection was made in the split mode of 1:50. Results are an average of two independent determinations with standard deviations less than 5%. Retention time of peaks were as follows: R-(−)-2-ethylhexanol, 22.72 min; S-(+)-2-ethylhexanol, 22.86 min; (+)-3,5,5-trimethylhexanol, 23.54 min, (−)-3,5,5-trimethylhexanol, 23.67 min. The enantioselectivity (E) was determined according to Chen’s formulas [25] for remaining chiral substrate: E = ln[(1 − c)(1 − ee(S)/ln[(1 − c)(1 + ee(S)] where c is the degree of conversion. The measurements of optical rotation of remaining alcohols separated from the reaction products through distillation were performed (in neat) using a Polamat A apparatus produced by Carl Zeiss Jenna Company. This enables the determination of the absolute configuration of individual enantiomers of 2-ethylhexanol on the bases of data reported previously [26]. The (−)-2-ethylhexanol possess R configuration.
3. Results and discussion
2.3. Biohydrolysis of esters
3.1. Sebacates
A mixture of 200 mg of ester to be hydrolysed, 50 mg of enzymatic preparation and 10 ml of phosphate buffer was placed in a round bottom flask. The reaction was conducted at 50 ◦ C. The reaction mixture was agitated on a rotary shaker rotated at a speed of 60 revolution/min. When the process was over, the products were cooled to room temperature and the organic components were extracted with
In Fig. 1, the conversion degree versus reaction time for di-2-ethylhexyl sebacate and di-3,5,5-trimethylhexyl sebacate synthesis catalysed by various lipases is demonstrated. As can be seen, the rate of the lipase catalysed transesterification reaction of dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol was fast. The degree of conversion was 84 and 94%, respectively, after 20 h of reaction
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S. Gryglewicz / Enzyme and Microbial Technology 33 (2003) 952–957 Table 2 Activity and enantioselectivity of lipases in sebacic and phthalic esters hydrolysis Substrate
Fig. 1. Kinetic of 2-ethylhexyl (—) and 3,5,5-trimethylhexyl (– –) sebacates synthesis catalysed by lipases: (䊉) Novozym 435, (䉱) Lipozyme IM, (䉬) PPL.
conducted in the presence Novozym 435. In the reaction catalysed by Lipozyme IM after 20 h 89% degree of conversion was achieved for 2-ethylhexanol and 78% for 3,5,5trimethylhexanol. The free lipase from Porcine pancreas gave the lowest conversion in the synthesis. Only 21 and 32% conversion degrees were observed, respectively. The above results indicate that 3,5,5-trimethylhexanol is a more reactive alcohol than 2-ethylhexanol in the studied reactions. The enantioselectivity of sebacates synthesis, as can be seen in Table 1, is relatively low. It is assumed that the reactions suitable for the resolution of enantiomers are characterised by the E value above 20, whereas nonselective reactions shows E close to 1. The enantioselectivity of the lipases used in sebacates synthesis did not exceed value of 1.7. Both the enanantioselectivity and the enantiomeric excess were the highest for the alcoholysis of dimethyl sebacate with 2-ethylhexanol catalysed by lipase from Porcine pancreas.
SeEH2 SeEH2 SeEH2 SeTMH2 SeTMH2 SeTMH2 m-PEH2 m-PEH2 m-PEH2 m-PTMH2 m-PTMH2 m-PTMH2 p-PEH2 p-PEH2 p-PEH2 p-PTMH2 p-PTMH2 p-PTMH2
Lipase
Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL
Reaction Time (h)
c (mol%)
ee (S) (%)
E
3 3 3 3 3 3 200 200 200 200 200 200 200 200 200 200 200 200
25.0 11.9 10.1 65.2 24.9 17.2 4.4 2.0 1.1 8.5 4.6 2.2 3.0 1.5 1.0 4.4 3.4 2.0
11.7 18.8 20.9 5.5 11.2 13.9 6.1 8.9 10.1 5.4 7.2 5.1 5.0 4.1 8.2 3.1 2.3 7.3
1.3 1.5 1.6 1.2 1.4 1.4 1.1 1.2 1.2 1.1 1.2 1.1 1.1 1.1 1.1 1.0 1.0 1.1
SeEH2 : di-2-ethylhexyl sebacate; SeTMH2 : di-3,5,5-trimethylhexyl sebacate; m,p-PEH2 : di-2-ethylhexyl m,p-phthalate; m,p-PTMH2 : di-3,5,5trimethylhexyl m,p-phthalate; c: conversion; ee (P): enantiomeric excess of (+)-2-ethylhexanol or (−)-3,5,5-trimethylhexanol.
The catalytic activity and enantioselectivity of the lipases used in the hydrolysis reaction of racemic di-2-ethylhexyl sebacate and di-3,5,5-trimethylhexyl sebacate are shown in Table 2. The hydrolysis of both sebacates proceeded as rapidly as their synthesis did. After 3 h of reaction, the degree of hydrolysis of di-3,5,5-trimethylhexyl sebacate and
Table 1 Activity and enantioselectivity of lipases in sebacic and phthalic esters synthesis Starting material
Reaction
Acid donor
Alcohol
Lipase
Time (h)
c (mol%)
ee (S) (%)
E
SeMe2 SeMe2 SeMe2 SeMe2 SeMe2 SeMe2 m-PMe2 m-PMe2 m-PMe2 m-PMe2 m-PMe2 m-PMe2 p-PMe2 p-PMe2 p-PMe2 p-PMe2 p-PMe2 p-PMe2
EH EH EH TMH TMH TMH EH EH EH TMH TMH TMH EH EH EH TMH TMH TMH
Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL
10 10 150 10 10 70 350 350 350 350 350 350 350 350 350 350 350 350
68.1 61.0 56.1 77.8 72.9 59.0 29.8 19.2 4.3 62.9 35.1 6.3 3.9 2.3 1.4 3.9 5.1 2.3
0.0 9.2 21.3 0.0 0.0 16.7 1.4 15.5 – 27.4 9.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0
1.0 1.2 1.7 1.0 1.0 1.4 1.4 5.5 – 1.4 1.6 − – – – – – –
EH: 2-ethylhexanol; TMH: 3,5,5-trimethylhexanol; SeMe2 : dimethyl sebacate; m-PMe2 : dimethyl m-phthalate; p-PMe2 : dimethyl p-phthalate; c: conversion; ee (S): enantiomeric excess of (−)-2-ethylhexanol or (+)-3,5,5-trimethylhexanol.
S. Gryglewicz / Enzyme and Microbial Technology 33 (2003) 952–957
Fig. 2. Kinetic of 2-ethylhexyl (—) and 3,5,5-trimethylhexyl (– –) m-phthalates synthesis catalyzed by lipases: (䊉) Novozym 435, (䉱) Lipozyme IM, (䉬) PPL.
di-2-ethylhexyl sebacate in the presence of Novozym 435 amounted to 65.2 and 25.0%, respectively. Again, PPL appeared to be the least active. The hydrolysis of di-2-ethylhexyl sebacate was slower than that of di-3,5,5-trimethylhexyl sebacate. Similarly as in the case of biosynthesis, this could be attributed to steric hindrance differences. The enantioselectivity of lipase-catalysed hydrolysis reactions was poor. The E value was not higher than 1.6. The synthesis of sebacates and their hydrolysis reactions showed a preference for (+)-2-ethylhexanol and (−)-3,5,5-trimethylhexanol. 3.2. Phthalates The results of biosynthesis and biohydrolysis of 3,5,5-trimethylhexyl and 2-ethylhexyl o-, m-, and p- phthalates are given in Tables 1 and 2. The catalytic activity of the lipases used in synthesis of phthalates by alcoholysis is illustrated in Fig. 2. It is worth noting that the rate of transesterification of dimethyl phthalates compared to dimethyl sebacate was lower. The highest conversion was achieved for the synthesis of m-phthalates, i.e., 63% for 3,5,5-trimethylhexanol and 30% for 2-ethylhexanol. The reaction time was 350 h. The results obtained in this work prove that lipase-catalysed alcoholysis of aryl acid esters compared to aliphatic acid esters was much slower. This is consistent with the finding previously reported [17]. In the alcoholysis reaction of dimethyl phthalates, 3,5,5-trimethylhexanol appeared to be more reactive than 2-ethylhexanol as was already observed for dimethyl sebacates. The lipase-catalysed alcoholysis of o-dimethyl phthalates proceeds for both alcohols used to a very small extent not higher then 4% despite long (350 h) of reaction time (data not displayed). It seems that vicinally placed ester groups in the aromatic ring can effectively retard the progress of reaction. It is rather certain that the difference in reactivity between o- and m-dimethyl phthalates did not arise from diffusional limitations because in both case the reagents form a solution of similar viscosity. Taking into account this fact,
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we can confirm a high regioselectivity of enzymatic catalysis. No similar effect was observed when the alcoholysis reaction was chemically catalysed with calcium methoxide. Such syntheses were also conducted, however, no difference in the reactivity between o- and m- dimethyl phthalates was observed. The alcoholysis of dimethyl p-phthalates with 3,5,5-trimethylhexanol and 2-eythylhexanol can hardly be said to have succeeded. For the synthesis of di-3,5,5-trimethylhexyl p-phthalate, the highest conversion was found to be only 5%. Taking into account the time of reaction, i.e. 350 h, the extent of conversion was very low. The possible reason for this is that p-dimethyl phthalate is a solid, hardly soluble in both 3,5,5-trimethylhexanol and 2-ethylhexanol and the enzyme preparation is also in a solid form at a reaction temperature, 50 ◦ C. Therefore, it is hard to expect a marked conversion where the reagents are in three separate phases. Moreover, the formed products are hardly soluble in the reaction mixture and as a result could block the active sites of enzyme. On the other hand, the alcoholysis of dimethyl p-phthalates catalysed with calcium methoxide was successfully conducted in the temperature range of 100–120 ◦ C. In this case the solubility of dimethyl p-phthalates in the reaction mixture was quite high and the rate of reaction was not hindered. To explain the difference in the reactivity of m- and p-dimethyl phthalates, the alcoholysis reaction of these esters by 2-ethylhexanol and 3,5,5-trimethylhexanol in toluene was carried out. The results were given in Table 3. As a result of using toluene as a solvent, the substrates formed a homogeneous solution. It was found that the reactivity of m- and p-dimethyl phthalates was comparable in these conditions. After 180 h for all reactions the conversion degree of 2-ethylhexanol and 3,5,5-trimethylhexanol was in the range of 35–45%. Prolongation of reaction time does not lead to an enhanced yield, probably, due to reaching the equilibrium by the reaction system. When toluene was used, the reactor was hermetically closed. Thus, the methanol formed could not leave the reaction environment. In the case of o-dimethyl phthalate, using toluene as solvent, nothing changed basically, the degree of conversion did not exceed 2%.
Table 3 Activity and enantioselectivity of Novozym 435 in phthalic esters synthesis performed in toluene solution Starting material
Reaction
Acid donor
Alcohol
Time (h)
c (mol%)
ee (S) (%)
E
m-PMe2 m-PMe2 p-PMe2 p-PMe2
EH TMH EH TMH
180 180 180 180
37.1 44.2 36.0 41.0
15.3 11.0 14.0 9.1
1.9 1.4 2.0 1.4
EH: 2-ethylhexanol; TMH: 3,5,5-trimethylhexanol; m-PMe2 : dimethyl m-phthalate; p-PMe2 : dimethyl p-phthalate; c: conversion; ee (S): enantiomeric excess of (−)-2-ethylhexanol or (+)-3,5,5-trimethylhexanol.
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S. Gryglewicz / Enzyme and Microbial Technology 33 (2003) 952–957
Generally, the enantioselectivity of the enzyme used in the phthalate ester synthesis is relatively low. The Lipozyme IM catalysed alcoholysis of m-dimethyl phthalate with 2-ethylhexanol was characterised by the highest enantioselectivity, 5.5. Di-3,5,5-trimethylhexyl and di-2-ethylhexyl phthalates exhibit very high resistance to enzymatic hydrolysis. Despite long reaction time (200 h), the hydrolysis degree which was calculated as the amount of alcohol released, did not exceed 8.5%. It is clearly seen from Table 2 that o-phthalates did not undergo enzymatic hydrolysis at all. Valiente et al. [27] came to the same conclusion while trying to intensify the hydrolysis of polyester containing o-phthalate units, using hydrolases from Chromobacterium viscosum. o-Diethyl phthalate used as a model compound exhibited the same behaviour. Esters of different isomers of salicylic acid were also resistant to enzymatic hydrolysis [28]. The resistance of o-salicylic acid esters to hydrolysis was 10–20 times higher compared to m- and p-isomers. For m-phthalates, a small extent of hydrolysis after 200 h was observed. A maximum conversion, 8.5%, was obtained for di-3,5,5-trimethylhexyl in the presence of Novozym 435. Moreover, the enzymatic hydrolysis of p-phthalates gave a much lower yield. This is probably a result of the multi-phase system, which is difficult to homogenise like in the case of synthesis. An insignificant enantioselectivity of enzymatic hydrolysis was observed. For all the hydrolysis reactions, the E value was close to 1. As expected the released enantiomers were preferentially that of (−)-3,5,5-trimethylhexanol and (+)-2-ethylhexanol.
4. Conclusion The enzyme preparations used, i.e. Novozym 435 and Lipozyme IM, were found to be effective catalysts in both the synthesis and the hydrolysis reactions of di-2-ethylhexyl sebacate and di-3,5,5-trimethylhexyl sebacate. On the contrary, the synthesis and hydrolysis of di-2-ethylhexyl phthalates and di-3,5,5-trimethylhexyl phthalates were characterised by rather slow reaction rate. Only the synthesis of di-3,5,5-trimethylhexyl m-phthalate and di-2-ethylhexyl m-phthalate were carried out with a mild satisfactory yield. Much lower rate of both the synthesis and hydrolysis of phthalates in comparison with sebacates is probably due to the resonance effect of aryl acid ester molecules. Moreover, the steric hindrances especially in o-phthalates and low solubility of p-phthalates also reduce the extent of both reactions. It was found that the reaction rate for both m- and p-dimethyl phthalates with alkanols was similar when the synthesis of dialkyl phthalates was carried out in toluene solution. Immobilised enzyme preparations, such as Novozym 435 and Lipozyme IM show much higher activity in alcoholysis reaction in organic environment than the free lipase from Porcine pancreas. The difference in catalytic activity of the
enzymes used was markedly reduced in the hydrolysis reaction carried out in an aqueous medium. Most reactions with 2-ethylhexanol were slower but more selective in comparison with reactions involving 3,5,5-trimethylhexanol. The favourite alcohols in alcoholysis reactions were S-(+)-2-ethylhexanol and (−)-3,5,5-trimethylhexanol. As expected, the same enantiomers were preferentially released in the process of hydrolysis.
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Lipase catalysed synthesis of sebacic and phthalic esters Stanisław Gryglewicz∗ Department of Chemistry, Wrocław University of Technology, ul. Gdañska7/9, 50-344 Wrocław, Poland Received 4 December 2002; received in revised form 7 July 2003; accepted 7 July 2003
Abstract The enzymatic synthesis and hydrolysis of alkyl sebacates and o-, m-, p-phthalates were studied. Biosyntheses were conducted through alcoholysis of dimethyl phthalates and dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol in a solvent-free medium, using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM) and Porcine pancreas (PPL). It was found that the synthesis and hydrolysis of sebacic acid esters were characterised by a satisfactory rate, however, by low enantioselectivity. The yield of synthesis of di-3,5,5-trimethylhexyl sebacate catalysed by Novozym 435 at 50 ◦ C was 84%, after 20 h of reaction. The degree of conversion, 62.9% after 350 h, was obtained for alcoholysis reaction of dimethyl m-phthalate with 3,5,5-trimethylhexanol. For the enzymes used, no activity was detected at all on both the synthesis and hydrolysis of di-2-ethylhexyl o-phthalate and di-3,5,5-trimethylhexyl o-phthalate. © 2003 Elsevier Inc. All rights reserved. Keywords: Alcoholysis; Candida antarctica; Hydrolysis; Phthalate; Porcine pancreas; Rhizomucor miehei; Sebacate
1. Introduction The use of biocatalysts has found widespread application in organic chemistry over the last decade [1]. Triacylglycerol hydrolases [2] known as lipases are of special interest. They are enzymes capable of catalysing cleavage ester bonds in water as well as in non-conventional medium as organic solvents, supercritical fluids and compressed gasses [3]. Lipases catalyse the hydrolysis reaction of synthetic esters as well as their synthesis and transesterification [4]. Many reactions catalysed by lipases are characterised by high stereospecificity, enantioselectivity and substrate specificity [5]. This allows to produce pure stereoisomers of definite compounds on a large scale [6] which is of great importance in the production of food [7,8], flavour materials [9,11] and modern insecticides [10] and in the pharmaceutical industry [12,13]. This is particularly significant considering the fact that different stereoisomers exhibit different physiological influences, some of which are undesirable. In many cases, lipases show high catalytic efficiency and require milder reaction conditions which reduces energy requirements and damage of reaction products. Usually, synthesis or transesterification of esters catalysed by enzymes are carried out in organic solvents. The type of solvent determines the rate and selectivity of the reaction ∗
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[14–16]. Physicochemical properties of substrates and products of the reaction often make it possible to take place in a solvent-free system [17]. This way the rate of reaction can be increased by increasing the concentration of substrates. Moreover, the product separation becomes easier. The selection of the acyl donor is also very important. It is not always possible to carry out a direct biosynthesis of esters starting from alcohol and carboxylic acid. Aromatic acids and short chain carboxylic acids for an example, can lead to the deactivation of enzymes [18,19]. Preparation of esters through the alcoholysis of carboxylic acid methyl esters by respective alcohols permits to overcome this drawback. Since no water is formed, the rate of the side reaction of hydrolysis is reduced. The volatile methanol leaving the reaction environment makes the process irreversible [20,21]. Transesterification of vinyl and oxime esters [22] as acyl donors gives a similar result. The aim of this work was to evaluate the suitability of lipases (EC 3.1.1.3) as catalysts in the synthesis and hydrolysis of di-2-ethylhexyl and 3,5,5-trimethylhexyl phthalates and sebacates. Esters of these chiral alcohols are produced on a large scale in the industry and are widely used as plasticizers of polymers [23]. The use of racemic compounds in particular, raises justified fears. R-(−)-2-Ethylhexanoic acid which is a natural metabolite of R-(−)-2-ethylhexanol exhibits a strong teratogenic activity in the organism of mammals. This implies a much interest in the asymmetric synthesis of S-(+)-2-ethylhexanol.
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2. Materials and methods
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2 ml of toluene. The obtained extracts were subjected to gas chromatographic analysis.
2.1. Materials 2.4. GC analysis Novozym 435 (Candida antarctica lipase) and Lipozyme IM (Rhizomucor miehei lipase) were a gift from Novo Nordisk. Type II crude lipase from Porcine pancreas (PPL) was obtained from Sigma. Dimethyl phthalates, dimethyl sebacate, isooctane, 2-ethylhexanol, 3,5,5-trimethylhexanol were purchased from Aldrich. The chemicals used were of analytical grade. Calcium methoxide was obtained by a direct reaction of metallic calcium with methanol. Di-2-ethylhexyl and di-3,5,5-trimethylhexyl sebacates and phthalates were synthesised by the transesterification of appropriate dimethyl esters with alcohols. The calcium methoxide was used as the catalyst in the alcoholysis reaction. The synthesis of these esters was conducted in a glass reactor stopped with a Dean-Stark cup. The formed methanol in the course of reaction, was continuously removed by azeotropic distillation with isooctane, in order to obtain a high yield. When the process was over, the solvent and unreacted substrates were separated by distillation (180 ◦ C, 5 mmHg). 2.2. Biosynthesis of esters The synthesis of esters catalysed by enzymes was carried out as follows: 0.05 mole of 2-ethylhexanol or 3,5,5-trimethylhexanol, 0.025 mole of appropriate dimethyl ester of dicarboxylic acid and 5 wt.% of a biocatalyst in relation to the mass of substrates were placed in an Erlenmeyer flask. The synthesis was conducted in a thermostatic shaker in open glass flask which enables the forming methanol to be removed from the reaction environment effectively. The reaction temperature was 50 ◦ C. In alcoholysis reactions of dimethyl phthalates conducted in toluene, a substrate ratio of 1:9 was maintained. In the course of the reaction, the samples were taken for gas chromatographic analysis at different time intervals to evaluate the progress of the reaction. The process was ended by removing the enzyme through filtration. Before reaction, the organic reagents and lipases were subjected to equilibration procedure of water activity to an aw = 0.9. For this purpose, the system was kept for 72 h in a closed vessel above a saturated solution of zinc sulphate of known water activity [24].
GC analysis was performed using a HP 5890II gas chromatograph with a capillary column (HP-5, 30 m × 0.32 mm) and flame ionisation detector (FID). Nitrogen was used as the carrier gas. The column temperature was programmed from 70 to 280 ◦ C at 20 ◦ C/min after an initial 1 min isothermal period. The split ratio was 5:1. The inlet and detector were set at 250 and 280 ◦ C, respectively. Each analysis was repeated three times. 2.5. Enantioselectivity The enantiomeric excess (ee) was determined by gas chromatography and expressed as the ratio of A − B/A + B, where A and B are peaks of individual enantiomers. A HP Chiral-20 capillary column (25 m × 0.25 mm) was used with nitrogen as the carrier gas. The column temperature was programmed from 50 to 150 ◦ C at 5 ◦ C/min after an initial 3 min isothermal period. Sample injection was made in the split mode of 1:50. Results are an average of two independent determinations with standard deviations less than 5%. Retention time of peaks were as follows: R-(−)-2-ethylhexanol, 22.72 min; S-(+)-2-ethylhexanol, 22.86 min; (+)-3,5,5-trimethylhexanol, 23.54 min, (−)-3,5,5-trimethylhexanol, 23.67 min. The enantioselectivity (E) was determined according to Chen’s formulas [25] for remaining chiral substrate: E = ln[(1 − c)(1 − ee(S)/ln[(1 − c)(1 + ee(S)] where c is the degree of conversion. The measurements of optical rotation of remaining alcohols separated from the reaction products through distillation were performed (in neat) using a Polamat A apparatus produced by Carl Zeiss Jenna Company. This enables the determination of the absolute configuration of individual enantiomers of 2-ethylhexanol on the bases of data reported previously [26]. The (−)-2-ethylhexanol possess R configuration.
3. Results and discussion
2.3. Biohydrolysis of esters
3.1. Sebacates
A mixture of 200 mg of ester to be hydrolysed, 50 mg of enzymatic preparation and 10 ml of phosphate buffer was placed in a round bottom flask. The reaction was conducted at 50 ◦ C. The reaction mixture was agitated on a rotary shaker rotated at a speed of 60 revolution/min. When the process was over, the products were cooled to room temperature and the organic components were extracted with
In Fig. 1, the conversion degree versus reaction time for di-2-ethylhexyl sebacate and di-3,5,5-trimethylhexyl sebacate synthesis catalysed by various lipases is demonstrated. As can be seen, the rate of the lipase catalysed transesterification reaction of dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol was fast. The degree of conversion was 84 and 94%, respectively, after 20 h of reaction
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S. Gryglewicz / Enzyme and Microbial Technology 33 (2003) 952–957 Table 2 Activity and enantioselectivity of lipases in sebacic and phthalic esters hydrolysis Substrate
Fig. 1. Kinetic of 2-ethylhexyl (—) and 3,5,5-trimethylhexyl (– –) sebacates synthesis catalysed by lipases: (䊉) Novozym 435, (䉱) Lipozyme IM, (䉬) PPL.
conducted in the presence Novozym 435. In the reaction catalysed by Lipozyme IM after 20 h 89% degree of conversion was achieved for 2-ethylhexanol and 78% for 3,5,5trimethylhexanol. The free lipase from Porcine pancreas gave the lowest conversion in the synthesis. Only 21 and 32% conversion degrees were observed, respectively. The above results indicate that 3,5,5-trimethylhexanol is a more reactive alcohol than 2-ethylhexanol in the studied reactions. The enantioselectivity of sebacates synthesis, as can be seen in Table 1, is relatively low. It is assumed that the reactions suitable for the resolution of enantiomers are characterised by the E value above 20, whereas nonselective reactions shows E close to 1. The enantioselectivity of the lipases used in sebacates synthesis did not exceed value of 1.7. Both the enanantioselectivity and the enantiomeric excess were the highest for the alcoholysis of dimethyl sebacate with 2-ethylhexanol catalysed by lipase from Porcine pancreas.
SeEH2 SeEH2 SeEH2 SeTMH2 SeTMH2 SeTMH2 m-PEH2 m-PEH2 m-PEH2 m-PTMH2 m-PTMH2 m-PTMH2 p-PEH2 p-PEH2 p-PEH2 p-PTMH2 p-PTMH2 p-PTMH2
Lipase
Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL
Reaction Time (h)
c (mol%)
ee (S) (%)
E
3 3 3 3 3 3 200 200 200 200 200 200 200 200 200 200 200 200
25.0 11.9 10.1 65.2 24.9 17.2 4.4 2.0 1.1 8.5 4.6 2.2 3.0 1.5 1.0 4.4 3.4 2.0
11.7 18.8 20.9 5.5 11.2 13.9 6.1 8.9 10.1 5.4 7.2 5.1 5.0 4.1 8.2 3.1 2.3 7.3
1.3 1.5 1.6 1.2 1.4 1.4 1.1 1.2 1.2 1.1 1.2 1.1 1.1 1.1 1.1 1.0 1.0 1.1
SeEH2 : di-2-ethylhexyl sebacate; SeTMH2 : di-3,5,5-trimethylhexyl sebacate; m,p-PEH2 : di-2-ethylhexyl m,p-phthalate; m,p-PTMH2 : di-3,5,5trimethylhexyl m,p-phthalate; c: conversion; ee (P): enantiomeric excess of (+)-2-ethylhexanol or (−)-3,5,5-trimethylhexanol.
The catalytic activity and enantioselectivity of the lipases used in the hydrolysis reaction of racemic di-2-ethylhexyl sebacate and di-3,5,5-trimethylhexyl sebacate are shown in Table 2. The hydrolysis of both sebacates proceeded as rapidly as their synthesis did. After 3 h of reaction, the degree of hydrolysis of di-3,5,5-trimethylhexyl sebacate and
Table 1 Activity and enantioselectivity of lipases in sebacic and phthalic esters synthesis Starting material
Reaction
Acid donor
Alcohol
Lipase
Time (h)
c (mol%)
ee (S) (%)
E
SeMe2 SeMe2 SeMe2 SeMe2 SeMe2 SeMe2 m-PMe2 m-PMe2 m-PMe2 m-PMe2 m-PMe2 m-PMe2 p-PMe2 p-PMe2 p-PMe2 p-PMe2 p-PMe2 p-PMe2
EH EH EH TMH TMH TMH EH EH EH TMH TMH TMH EH EH EH TMH TMH TMH
Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL Novozym 435 Lipozyme IM PPL
10 10 150 10 10 70 350 350 350 350 350 350 350 350 350 350 350 350
68.1 61.0 56.1 77.8 72.9 59.0 29.8 19.2 4.3 62.9 35.1 6.3 3.9 2.3 1.4 3.9 5.1 2.3
0.0 9.2 21.3 0.0 0.0 16.7 1.4 15.5 – 27.4 9.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0
1.0 1.2 1.7 1.0 1.0 1.4 1.4 5.5 – 1.4 1.6 − – – – – – –
EH: 2-ethylhexanol; TMH: 3,5,5-trimethylhexanol; SeMe2 : dimethyl sebacate; m-PMe2 : dimethyl m-phthalate; p-PMe2 : dimethyl p-phthalate; c: conversion; ee (S): enantiomeric excess of (−)-2-ethylhexanol or (+)-3,5,5-trimethylhexanol.
S. Gryglewicz / Enzyme and Microbial Technology 33 (2003) 952–957
Fig. 2. Kinetic of 2-ethylhexyl (—) and 3,5,5-trimethylhexyl (– –) m-phthalates synthesis catalyzed by lipases: (䊉) Novozym 435, (䉱) Lipozyme IM, (䉬) PPL.
di-2-ethylhexyl sebacate in the presence of Novozym 435 amounted to 65.2 and 25.0%, respectively. Again, PPL appeared to be the least active. The hydrolysis of di-2-ethylhexyl sebacate was slower than that of di-3,5,5-trimethylhexyl sebacate. Similarly as in the case of biosynthesis, this could be attributed to steric hindrance differences. The enantioselectivity of lipase-catalysed hydrolysis reactions was poor. The E value was not higher than 1.6. The synthesis of sebacates and their hydrolysis reactions showed a preference for (+)-2-ethylhexanol and (−)-3,5,5-trimethylhexanol. 3.2. Phthalates The results of biosynthesis and biohydrolysis of 3,5,5-trimethylhexyl and 2-ethylhexyl o-, m-, and p- phthalates are given in Tables 1 and 2. The catalytic activity of the lipases used in synthesis of phthalates by alcoholysis is illustrated in Fig. 2. It is worth noting that the rate of transesterification of dimethyl phthalates compared to dimethyl sebacate was lower. The highest conversion was achieved for the synthesis of m-phthalates, i.e., 63% for 3,5,5-trimethylhexanol and 30% for 2-ethylhexanol. The reaction time was 350 h. The results obtained in this work prove that lipase-catalysed alcoholysis of aryl acid esters compared to aliphatic acid esters was much slower. This is consistent with the finding previously reported [17]. In the alcoholysis reaction of dimethyl phthalates, 3,5,5-trimethylhexanol appeared to be more reactive than 2-ethylhexanol as was already observed for dimethyl sebacates. The lipase-catalysed alcoholysis of o-dimethyl phthalates proceeds for both alcohols used to a very small extent not higher then 4% despite long (350 h) of reaction time (data not displayed). It seems that vicinally placed ester groups in the aromatic ring can effectively retard the progress of reaction. It is rather certain that the difference in reactivity between o- and m-dimethyl phthalates did not arise from diffusional limitations because in both case the reagents form a solution of similar viscosity. Taking into account this fact,
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we can confirm a high regioselectivity of enzymatic catalysis. No similar effect was observed when the alcoholysis reaction was chemically catalysed with calcium methoxide. Such syntheses were also conducted, however, no difference in the reactivity between o- and m- dimethyl phthalates was observed. The alcoholysis of dimethyl p-phthalates with 3,5,5-trimethylhexanol and 2-eythylhexanol can hardly be said to have succeeded. For the synthesis of di-3,5,5-trimethylhexyl p-phthalate, the highest conversion was found to be only 5%. Taking into account the time of reaction, i.e. 350 h, the extent of conversion was very low. The possible reason for this is that p-dimethyl phthalate is a solid, hardly soluble in both 3,5,5-trimethylhexanol and 2-ethylhexanol and the enzyme preparation is also in a solid form at a reaction temperature, 50 ◦ C. Therefore, it is hard to expect a marked conversion where the reagents are in three separate phases. Moreover, the formed products are hardly soluble in the reaction mixture and as a result could block the active sites of enzyme. On the other hand, the alcoholysis of dimethyl p-phthalates catalysed with calcium methoxide was successfully conducted in the temperature range of 100–120 ◦ C. In this case the solubility of dimethyl p-phthalates in the reaction mixture was quite high and the rate of reaction was not hindered. To explain the difference in the reactivity of m- and p-dimethyl phthalates, the alcoholysis reaction of these esters by 2-ethylhexanol and 3,5,5-trimethylhexanol in toluene was carried out. The results were given in Table 3. As a result of using toluene as a solvent, the substrates formed a homogeneous solution. It was found that the reactivity of m- and p-dimethyl phthalates was comparable in these conditions. After 180 h for all reactions the conversion degree of 2-ethylhexanol and 3,5,5-trimethylhexanol was in the range of 35–45%. Prolongation of reaction time does not lead to an enhanced yield, probably, due to reaching the equilibrium by the reaction system. When toluene was used, the reactor was hermetically closed. Thus, the methanol formed could not leave the reaction environment. In the case of o-dimethyl phthalate, using toluene as solvent, nothing changed basically, the degree of conversion did not exceed 2%.
Table 3 Activity and enantioselectivity of Novozym 435 in phthalic esters synthesis performed in toluene solution Starting material
Reaction
Acid donor
Alcohol
Time (h)
c (mol%)
ee (S) (%)
E
m-PMe2 m-PMe2 p-PMe2 p-PMe2
EH TMH EH TMH
180 180 180 180
37.1 44.2 36.0 41.0
15.3 11.0 14.0 9.1
1.9 1.4 2.0 1.4
EH: 2-ethylhexanol; TMH: 3,5,5-trimethylhexanol; m-PMe2 : dimethyl m-phthalate; p-PMe2 : dimethyl p-phthalate; c: conversion; ee (S): enantiomeric excess of (−)-2-ethylhexanol or (+)-3,5,5-trimethylhexanol.
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S. Gryglewicz / Enzyme and Microbial Technology 33 (2003) 952–957
Generally, the enantioselectivity of the enzyme used in the phthalate ester synthesis is relatively low. The Lipozyme IM catalysed alcoholysis of m-dimethyl phthalate with 2-ethylhexanol was characterised by the highest enantioselectivity, 5.5. Di-3,5,5-trimethylhexyl and di-2-ethylhexyl phthalates exhibit very high resistance to enzymatic hydrolysis. Despite long reaction time (200 h), the hydrolysis degree which was calculated as the amount of alcohol released, did not exceed 8.5%. It is clearly seen from Table 2 that o-phthalates did not undergo enzymatic hydrolysis at all. Valiente et al. [27] came to the same conclusion while trying to intensify the hydrolysis of polyester containing o-phthalate units, using hydrolases from Chromobacterium viscosum. o-Diethyl phthalate used as a model compound exhibited the same behaviour. Esters of different isomers of salicylic acid were also resistant to enzymatic hydrolysis [28]. The resistance of o-salicylic acid esters to hydrolysis was 10–20 times higher compared to m- and p-isomers. For m-phthalates, a small extent of hydrolysis after 200 h was observed. A maximum conversion, 8.5%, was obtained for di-3,5,5-trimethylhexyl in the presence of Novozym 435. Moreover, the enzymatic hydrolysis of p-phthalates gave a much lower yield. This is probably a result of the multi-phase system, which is difficult to homogenise like in the case of synthesis. An insignificant enantioselectivity of enzymatic hydrolysis was observed. For all the hydrolysis reactions, the E value was close to 1. As expected the released enantiomers were preferentially that of (−)-3,5,5-trimethylhexanol and (+)-2-ethylhexanol.
4. Conclusion The enzyme preparations used, i.e. Novozym 435 and Lipozyme IM, were found to be effective catalysts in both the synthesis and the hydrolysis reactions of di-2-ethylhexyl sebacate and di-3,5,5-trimethylhexyl sebacate. On the contrary, the synthesis and hydrolysis of di-2-ethylhexyl phthalates and di-3,5,5-trimethylhexyl phthalates were characterised by rather slow reaction rate. Only the synthesis of di-3,5,5-trimethylhexyl m-phthalate and di-2-ethylhexyl m-phthalate were carried out with a mild satisfactory yield. Much lower rate of both the synthesis and hydrolysis of phthalates in comparison with sebacates is probably due to the resonance effect of aryl acid ester molecules. Moreover, the steric hindrances especially in o-phthalates and low solubility of p-phthalates also reduce the extent of both reactions. It was found that the reaction rate for both m- and p-dimethyl phthalates with alkanols was similar when the synthesis of dialkyl phthalates was carried out in toluene solution. Immobilised enzyme preparations, such as Novozym 435 and Lipozyme IM show much higher activity in alcoholysis reaction in organic environment than the free lipase from Porcine pancreas. The difference in catalytic activity of the
enzymes used was markedly reduced in the hydrolysis reaction carried out in an aqueous medium. Most reactions with 2-ethylhexanol were slower but more selective in comparison with reactions involving 3,5,5-trimethylhexanol. The favourite alcohols in alcoholysis reactions were S-(+)-2-ethylhexanol and (−)-3,5,5-trimethylhexanol. As expected, the same enantiomers were preferentially released in the process of hydrolysis.
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