CA2064676A1 - Immobilized biocatalyst, its preparation and use for ester synthesis in a column reactor - Google Patents
Immobilized biocatalyst, its preparation and use for ester synthesis in a column reactorInfo
- Publication number
- CA2064676A1 CA2064676A1 CA 2064676 CA2064676A CA2064676A1 CA 2064676 A1 CA2064676 A1 CA 2064676A1 CA 2064676 CA2064676 CA 2064676 CA 2064676 A CA2064676 A CA 2064676A CA 2064676 A1 CA2064676 A1 CA 2064676A1
- Authority
- CA
- Canada
- Prior art keywords
- immobilized
- immobilized biocatalyst
- lipase
- carrier
- biocatalyst
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
Abstract
Abstract of the disclosures An immobilized biocatalyst, its preparation and use for ester synthesis in a column reactor The invention relates to a process for the acylation of alcohols, with the exception of tertiary alcohols, using lipases from Pseudomonas, Mucor, Candida, Rhizopus, Penicillium or pig pancreas, which are immobilized on inert carriers (seasand, powdered rock, powdered brick, glass, ceramic powders).
Description
20~76 H~ECHST AKTIENGESELLSCHAFT HOE 91/F 100 Dr. Si/rh Description An immobilized biocatalyst, its preparation and use for ester synthesis in a column reactor The synthesis of esters from carboxylic acids or car-boxylic acid derivatives and alcohols is a task which is central within the scope of organic chemistry. For example, esters are used as fats, oils, emulsifiers, fragrances, pharmaceuticals, plant protection agents, liquid crystal components etc. Ester synthesis generally takes place in the presence of a catalyst. It is known that the catalytic function can be carried out by enzymes.
Many of the abovementioned substances are optically active. To synthesize them it is often simply necessary to have enantiomerically pure starting materials. Theæe can be obtained in some cases from optically pure natural substances but it is o ten necessary to take the route of enantiodifferentiating synthesis starting from racemic or prochiral compounds. Since, in particular, optically active alcohols, carboxylic acids and carboxylic esters have great economic importance, for this reason an economic preparation process for the enantiodifferentiat-ing acylation of alcohols or with carboxylic acids has great importance. The same applies to the stereo- and~or regioselective acylation of mono-, di- or polyols (for example carbohydrates).
Furthermore, enzymes have the advantage that, besides unbranched alcohols and carboxylic acid~, it i8 alRo possible to acylate racemic or prochiral alcohol~ or carboxylic acids regio-, enantio- and diastereoselective-ly under extremely mild conditions so that, for example, the acylation of alcohols takes place in the sen~e of racemate resolution, or that prochiral alcohols undergo 2 ~ 7 ~
asymmetric acylation.
It has already been disclosed that vinyl esters can be transesterified with the addition of alcohols in the presence of solvents such as, for example, tetrahydro-furan under enzymatic catalysis (M. Degueil-Castaing et al., Tetrahedron Letters, Vol. 28, No. 9, pages 953-954, lg87). Lipase from pig pancreas was used as enzyme. No stereoselectivity was observed.
The enzymatic separation of racemic alcohols on the basis of a selective, enzyme-catalyzed transesterification reaction with vinyl esters in the absence of solvents has also been disclosed. The enzymes used are immobilized lipases from pig liver and pancreas, and from the micro-organisms Pseudomonas, Candida, Mucor, Rhizopus and Penicillium (EP 0 321 918).
It is possible to employ continuous enzymatic processes particularly advantageously. The enzymes are generally employed for this in immobilized form. This has the advantage of wide applicability and virtually unlimited scale-up of the process. The prerequisite for this is economic access to suitable carrier materials, and a simple, rapid immobilization process which can be carried out at reasonable cost.
Thus, for example, it has also been disclosed that lipases can be employed for the hydrolysis and trans-esterification of fats, oils and similar compounds immokilized on anionic ion exchangers [M. Mittelbach, J. Am. Oil Chemist's Society, 67, 168-170 (90)].
Certain standard processes are usually employed for the immobilization of enzymes. The enzyme is immobilized on a suitable carrier by adsorption, or ionically or co-valently (I. Chibata, Immobilized Enzymes: Research and Development, Halsted Press, Wiley, New York, 1978 page 5-7; W. Hartmeier: Immobilisierte Biokatalysatoren, Springer ~0~7~
Verlag Berlin Heidelberg, New York, Tokyo 1986 page 14-16).
It has now been found that both the immobilization of hydrolases on inert, inorganic carriers, and the use of the immobilizates for ester synthesi6 in an organic reaction medium can be carried out considerably more simply and economically than with known processes by mixing together the inert, inorganic carrier and the hydrolase in simple form. Inert carriers are defined as tho~e which have no active surface which is necessary in conventional processe~ for the enzyme-carrier connection (ionic or covalent bonding). Thi~ ~imple process has not hitherto been described and has the advantage that the catalytic center of the enzyme i5 not blocked by bond formation.
Hence the invention relates to:
l. An immobilized biocatalyst composed of an inert, inorganic carrier and of a hydrolase which i8 immobilized on this carrier.
2. A biocatalyst carrier as claimed in claim l, com-posed of the materials seasand, powdered rock or brick, glass or ceramic powder.
Many of the abovementioned substances are optically active. To synthesize them it is often simply necessary to have enantiomerically pure starting materials. Theæe can be obtained in some cases from optically pure natural substances but it is o ten necessary to take the route of enantiodifferentiating synthesis starting from racemic or prochiral compounds. Since, in particular, optically active alcohols, carboxylic acids and carboxylic esters have great economic importance, for this reason an economic preparation process for the enantiodifferentiat-ing acylation of alcohols or with carboxylic acids has great importance. The same applies to the stereo- and~or regioselective acylation of mono-, di- or polyols (for example carbohydrates).
Furthermore, enzymes have the advantage that, besides unbranched alcohols and carboxylic acid~, it i8 alRo possible to acylate racemic or prochiral alcohol~ or carboxylic acids regio-, enantio- and diastereoselective-ly under extremely mild conditions so that, for example, the acylation of alcohols takes place in the sen~e of racemate resolution, or that prochiral alcohols undergo 2 ~ 7 ~
asymmetric acylation.
It has already been disclosed that vinyl esters can be transesterified with the addition of alcohols in the presence of solvents such as, for example, tetrahydro-furan under enzymatic catalysis (M. Degueil-Castaing et al., Tetrahedron Letters, Vol. 28, No. 9, pages 953-954, lg87). Lipase from pig pancreas was used as enzyme. No stereoselectivity was observed.
The enzymatic separation of racemic alcohols on the basis of a selective, enzyme-catalyzed transesterification reaction with vinyl esters in the absence of solvents has also been disclosed. The enzymes used are immobilized lipases from pig liver and pancreas, and from the micro-organisms Pseudomonas, Candida, Mucor, Rhizopus and Penicillium (EP 0 321 918).
It is possible to employ continuous enzymatic processes particularly advantageously. The enzymes are generally employed for this in immobilized form. This has the advantage of wide applicability and virtually unlimited scale-up of the process. The prerequisite for this is economic access to suitable carrier materials, and a simple, rapid immobilization process which can be carried out at reasonable cost.
Thus, for example, it has also been disclosed that lipases can be employed for the hydrolysis and trans-esterification of fats, oils and similar compounds immokilized on anionic ion exchangers [M. Mittelbach, J. Am. Oil Chemist's Society, 67, 168-170 (90)].
Certain standard processes are usually employed for the immobilization of enzymes. The enzyme is immobilized on a suitable carrier by adsorption, or ionically or co-valently (I. Chibata, Immobilized Enzymes: Research and Development, Halsted Press, Wiley, New York, 1978 page 5-7; W. Hartmeier: Immobilisierte Biokatalysatoren, Springer ~0~7~
Verlag Berlin Heidelberg, New York, Tokyo 1986 page 14-16).
It has now been found that both the immobilization of hydrolases on inert, inorganic carriers, and the use of the immobilizates for ester synthesi6 in an organic reaction medium can be carried out considerably more simply and economically than with known processes by mixing together the inert, inorganic carrier and the hydrolase in simple form. Inert carriers are defined as tho~e which have no active surface which is necessary in conventional processe~ for the enzyme-carrier connection (ionic or covalent bonding). Thi~ ~imple process has not hitherto been described and has the advantage that the catalytic center of the enzyme i5 not blocked by bond formation.
Hence the invention relates to:
l. An immobilized biocatalyst composed of an inert, inorganic carrier and of a hydrolase which i8 immobilized on this carrier.
2. A biocatalyst carrier as claimed in claim l, com-posed of the materials seasand, powdered rock or brick, glass or ceramic powder.
3. A proce6s for the preparation of an immobilized biocatalyst, which comprises the hydrolase being mixed with an inert, inorganic carrier composed of seasand, powdered rock, powdered brick, glas~ or ceramic powder snd being immobilized thereon.
4. The use of the process for ester synthesis in a column reactor.
The invention is described in detail herein~fter. The invention is furthermore defined by the contents of the cl~ims.
2~6~
The process can be applied to all reactions, i.e. acyl transfer reactions, which can be catalyzed by hydrolases in organic solvents. The possible reactions are either disclosed in the literature or can easily be determined by preliminary tests.
Used as carrier materials for the enzyme immobilization are inert, inorganic materials such as powdered rock, powdered brick, ceramic powders, but preferably seasand.
All the materials are commercially available or c~n be ob~ained very straightforwardly and do not need pre- or after-treatment. However, it is preferable for the carriers also to be comminuted and graded (standard process). Seasand can be bought in the calcined state (Riedel-de Haen).
The enzymes which are used to catalyze the esterification are hydrolases, especially lipases from Mucor, Rhizopus, Penicillium, Pseudomonas or the lipase from pig pancreas, but preferably the lipase from Pseudomonas (lipase P, also called FP or PS, Amano Pharmaceuticals, Nagoya, Japan) or from Candida (for example Sigma Chemicals Co., St. Louis, MO, USA or lipase OF (Meito Sangyo, Nagoya, Japan)).
To immobilize the enzyme, the hydrolase and carrier are mixed, preferably in the dry state.
The particle size of the carrier is a maximum of 2 mm, preferably 0.01 - 2 mm, but particularly 0.1 - 1 ~m, but does not have to be uniform for carrying out the process according to the invention.
The amount of carrier to be loaded with enzyme is chosen depending on the size of the batch, on the reaction time to be expected and on the level of conversion. It can easily be determined by preliminary tests.
The immobilized hydrolase i5 then stable for a surpris-ingly long time with full ens:yme activity under dry storage conditions at room temperature~ The half-life of the immobilized enzyme on use in the laboratory is 2 200 days.
In the acyl transfer reaction, the acyl component which acts as solvent or is dissolved in another organic solvent is cleaved to a ketone, aldehyde, alcohol or water and an acyl-enzyme complex, and the latter reacts with the alcohol (substrate) to be added.
Employed as acyl donors are branched and unbranched carboxylic acids, carboxylic esters, carboxylic anhy-drides, but preferably vinyl esters. The reaction takes place as an acyl transfer. If the acyl donor or the alcohol contains stereogenic centers, the reaction takes place as enantio-, regio- or diastereodifferentiating acyl transfer.
The vinyl esters can easily be prepared by processes known to the person skilled in the art or are commercial-ly available.
The same applies to the carboxylic esters, the carboxylicacids and the cyclic carboxylic anhydrides.
It is possible to employ branched and unbranched alco-hols, with the exception of the tertiary alcohols, as alcohols to be acylated. Those alcohols which cannot be bought are obtained, for example, by reduction from the corresponding ketones or carboxylic esters, most of which can be bought, or by ~-halogenation of the corresponding ketones with subsequent reduction to the alcohol. Other compounds which cannot be bought can be easily prepared by processes known from the literature, for example by Grignard or other conventional addition reactions.
The following are preferably employed for the acylation 2~fi7~
reaction:
1-phenylethanol, l-phenylpropanol, 2-chloro-1-phenyl-ethanol/ (+)~dihydro-4,4-dimethyl-3-hydroxy-2(3H)-fura-none (pantolactone), 2-hydroxypropanal dimethyl acetal, 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopentenone (allethrolone), 2-(6-acetoxynaphthyl)ethanol, 1-octen-3-ol, 2-(1-naphthylmethyl)-1,3-propanediol, 1-(4-isobutyl-phenyl)ethanol, 1,5-anhydro-D-arabino-l-hexenitol (glucal).
The alcohol to be acylated or the carboxylic acid to be esteriied are employed in concentrations of 0.05 - 200 %
based on the volume of the acyl components to be employed or the solution thereof in an inert organic solvent. A
wide concentration range is possible and depends on the reactivity of the substrate to be reacted.
The acylation is carried out in a column operated batch-wise or continuou~ly. The principle of column operation is a standard process.
To run the column, a temperature-controllable jacketed glass column with frit is packed first with carrier, then with the enzyme/carrier mixture and finally with carrier.
Enzyme and carrier are employed in the ratio 1:100 to 1:2, preferably 1:20 - 1:5 (weight/weight).
The column dimension can be chosen depending on the ~5 reactivity of the substrate and the amount of substrate to be reacted. Because the Lmmobilized biocatalyst is simple to prepare by mixing enzyme and inert, inorganic material, the process is paxticularly suitable for industrial applications.
The temperature of the column is -10 to ~80C, preferably lO - 40C and depends, in particular, on the expected reaction rate, the xequired selectivity and the stability of the enzyme.
2 ~ 6 For enzymatic acylation in a column operated batchwise, the alcohol or the carboxylic acid is taken up in the acyl donor or alcohol or in a solution of the acyl donor or of the alcohol, and the mixture is continuously pumped round by means of a pump.
Operating the column batchwise has the advantage that conversion can be checked easily and, moreover, allows the level of conversion to be adjusted at any tLme.
When the column is operated continuously, the appropriate solution passes through, with or without a pumping system, the column which is packed with immobilized biocatalyst.
The level of the conversion in the continuous process can be chosen virtually as required by adjusting the dropping rate and must be determined beforehand by prelLminary tests.
The space-time yields in both processes depend directly on the absolute values of the abovementioned parameters, but particularly on the Lmmobilized amount of enzyme.
Subsequently, the solvent is removed by distillation from the product solution obtained in the batchwise or con-tinuous process.
The purity is checked and the conversion is determined using conventional analytical methods, for example TLC, GC, lH-NMR spectroscopy and by measurements of optical rotation.
The invention is explained in detail in the examples which follow.
1) Preparation of the immobilized biocatalyst The inert, inorganic carrier is employed after 2~6d~76 comminution, calcining and grading or directly (seasand).
A homogeneous mixture of 5 g of lipase P with 50 ml of commercially available calcined seasand is prepared by mixing.
The temperature-controllable jacketed glass col D
is then packed with 25 ml of seasand, a layer of 55 ml of ~he enzyme/seasand mixture is placed on the seasand and once more covered with a layer of 20 ml of seasand.
2) Use for acylation in batchwise operation 30 g of racemic 4-hydro~y-3-methyl-2-(2-propenyl)-2-cyclopentenone (allethrolone) are taken up in 180 ml of vinyl acetate and continuously pumped round for 23 h (volume delivered: 6 - 10 l/h). The temperature of the reservoir containing the sub-strate is likewise controlled throughout the process so that the temperatures in the column and reservoir are identical.
After the end of the reaction time (= duration of pumping round: 23 h), the substrate solution is pumped off and the column is then washed three times with 50 ml of vinyl acetate each time.
To remove the solvent quantitatively, the substrate solution is distilled initially at 60C under 400 mbar for 2 h and then once more at 60C under 50 mbar for 2 h.
Determinations of conversion by GC: 35 % acetate 65 % alcohol Yield: (Determination of the enantiomeric excesses by lH-NMR shift experiments with Eu~hfc)3and measurements of optical rotation) 2 ~ 7 ~
Acetate- ee 2 9 ej ~
Optical rotation: -31.4 (1-2 %
strength solution inchloroform) Alcohol: ee - 4t) ~
Optical rotation: +5.3 (1-2 %
strength solution inchloroform) Optical rotation of the pure compounds:
Acetate: -32.3 Alcohol: -13.9 The reaction is then carried out 159 x in the same manner. The conversion falls to 18 ~ during this.
3) Use for acylation with continuous operation of the column A solution of 1.2 kg (6.74 mol) of 1-(4-isobutyl-phenyl)ethanol in 1.2 1 of vinyl acetate is passed through a biocatalyst column at a dropping rate of 16 ml/h. The column (diameter: 8 cm; length: 40 cm) was packed with 150 ml of seasand onto which a mixture of 250 ml of æeasand and 50 g of lipase P
was placed and subsequently covered with a layer of 100 ml of seasand. Excess vinyl acetate in the product solution is removed in a rotary evaporator, and the product mixture is examined for conversion by lH-NMR.
Conversion: 50 %
The product mixture is fractionated by column chromatography on silica gel (mobile phase hexane/
ethyl acetate, 50:1 vol/vol).
Yield: 650 g of acetate 44 %
580 g of alcohol 48 %
2~6~7~
Acetate: Optical rotation [~]DO = +97.6 (c = 1, CHCl3~
clear liquid ee 2 95 % (lH-NMR + shift) S Alcohol: Optical rotation [~] 20 = -41.69 (c = 1, CHCl3) Melting point: 33C
ee 2 95 ~ (lH-NMR + shift) 4) Regioselective diacetylation of D-glucal (1,5-anhydro-D-arabino-1-hexenitol) to 3,6-di-O-acetyl-D-g}ucal (column operated continuously) A solution of 5.21 g of glucal in 50 ml of di-methoxyethane (DME) is mixed with 500 ml of vinyl acetate and pumped round in a batchwise process for 20 h (12 g of lipase P on 120 ml of seasand).
7.39 g of the desired 3,6-diacetate (90 %), which is pure by TLC and lH-NMR, are obtained.
The invention is described in detail herein~fter. The invention is furthermore defined by the contents of the cl~ims.
2~6~
The process can be applied to all reactions, i.e. acyl transfer reactions, which can be catalyzed by hydrolases in organic solvents. The possible reactions are either disclosed in the literature or can easily be determined by preliminary tests.
Used as carrier materials for the enzyme immobilization are inert, inorganic materials such as powdered rock, powdered brick, ceramic powders, but preferably seasand.
All the materials are commercially available or c~n be ob~ained very straightforwardly and do not need pre- or after-treatment. However, it is preferable for the carriers also to be comminuted and graded (standard process). Seasand can be bought in the calcined state (Riedel-de Haen).
The enzymes which are used to catalyze the esterification are hydrolases, especially lipases from Mucor, Rhizopus, Penicillium, Pseudomonas or the lipase from pig pancreas, but preferably the lipase from Pseudomonas (lipase P, also called FP or PS, Amano Pharmaceuticals, Nagoya, Japan) or from Candida (for example Sigma Chemicals Co., St. Louis, MO, USA or lipase OF (Meito Sangyo, Nagoya, Japan)).
To immobilize the enzyme, the hydrolase and carrier are mixed, preferably in the dry state.
The particle size of the carrier is a maximum of 2 mm, preferably 0.01 - 2 mm, but particularly 0.1 - 1 ~m, but does not have to be uniform for carrying out the process according to the invention.
The amount of carrier to be loaded with enzyme is chosen depending on the size of the batch, on the reaction time to be expected and on the level of conversion. It can easily be determined by preliminary tests.
The immobilized hydrolase i5 then stable for a surpris-ingly long time with full ens:yme activity under dry storage conditions at room temperature~ The half-life of the immobilized enzyme on use in the laboratory is 2 200 days.
In the acyl transfer reaction, the acyl component which acts as solvent or is dissolved in another organic solvent is cleaved to a ketone, aldehyde, alcohol or water and an acyl-enzyme complex, and the latter reacts with the alcohol (substrate) to be added.
Employed as acyl donors are branched and unbranched carboxylic acids, carboxylic esters, carboxylic anhy-drides, but preferably vinyl esters. The reaction takes place as an acyl transfer. If the acyl donor or the alcohol contains stereogenic centers, the reaction takes place as enantio-, regio- or diastereodifferentiating acyl transfer.
The vinyl esters can easily be prepared by processes known to the person skilled in the art or are commercial-ly available.
The same applies to the carboxylic esters, the carboxylicacids and the cyclic carboxylic anhydrides.
It is possible to employ branched and unbranched alco-hols, with the exception of the tertiary alcohols, as alcohols to be acylated. Those alcohols which cannot be bought are obtained, for example, by reduction from the corresponding ketones or carboxylic esters, most of which can be bought, or by ~-halogenation of the corresponding ketones with subsequent reduction to the alcohol. Other compounds which cannot be bought can be easily prepared by processes known from the literature, for example by Grignard or other conventional addition reactions.
The following are preferably employed for the acylation 2~fi7~
reaction:
1-phenylethanol, l-phenylpropanol, 2-chloro-1-phenyl-ethanol/ (+)~dihydro-4,4-dimethyl-3-hydroxy-2(3H)-fura-none (pantolactone), 2-hydroxypropanal dimethyl acetal, 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopentenone (allethrolone), 2-(6-acetoxynaphthyl)ethanol, 1-octen-3-ol, 2-(1-naphthylmethyl)-1,3-propanediol, 1-(4-isobutyl-phenyl)ethanol, 1,5-anhydro-D-arabino-l-hexenitol (glucal).
The alcohol to be acylated or the carboxylic acid to be esteriied are employed in concentrations of 0.05 - 200 %
based on the volume of the acyl components to be employed or the solution thereof in an inert organic solvent. A
wide concentration range is possible and depends on the reactivity of the substrate to be reacted.
The acylation is carried out in a column operated batch-wise or continuou~ly. The principle of column operation is a standard process.
To run the column, a temperature-controllable jacketed glass column with frit is packed first with carrier, then with the enzyme/carrier mixture and finally with carrier.
Enzyme and carrier are employed in the ratio 1:100 to 1:2, preferably 1:20 - 1:5 (weight/weight).
The column dimension can be chosen depending on the ~5 reactivity of the substrate and the amount of substrate to be reacted. Because the Lmmobilized biocatalyst is simple to prepare by mixing enzyme and inert, inorganic material, the process is paxticularly suitable for industrial applications.
The temperature of the column is -10 to ~80C, preferably lO - 40C and depends, in particular, on the expected reaction rate, the xequired selectivity and the stability of the enzyme.
2 ~ 6 For enzymatic acylation in a column operated batchwise, the alcohol or the carboxylic acid is taken up in the acyl donor or alcohol or in a solution of the acyl donor or of the alcohol, and the mixture is continuously pumped round by means of a pump.
Operating the column batchwise has the advantage that conversion can be checked easily and, moreover, allows the level of conversion to be adjusted at any tLme.
When the column is operated continuously, the appropriate solution passes through, with or without a pumping system, the column which is packed with immobilized biocatalyst.
The level of the conversion in the continuous process can be chosen virtually as required by adjusting the dropping rate and must be determined beforehand by prelLminary tests.
The space-time yields in both processes depend directly on the absolute values of the abovementioned parameters, but particularly on the Lmmobilized amount of enzyme.
Subsequently, the solvent is removed by distillation from the product solution obtained in the batchwise or con-tinuous process.
The purity is checked and the conversion is determined using conventional analytical methods, for example TLC, GC, lH-NMR spectroscopy and by measurements of optical rotation.
The invention is explained in detail in the examples which follow.
1) Preparation of the immobilized biocatalyst The inert, inorganic carrier is employed after 2~6d~76 comminution, calcining and grading or directly (seasand).
A homogeneous mixture of 5 g of lipase P with 50 ml of commercially available calcined seasand is prepared by mixing.
The temperature-controllable jacketed glass col D
is then packed with 25 ml of seasand, a layer of 55 ml of ~he enzyme/seasand mixture is placed on the seasand and once more covered with a layer of 20 ml of seasand.
2) Use for acylation in batchwise operation 30 g of racemic 4-hydro~y-3-methyl-2-(2-propenyl)-2-cyclopentenone (allethrolone) are taken up in 180 ml of vinyl acetate and continuously pumped round for 23 h (volume delivered: 6 - 10 l/h). The temperature of the reservoir containing the sub-strate is likewise controlled throughout the process so that the temperatures in the column and reservoir are identical.
After the end of the reaction time (= duration of pumping round: 23 h), the substrate solution is pumped off and the column is then washed three times with 50 ml of vinyl acetate each time.
To remove the solvent quantitatively, the substrate solution is distilled initially at 60C under 400 mbar for 2 h and then once more at 60C under 50 mbar for 2 h.
Determinations of conversion by GC: 35 % acetate 65 % alcohol Yield: (Determination of the enantiomeric excesses by lH-NMR shift experiments with Eu~hfc)3and measurements of optical rotation) 2 ~ 7 ~
Acetate- ee 2 9 ej ~
Optical rotation: -31.4 (1-2 %
strength solution inchloroform) Alcohol: ee - 4t) ~
Optical rotation: +5.3 (1-2 %
strength solution inchloroform) Optical rotation of the pure compounds:
Acetate: -32.3 Alcohol: -13.9 The reaction is then carried out 159 x in the same manner. The conversion falls to 18 ~ during this.
3) Use for acylation with continuous operation of the column A solution of 1.2 kg (6.74 mol) of 1-(4-isobutyl-phenyl)ethanol in 1.2 1 of vinyl acetate is passed through a biocatalyst column at a dropping rate of 16 ml/h. The column (diameter: 8 cm; length: 40 cm) was packed with 150 ml of seasand onto which a mixture of 250 ml of æeasand and 50 g of lipase P
was placed and subsequently covered with a layer of 100 ml of seasand. Excess vinyl acetate in the product solution is removed in a rotary evaporator, and the product mixture is examined for conversion by lH-NMR.
Conversion: 50 %
The product mixture is fractionated by column chromatography on silica gel (mobile phase hexane/
ethyl acetate, 50:1 vol/vol).
Yield: 650 g of acetate 44 %
580 g of alcohol 48 %
2~6~7~
Acetate: Optical rotation [~]DO = +97.6 (c = 1, CHCl3~
clear liquid ee 2 95 % (lH-NMR + shift) S Alcohol: Optical rotation [~] 20 = -41.69 (c = 1, CHCl3) Melting point: 33C
ee 2 95 ~ (lH-NMR + shift) 4) Regioselective diacetylation of D-glucal (1,5-anhydro-D-arabino-1-hexenitol) to 3,6-di-O-acetyl-D-g}ucal (column operated continuously) A solution of 5.21 g of glucal in 50 ml of di-methoxyethane (DME) is mixed with 500 ml of vinyl acetate and pumped round in a batchwise process for 20 h (12 g of lipase P on 120 ml of seasand).
7.39 g of the desired 3,6-diacetate (90 %), which is pure by TLC and lH-NMR, are obtained.
5) Regioselective monoacetylation of D-glucal to 6-O-acetyl-D-glucal (column operated continuously) A column is prepared from 20 g of lipase OF (Candida Cylindracaea) and 200 ml of seasand. 5 g of D-glucal are dissolved in 50 ml of dimethoxyethane and passed together with 500 ml of vinyl acetate through the column. The resulting heterogeneous mixture i6, after a single passage through the column (~ 5 h), circulated by pumping for a further 20 h.
Removal of the solvent by distillation in vacuo and silica gel chromatography result in 5.07 g (79 ~ of the desired monoacetate which is pure by TLC
andlH-NMR.
Removal of the solvent by distillation in vacuo and silica gel chromatography result in 5.07 g (79 ~ of the desired monoacetate which is pure by TLC
andlH-NMR.
6) Lipase OF-catalyzed preparation of geranyl laurate 2 ~ 7 ~
(column operated continuously) The column packing is composed of 20 ml of seasand and 2 g of lipase OF. 200 mg of dodecanoic acid and 154 mg of geraniol are dissolved in 100 ml of hexane and passed through the enzyme co]umn (flow rate 10 ml/min). The reaction mixture contains about 95 % product and 5 % precursors (TLC~ and is puri-fied by silica gel chromatography. 222 mg of geranyl laurate which is pure by TLC and lH-NMR are obtained.
Further examples are listed in Table 1.
All the experiments carried out in Table 1 are carried out in the continuous process using the Pseudomonas lipase.
(column operated continuously) The column packing is composed of 20 ml of seasand and 2 g of lipase OF. 200 mg of dodecanoic acid and 154 mg of geraniol are dissolved in 100 ml of hexane and passed through the enzyme co]umn (flow rate 10 ml/min). The reaction mixture contains about 95 % product and 5 % precursors (TLC~ and is puri-fied by silica gel chromatography. 222 mg of geranyl laurate which is pure by TLC and lH-NMR are obtained.
Further examples are listed in Table 1.
All the experiments carried out in Table 1 are carried out in the continuous process using the Pseudomonas lipase.
7 ~
~ O I A A A A
al 0 0 -- I A A A A I A I A
ll O (-') t~ a) I +
~ l ! 1, 1l , ~ I ~ I I
o I ~ ~ ~ ~ I ~ I ~, I
o ,, I I I I
~, ~ I I I I
I o o o o I o I o a) I ~ o o o I ~ I u~ I
o o a N
I ~ ~ O U~
l~i I 1~-) 1 I I
S-l I I I I
~i I O O O O I O I O
I I d~ I o\o I
I df' d~ ~P dP I I I
o I ~ ul o u7 ~ . I N
~_) I --I I O I O
O ~ I li i o o Ir) I u-) I IJ
o I . I
I O IJ') I O I O
~4 1 1 1 1 S i i ~ ~ ~ ~ I ~ I ~ i s i I ~ i 3 ,i I ~i I ~i I
o i ~ O il ~ ~o I oi ~ O
~, Z I ~ I ~ I o~
2 ~ 7 ~
-- 13 -- .
~ I I . I. I
o Ia dPI . I. I
O
a) u -- I~ ~ I I ~P l a) u ~ ~ I o dP I '~ I n I
A ~
t: --`I I I I
O ~ l l l l .~ _ I I I I
J~ I I I I
u a) ~ ~ I I I +I +
a) la ~; h ,_ I ,_ I I I
oP U I d~ I dP I
a) I o ~o o I l` I ~ I
~ O I _ I
O ,1 ~ ~n I I I I
Io o I O
~ O IO O I O I o ,1 u I--I o I In I u~ I
a~ I I I I
E~ I I I I
:~ ItJ` ~ I tP I t51 1 N
~ I11'1 0 1 It'~ I Il~ I
P~ I~r) I I I
Sl IO O I O I O
U I dl~ dP I dP I oY> l O I. . I
l O O
O
tO I I I I
_i I I I
Pl I O I I O
~i Ia 111 I tll 1 1~1 l 11~ I~ a) I a) I a) I
C~ IIn m I ~n I m I
i I o ~ ~ I
a I ~
o Io o I ~ ~ ~ ~ I ~ I
I ~ ~U ~ ~ oP' ~
o I 0 ~ I I h ~1 U 1 ~1 0 I O I --I I N
ZI ~ I ~
- 14 - 2~
I
a) ~ , dP I ~ I I
.
a ~ ' I ' +
ll ll l l I
o I ~ I Ul ~, U~ I I I I
~, ~ , o , ,, I o , ~ I ~ I Ul I ~ I
a I ~ I
o I o ~ o N I t~
S-l , 3 ~a ~ O , ~ ~ o o o ~ ~ C
~0 110~I11 1 ~a?~
0 1 ~1 ~J I N
X Z I ~
15 - 2 ~ 7 ~
-Table 1 explanations:
n.d.: not determined (1) In Example 7 (l-phenylethanol), the ee was also determined by HPLC (~Chiralcel OB).
(2) The ee was determined by lH-NMR shift experiments with Eu(hfc)3 (acetates) or in the same manner after the chemical acetylation of the alcohols.
(3) The reaction rates were justified as follows:
+ = fast i.e. > 1 gtl/h - = slow i.e. < 1 g/l/h (4) Conversion determined by ~C or lH-NMR.
The advantages of the invention are 1. the immobilized biocatalyst is prepared simply by mixing enzyme and carrier together without previous derivatization of enzyme and carrier being necessary, 2. the immobilized biocatalyst ~as a long half life, because the long useful lives of the catalyst columns save work and costs, 3. the enzyme is not washed out by solvents, which in turn guarantees the ability for frequent reuse and long useful lives and 4. the immobilized biocatalyst can be disposed of easily and at reasonable cost because the carriers are natural materials.
~ O I A A A A
al 0 0 -- I A A A A I A I A
ll O (-') t~ a) I +
~ l ! 1, 1l , ~ I ~ I I
o I ~ ~ ~ ~ I ~ I ~, I
o ,, I I I I
~, ~ I I I I
I o o o o I o I o a) I ~ o o o I ~ I u~ I
o o a N
I ~ ~ O U~
l~i I 1~-) 1 I I
S-l I I I I
~i I O O O O I O I O
I I d~ I o\o I
I df' d~ ~P dP I I I
o I ~ ul o u7 ~ . I N
~_) I --I I O I O
O ~ I li i o o Ir) I u-) I IJ
o I . I
I O IJ') I O I O
~4 1 1 1 1 S i i ~ ~ ~ ~ I ~ I ~ i s i I ~ i 3 ,i I ~i I ~i I
o i ~ O il ~ ~o I oi ~ O
~, Z I ~ I ~ I o~
2 ~ 7 ~
-- 13 -- .
~ I I . I. I
o Ia dPI . I. I
O
a) u -- I~ ~ I I ~P l a) u ~ ~ I o dP I '~ I n I
A ~
t: --`I I I I
O ~ l l l l .~ _ I I I I
J~ I I I I
u a) ~ ~ I I I +I +
a) la ~; h ,_ I ,_ I I I
oP U I d~ I dP I
a) I o ~o o I l` I ~ I
~ O I _ I
O ,1 ~ ~n I I I I
Io o I O
~ O IO O I O I o ,1 u I--I o I In I u~ I
a~ I I I I
E~ I I I I
:~ ItJ` ~ I tP I t51 1 N
~ I11'1 0 1 It'~ I Il~ I
P~ I~r) I I I
Sl IO O I O I O
U I dl~ dP I dP I oY> l O I. . I
l O O
O
tO I I I I
_i I I I
Pl I O I I O
~i Ia 111 I tll 1 1~1 l 11~ I~ a) I a) I a) I
C~ IIn m I ~n I m I
i I o ~ ~ I
a I ~
o Io o I ~ ~ ~ ~ I ~ I
I ~ ~U ~ ~ oP' ~
o I 0 ~ I I h ~1 U 1 ~1 0 I O I --I I N
ZI ~ I ~
- 14 - 2~
I
a) ~ , dP I ~ I I
.
a ~ ' I ' +
ll ll l l I
o I ~ I Ul ~, U~ I I I I
~, ~ , o , ,, I o , ~ I ~ I Ul I ~ I
a I ~ I
o I o ~ o N I t~
S-l , 3 ~a ~ O , ~ ~ o o o ~ ~ C
~0 110~I11 1 ~a?~
0 1 ~1 ~J I N
X Z I ~
15 - 2 ~ 7 ~
-Table 1 explanations:
n.d.: not determined (1) In Example 7 (l-phenylethanol), the ee was also determined by HPLC (~Chiralcel OB).
(2) The ee was determined by lH-NMR shift experiments with Eu(hfc)3 (acetates) or in the same manner after the chemical acetylation of the alcohols.
(3) The reaction rates were justified as follows:
+ = fast i.e. > 1 gtl/h - = slow i.e. < 1 g/l/h (4) Conversion determined by ~C or lH-NMR.
The advantages of the invention are 1. the immobilized biocatalyst is prepared simply by mixing enzyme and carrier together without previous derivatization of enzyme and carrier being necessary, 2. the immobilized biocatalyst ~as a long half life, because the long useful lives of the catalyst columns save work and costs, 3. the enzyme is not washed out by solvents, which in turn guarantees the ability for frequent reuse and long useful lives and 4. the immobilized biocatalyst can be disposed of easily and at reasonable cost because the carriers are natural materials.
Claims (13)
1. An immobilized biocatalyst which is composed of an inert, inorganic carrier and of a hydrolase which is immobilized on this carrier.
2. An immobilized biocatalyst as claimed in claim 1, wherein the carrier is seasand, powdered rock or brick, glass or ceramic powder.
3. An immobilized biocatalyst as claimed in claim 1, wherein the hydrolase is a lipase.
4. An immobilized biocatalyst as claimed in claim 1 and 3, wherein a lipase from Pseudomonas, Mucor, Candida, Rhizopus, Penicillium, or the lipase from pig pancreas is employed as hydrolase.
5. An immobilized biocatalyst as claimed in claim 4, wherein Pseudomonas lipase P/FP/PS or Candida lipase is used.
6. An immobilized biocatalyst as claimed in claim 1, wherein the particle size of the carriers does not exceed 2 mm.
7. A process for the preparation of an immobilized biocatalyst, which comprises a hydrolase being mixed with an inert, inorganic carrier, preferably composed of seasand, powdered rock or brick, glass or ceramic powder, and being immobilized thereon.
8. The process as claimed in claim 7, wherein the reac-tion is carried out in a batchwise process.
9. The process as claimed in claim 7, wherein the reac-tion is carried out in a continuous process.
10. The process as claimed in one or more of claims 7 to 9, wherein the carrier is employed untreated for the immobilization.
11. The use of the catalyst as claimed in claims 1 to 6 or of the immobilized biocatalyst obtainable as claimed in claims 7 - 10 for ester synthesis in a column reactor.
12. The use of the catalyst as claimed in claims 1 to 6 or of the immobilized biocatalyst obtainable as claimed in claims 7 - 10 for enantio-, diastereo- and regiodif-ferentiating esterification by acylation of alcohols with carboxylic acids and carboxylic acid derivatives.
13. The use of the immobilized biocatalyst as claimed in claim 12, where the following alcohols and carboxylic acids are employed for the acylation reaction:
1-phenylethanol, 1-phenylpropanol, 2-chloro-1-phenyl-ethanol, (+)-dihydro-4,4-dimethyl-3-hydroxy-2(3H)-fura-none (pantolactone), 2-hydroxypropanal dimethyl acetal, 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopentenone, 2-(6-acetoxynaphthyl)ethanol, 1-octen-3-ol, 2-(1-naphthyl-methyl)-1,3-propanediol, 1-(4-isobutylphenyl)ethanol, geraniol, dodecanoic acid or 1,5-anhydro-D-arabino-1-hexenitol (glucal).
1-phenylethanol, 1-phenylpropanol, 2-chloro-1-phenyl-ethanol, (+)-dihydro-4,4-dimethyl-3-hydroxy-2(3H)-fura-none (pantolactone), 2-hydroxypropanal dimethyl acetal, 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopentenone, 2-(6-acetoxynaphthyl)ethanol, 1-octen-3-ol, 2-(1-naphthyl-methyl)-1,3-propanediol, 1-(4-isobutylphenyl)ethanol, geraniol, dodecanoic acid or 1,5-anhydro-D-arabino-1-hexenitol (glucal).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4110637 | 1991-04-02 | ||
| DEP4110637.7 | 1991-04-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2064676A1 true CA2064676A1 (en) | 1992-10-03 |
Family
ID=6428655
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2064676 Abandoned CA2064676A1 (en) | 1991-04-02 | 1992-04-01 | Immobilized biocatalyst, its preparation and use for ester synthesis in a column reactor |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0507278A3 (en) |
| JP (1) | JPH05192145A (en) |
| AU (1) | AU647603B2 (en) |
| CA (1) | CA2064676A1 (en) |
| TW (1) | TW246689B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6998258B1 (en) | 1999-10-29 | 2006-02-14 | Basf Aktiengesellschaft | L-pantolactone-hydrolase and a method for producing D-pantolactone |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19505672A1 (en) * | 1995-02-20 | 1996-08-22 | Hoechst Ag | Process for the enzymatic acylation of alcohols with alkoxy vinyl acetates by transesterification |
| DE19809649A1 (en) | 1998-03-06 | 1999-09-09 | Hoechst Marion Roussel De Gmbh | Process for the enzymatic separation of enantiomers of 3 (R) - and 3 (S) -hydroxy-1-methyl-4- (2,4,6-trimethoxyphenyl) -1,2,3,6-tetrahydro-pyridine or the Carboxylic acid esters |
| US20080306144A1 (en) * | 2007-06-08 | 2008-12-11 | Stephanie Kay Clendennen | Hydroxybenzyl or hydroxypyranonemethyl esters as tyrosinase inhibitors |
| MX2017013383A (en) | 2015-04-20 | 2017-12-07 | Tolero Pharmaceuticals Inc | Predicting response to alvocidib by mitochondrial profiling. |
| KR102608921B1 (en) | 2015-05-18 | 2023-12-01 | 스미토모 파마 온콜로지, 인크. | Albocidip prodrug with increased bioavailability |
| MX2018001289A (en) | 2015-08-03 | 2018-04-30 | Tolero Pharmaceuticals Inc | Combination therapies for treatment of cancer. |
| US11279694B2 (en) | 2016-11-18 | 2022-03-22 | Sumitomo Dainippon Pharma Oncology, Inc. | Alvocidib prodrugs and their use as protein kinase inhibitors |
| WO2019055579A1 (en) | 2017-09-12 | 2019-03-21 | Tolero Pharmaceuticals, Inc. | Treatment regimen for cancers that are insensitive to bcl-2 inhibitors using the mcl-1 inhibitor alvocidib |
| MX2021006544A (en) | 2018-12-04 | 2021-07-07 | Sumitomo Pharma Oncology Inc | Cdk9 inhibitors and polymorphs thereof for use as agents for treatment of cancer. |
| WO2020191326A1 (en) | 2019-03-20 | 2020-09-24 | Sumitomo Dainippon Pharma Oncology, Inc. | Treatment of acute myeloid leukemia (aml) with venetoclax failure |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1293215C (en) * | 1985-07-10 | 1991-12-17 | Robert Clyde | Process and apparatus for enhancing biological and chemical reactions from high area inorganic base silica on fibers |
| AU618277B2 (en) * | 1987-08-28 | 1991-12-19 | Royal Melbourne Institute Of Technology Limited | Production of organic acid esters |
| MY103640A (en) * | 1987-12-09 | 1993-08-28 | Kao Corp | Immobilized enzyme and esterification and interesterification therewith |
| GB8729890D0 (en) * | 1987-12-22 | 1988-02-03 | Unilever Plc | Improvements in & relating to fat processes |
| DE3743824C2 (en) * | 1987-12-23 | 1997-03-06 | Hoechst Ag | Process for the enzymatic resolution of racemic alcohols with / in vinyl esters by transesterification |
| JPH0220289A (en) * | 1988-07-08 | 1990-01-23 | Sagami Chem Res Center | Production of farnesylacetic acid geranyl ester |
-
1992
- 1992-04-01 EP EP19920105616 patent/EP0507278A3/en not_active Withdrawn
- 1992-04-01 JP JP7951892A patent/JPH05192145A/en active Pending
- 1992-04-01 AU AU13937/92A patent/AU647603B2/en not_active Ceased
- 1992-04-01 CA CA 2064676 patent/CA2064676A1/en not_active Abandoned
- 1992-04-06 TW TW81102577A patent/TW246689B/zh active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6998258B1 (en) | 1999-10-29 | 2006-02-14 | Basf Aktiengesellschaft | L-pantolactone-hydrolase and a method for producing D-pantolactone |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0507278A2 (en) | 1992-10-07 |
| AU1393792A (en) | 1992-10-08 |
| TW246689B (en) | 1995-05-01 |
| JPH05192145A (en) | 1993-08-03 |
| EP0507278A3 (en) | 1993-12-15 |
| AU647603B2 (en) | 1994-03-24 |
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