CZ20001235A3 - Process for preparing lactam compounds. - Google Patents

Abstract

Compounds, especially lactams, for example 6-aminopenicillanic acid acid (6-APA), may be prepared by catalytic, in particular, by enzymatic conversion of a compound such as penicillin G, in a solvent mixture containing water and non-aqueous an organic solvent, especially 1,1,1,2-tetrafluoroethane. 6- The APA is precipitated, isolated by filtration and optionally derivatized the desired compound. Enzymatic by-product the conversion, which is phenylacetic acid, is isolated by extraction solvent, suitably using a solvent which also contains 1,1,1,2-tetrafluoroethane.

Description

Process for the preparation of lactam compounds

Technical field

The present invention relates to a process for preparing a lactam compound, in particular a pharmaceutically active lactam compound. In particular, but not exclusively, the invention relates to a process for the preparation of lactams, for example penicillins and cephalosporins and / or derivatives thereof.

BACKGROUND OF THE INVENTION

6-Aminopenicillanic acid (6-APA) and 7-aminodesacetoxycephalosporanic acid (7-ADCA) are intermediates used in the manufacture of most semi-synthetic β-lactam antibiotics. A commercially preferred process for the production of 6-APA is carried out by biochemical deacylation of benzylpenicillin, commonly known as Pen-G, or by equivalent enzymatic deacylation of phenoxymethylpenicillin, commonly known as Pen-V. The method is carried out using an enzyme such as penicillin acylase that is immobilized on an insoluble matrix such as polystyrene or polystyrene.

And polyacrylate polymers or copolymers.

Various methods using this technique are described in the scientific literature. In these methods, penicillin-G is isolated from the fermentation liquid as a solid intermediate using known methods and then dissolved in water as a relatively dilute solution (e.g., 5% w / v). The enzymatic reaction (illustrated in Scheme 1 below, wherein R is a potassium or sodium ion) is performed at an elevated temperature (e.g., 35 to 40 ° C). The phenylacetic acid (PAA) obtained is neutralized by the continuous addition of dilute aqueous sodium hydroxide solution (e.g. 5% w / v) to maintain the pH at about 8.0. Isolation of 6-APA is typically accomplished by concentrating the enzymatic reaction liquid to, for example,% relative to 6-APA, thereby maximizing yield, then feefefefef feefefefefe feefefefefe · · · ·

•. Followed by precipitation with a dilute inorganic acid such as 5% nitric acid or sulfuric acid. PAA is removed by extraction into a immiscible organic solvent such as methyl isobutyl ketone or butyl acetate. The 6-APA is finally filtered off, washed with acetone and then dried under vacuum. Acceptable standard conversion for the described deacylation process is 94 to 96% and is 82 and 86 for the precipitation and isolation step.

Scheme 1

PENICILIN-G

H H ENZYM ^H 2 ^N V ' ³ (A ->. NaOH f— ⁿ -V \ h ₃ 0 COOR

6-APA

PAA

The immobilized enzymes used in the above-described process may be sensitive to inhibition by the product. Therefore, the reaction is conveniently carried out in relatively dilute solutions. The disadvantage of this method is that the solubility of 6-APA in water is about 2%, which means that a thickening step is necessary to minimize losses in the mother liquors. Therefore, an expensive concentration step is used, thereby increasing the content of 6-APA to 12-16%.

PAA is one of the components commonly used in penicillin-G fermentation. It is therefore commercially advantageous to recover PAA 'from the reaction medium so that it can be recycled. Conventional methods that are commercially used for PAA isolation utilize a multi-step process involving a combination of two or more of the following techniques: vacuum distillation, purification (e.g., charcoal treatment), aqueous phase extraction, chromatography, precipitation, filtration and drying. These techniques are relatively expensive and cause environmental problems.

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It is an object of the present invention to solve the problems described above.

SUMMARY OF THE INVENTION

According to a first embodiment, the present invention provides a method for preparing a second compound by catalytic conversion of a first compound, the method comprising contacting said first compound with a catalyst in a solvent mixture comprising water and a first non-aqueous solvent.

Unless otherwise indicated, or unless the context requires otherwise, a reference to any compound of the present invention also includes a reference to a salt of the compound.

Said catalyst is preferably an enzyme. Said enzyme may be sensitive to said second compound, for example in the sense that relatively high concentrations of said second compound may reduce the ability of the enzyme to effect conversion - a phenomenon known as "inhibition by a product."

Said enzyme is preferably capable of catalyzing a deacylation reaction. It may suitably include acylase, which may be produced by any penicillin acylase-producing microorganism such as Escherichia, in particular Escherichia coli, Pseudomonas, Streptomyces, Proteus and Micrococus. Said enzyme is preferably mobilized, suitably by physical absorption or binding to a solid, insoluble matrix.

Said first compound preferably has the general formula R ¹ NHQ, wherein R ¹ is an optionally substituted alkylcarbonyl group and Q is an optionally substituted cyclic group, particularly an optionally substituted lactam group, for example a β-lactam group.

Unless otherwise indicated, an optionally substituted alkyl group may contain up to 12, preferably up to 8, more preferably up to 6, especially up to 4, carbon atoms. Optional substituents of said alkyl group are an optionally substituted aryl group.

• o · o · o · o · o · o

alkenyl, alkynyl, acyl, nitro, cyano, alkoxy, hydroxyl, amino, alkylamino, sulfinyl, alkylsulfinyl, sulfonyl, alkylsulfonyl, sulfonate, amido, alkylamidoalkyl, halocarbonyl and alkoxycarbonyl a halogen atom, in particular a fluorine atom, a chlorine atom or a bromine atom.

Preferred optional substituents of said alkoxycarbonyl group include optionally substituted, especially unsubstituted, phenyl, carboxyl and amino groups.

In a preferred embodiment said R ¹ group may be an optionally substituted benzylcarbonyl group or a C (COOH) (NH ₂ ) HCH ₂ CH ₂ CH ₂ C (O) - or a salt thereof.

Said group Q may contain a lactam fused to another optionally substituted cyclic group, which may be a five or six membered ring.

Said first compound may be a natural product, in particular a fermentation reaction product or a derivative thereof. Said first compound is preferably an antibiotic. The method of the first embodiment may comprise the step of preparing said first compound by means of a biochemical, in particular a fermentation process.

The weight ratio of said first solvent to water in said solvent mixture may. be at least 2, preferably at least 5, more preferably at least 7, especially at least 10. In some cases, it may be at least 15 or 20. Said ratio may be less than 100, preferably less than 50, more preferably less than 30, especially 20 or less. . This ratio is suitably

i i

f f

This at any time during the conversion reaction, but preferably this ratio relates to the start of the reaction.

The ratio (% w / v) of said first compound to water present in the composition may be at least 10, suitably at least 20, preferably at least 30, more preferably at least 40, especially 50 or higher. The ratio may be less than 100, suitably less than. 90, preferably 80 or less. Said ratio is suitably present at any time during the conversion reaction, but is preferably present at the beginning of the reaction.

Said first non-aqueous solvent may have a boiling point at atmospheric pressure of less than 80 ° C, suitably less than 60 ° C, preferably less than 40 ° C, more preferably less than 20 ° C. Particularly preferred solvents have a boiling point below 0 ° C, preferably below -10 ° C. The boiling point may be above -90 ° C, preferably above -70 ° C, more preferably above -50 ° C.

Said solvent is preferably organic. It may be aromatic, but is preferably aliphatic. It may contain less than 10, suitably less than 8, preferably less than 6, more preferably less 4, especially 2 or less carbon atoms. It may be an optionally substituted alkane, alkene or alkyne. Optionally substituted alkanes having 1 to 4 carbon atoms are preferred. Preferably, the solvent is halogenated. It may contain less than 10, suitably less than 8, preferably less than 6, more preferably less than 5, especially 4 or less halogen atoms. Preferred halogen atoms include fluorine, chlorine, and bromine, with fluorine and chlorine being preferred and atom. fluorine is particularly preferred.

Preferably, the first solvent is non-chlorinated. Preferably it contains one or more carbon atoms, and only fluorine atoms and hydrogen atoms. .

 • 9 0 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · · · Φφ · · · «

Preferably, the first solvent is tetrafluoroethane, with 1,1,1,2-tetrafluoroethane being preferred.

Said solvent mixture may contain other solvents.

Said second compound, especially when in free acid form (i.e., not in salt form), may be at least 0.1%, suitably at least 0.5%, preferably at least 1.0. %, more preferably at least 1.5%, especially 2% water-soluble. The above solubilities are preferably measured at 5 ° C.

Said second salt form, for example in the form of an alkali metal salt, may have a water solubility of at least 5% w / v, preferably at least 10% w / v, more preferably 15% w / v.

Preferably, the method comprises the step of adjusting the pH of the reaction mixture during the conversion reaction, wherein the pH is suitably maintained within a physiologically acceptable pH range. The pH is preferably in the range of 7 to 9, more preferably in the range of 7.8 to 8.2.

According to a first embodiment, the method comprises isolating the second compound from other compounds / solvents. Isolation may include precipitation of the second compound and subsequent isolation of the precipitate by filtration. Precipitation can be induced by adjusting the pH to a value close to the pKa of said second compound. A suitable pH may. The pH may be greater than 2.5, preferably greater than 3.5. Preferably, the substantially insoluble form of the second compound is formed. The pH can usually be adjusted by the addition of an aqueous acid solution such as 1M or 2M nitric acid or preferably 1M or 2M sulfuric acid. The filtered second compound may be washed with a second solvent. Traces of the second solvent can be substantially or completely removed from the filter product by evaporation.

According to a second embodiment, the method may comprise adjusting the pH in the presence of the second solvent to a value at which the name is

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0 0 0 0

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0 0 0 • 0 0 0 0

0 · 0 0 0

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The second compound is substantially soluble in the aqueous phase of the solvent mixture while the by-products of the reaction are substantially soluble in the immiscible said second solvent of the solvent mixture. A suitable pH may be less than 2.5, more preferably less than 2. The pH may suitably be 1 or higher. The solution of said second solvent may be separated from the aqueous phase containing said second compound by settling and physical separation, to obtain an aqueous reaction solution containing said second compound. Said reaction solution may be washed with said second solvent to remove traces of impurities from the reaction by-products. Said second compound may precipitate from said reaction solution by adjusting the pH with a suitable base to form an almost insoluble form of said second compound. A suitable base may be an aqueous solution of sodium hydroxide, potassium hydroxide or ammonium hydroxide. The pH may suitably be adjusted to a value greater than 2.5, more preferably greater than 3.5. The pH may suitably be less than 5, preferably less than 4. The yield of said second compound of both the first and second embodiments may be maximized by stirring at a reduced temperature, preferably at 2 to 10 ° C. Said second compound can be isolated by filtration, washing and drying.

Said second solvent used in the first and second embodiments described preferably comprises said first non-aqueous solvent, preferably in combination with another solvent. Said further solvent may comprise another first solvent of the type described herein. Preferably, however, it is a different type of solvent. Said further solvent is selected to influence the boiling point and / or the dissolution properties of the solvent relative to the first substance. The boiling point of said additional solvent may be less than 60 ° C, preferably less than 30 ° C, more preferably less than 15 ° C, especially less than 5 ° C. The boiling point of said additional solvent

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said first solvent in combination with a minor portion of said further solvent. Preferably at least 90% by weight, more preferably at least 93% by weight, in particular at least 97% by weight of said second solvent is contained in said first non-aqueous solvent, in particular a hydrofluorocarbon solvent. Preferably, the balance comprises one or more of the other solvents described.

Said further solvent may be selected from hydrocarbons and ethers. Preferred hydrocarbons contain up to six carbon atoms. They may be alicyclic or preferably aliphatic. They are preferably alkanes, with methane, ethane, propane and butane being preferred. Preferred ethers are dialkyl ethers, for example dialkyl ethers having 1 to 4 carbon atoms in the alkyl moieties, with dimethyl ether being particularly preferred.

When purifying the second compound using said second solvent, said second compound may precipitate, suitably by adjusting the pH as described above.

In the process of the first embodiment, said catalytic can be conducted. conversion to prepare said second compound and a third compound. Said enzyme may be sensitive to the third compound, for example in the sense that relatively high concentrations of said third compound may reduce the ability of the enzyme to effect conversion. If said first compound has the formula R ¹ NHQ, said third compound may have the formula R ¹ COOH or it may be a salt thereof. In this case, the latter compound may have the formula H ₂ NQ or a salt thereof. The method of the first embodiment may comprise the step of separating the second and third compounds from each other. It may include the use of a solvent (which may be the described first or second solvent),

 • ·· ♦ · <· 9 9 · 4 4 4 4 4 4 4 4 44 44 44 4 4 4 4 4 4

4 ·> 4 4 4

44444 wherein said second and third compounds (or their salts) have different solubilities and / or have different partition coefficients and utilize these properties to achieve partitioning. The second and third compounds may be separated using a solvent / water mixture.

Said third compound, in particular its free acid, is preferably substantially soluble in the solvent used for partitioning.

After isolation, said second compound can be derivatized, suitably to form an antibiotic.

Said first, the compound may be natural penicillin or a biosynthetic penicillin prepared by adding a precursor to a penicillin fermentation broth or cephalosporin. Preferably said first compound is selected from the group consisting of Penícilin-G, Penicillin-X (p-hydroxyphenylpenicillin), Penicillin-V (phenoxymethylpenicillin) and cephalosporin G. Preferably, said second compound is 6-aminopenicillanic acid or 7-aminodesacetoxycephalosporanic acid. ; and said third compound may be optionally substituted, especially unsubstituted, phenylacetic acid.

In one embodiment of the present invention, the penieilinacylase enzyme, which is anchored on an insoluble matrix, is charged into a jacketed reaction vessel, then a suitable volume of water is added and, with stirring, penicillin-G is added. The reaction vessel is sealed and evacuated to a pressure of 1 kPa or less. The first solvent is charged into the reaction vessel. An aqueous sodium hydroxide solution (2.5 to 20% w / v or more) is added to maintain the pH in the range of 7.0 to 9.0, in particular around 8.0. The reaction temperature is maintained approximately constant at all times. The preferred temperature range is between 20 and 50 ° C, more preferably between 30 and 40 ° C.

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0 0000 0 · I · <4 0 0 000000 0 0 0 0 · · 0 · 000 ···· · ··. ♦ »« »

After completion of the reaction, which is determined by the pH remaining constant at the pH value without further addition of sodium hydroxide, the reaction solutions are transferred from the bottom of the reaction vessel via an in-line filter to a second jacketed vessel (evaporator vessel). In this way the anchored enzyme is removed, washed with water and stored for further use. '

The clear filtrate solution is cooled by passing the cooling medium through the jacket. The preferred temperature is between 0 and 20 ° C. more preferably 2 to 10 ° C. While stirring, an aqueous solution of an inorganic acid (such as 1 to 4 M nitric acid or 1 to 4 M sulfuric acid) is slowly added until a pH of 3.5 to 4.0 is reached, ideally a pH of 3.8. During this operation, the phenylacetic acid (PAA) present as the sodium salt is converted into the free acid form which is soluble in the first solvent. While PAA is extracted into the first solvent, 6-APA (as the free acid) precipitates and is now. present as a suspension. The reaction mixture may be stirred further. 30 minutes to 1 hour, maintaining a constant pH and temperature.

The 6-APA can be conveniently and simply isolated by filling the reaction mixture from the bottom of the evaporation vessel back into the reaction vessel via a filter. Optionally, the filter element may be attached to the outlet of the bottom of the container as a wire mesh filter or a glass sinter filter.

According to a second embodiment, the present invention provides a method of removing a third compound (preferably an acid or salt, in particular PAA) as described herein from the mass of the compound-containing material, the method comprising:

(a) contacting the mass of the material with a solvent (e.g., said first solvent or, in particular, said second solvent described according to the invention) by saturating the solvent with said third compound; and

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Μ · 0 0 · f · f 0 0 000 0 · 0 ft

(B) separating a saturated solvent from the remainder of said material mass.

Said third compound contacted according to the method is preferably a free acid.

Any feature of any embodiment of any invention or embodiment described herein may be combined with any feature of any embodiment of any invention or embodiment described herein.

Specific embodiments of the present invention will be further described by way of example.

In addition, the following abbreviations are used:

Pen-G - means penicillin-G as illustrated in Scheme 1.

6-PAA - means 6-aminopenicillanic acid.

PAA - means phenylacetic acid.

MIBK - means methylisobutylketone.

Phytosol means 1,1,1,2-tetrafluoroethane.

Phytosol D - means a mixture containing dimethyl ether (10% by weight) and 1,1,1,2-tetrafluoroethane (90% by weight).

All analyzes mentioned below were performed using HPLC as follows:

Mobile phase: 25 mM ammonium acetate in 1: 1 methanol / water + acetic acid to pH 6.0. Column: .3,9 x 300 mm column, reverse phase cartridge 10 mikron C18. Detection: 23 0 nm Injection: 10 μΐ.

Examples containing the letter “C” are comparative.

 * «· ·. * * * *......

»· · · · · · · · ♦ · · · · · · · · · · · · · · · · · · ·

Examples of external design

Example C1 - Standard (Known) Preparation of 6-APA (i) Enzymatic Deacylation of Pen-G

Water (480 mL) and Penicillin-G (30 g) were charged into a beaker in a water bath set at 37 ° C. The mixture is gently stirred until the temperature has stabilized at 37 ° C. Enzyme resin (38 g) containing penicillin acylase was added to the polymer resin matrix followed by 5% sodium hydroxide solution to pH 8.0. Stirring is continued while maintaining the pH at 8.0 and at 37 ° C until a stable state is reached. This process takes about 2 hours. The total volume of the 5% sodium hydroxide solution used is about 65 ml. The enzyme resin is filtered through a sinter funnel and the resin is washed with water and stored in a refrigerator for further use. The solution containing 6-APA and PAA is treated as described in step (ii) below.

(ii) Isolation of 6-APA

The enzyme solution containing 6-APA and PAA is concentrated four times, then cooled to 5 ° C and an equal volume of MIBK is added. Add 2M nitric acid dropwise to pH 3.8. The pH is maintained at 3.8 1 hour during which 6APA free acid precipitates. The precipitate is then filtered off, washed with MIBK, then with acetone and dried.

Example 1 - General Procedure of the Invention

Methods (iii) and (iv) below are alternative and both can be used to isolate 6-APA.

(i). Equipment

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The apparatus for carrying out the method comprises a reaction vessel and an evaporation vessel, both of which are jacketed to control the temperature. Furthermore, both vessels are equipped with burettes for reagent addition and equipment for mixing, temperature and pH measurements. Both containers are connected to each other and to the vacuum pump and gas compressor so that. For example, the reaction streams may be transferred from one vessel to another and the volatile solvent used (described in accordance with the invention) may be transferred to and from both vessels and a suitable solvent storage container. In-line filters, one-way valves and pressure relief valves can be incorporated to provide a suitable and safe apparatus for carrying out this method.

(ii) Enzymatic deacylation of Pen-G

Charge the reaction vessel with water (100 mL) and Pen-G (40 g), followed by an enzyme resin (50.6 g). The vessel is sealed and evacuated to a pressure of less than 1 kPa and stirring is initiated. Phytosol is added. A (1-2 kg) and the temperature is stabilized at 37 ° C by passing hot water through the jacket. The pH is maintained at 8.0 by the addition of 5% sodium hydroxide solution using the addition burette until the reagent is stable. The total volume of sodium hydroxide solution needed is about 90 ml.

(iii) Isolation of 6-APA

The enzyme liquid containing 6-APA and PAA is refilled into the reaction vessel. Phytosol D (1-2 kg) was added and stirring was started. Cooling is performed by passing cold water through the jacket. Using a reagent burette, 2M nitric acid is slowly added to pH 3.8 and stirring is continued for 1 hour while maintaining the pH at 3.8. During this time, the free acid precipitates 6-APA, while PAA remains dissolved in Phytosol D.

 · · (((• • • • • • • • • • • • • • • • • • • • • • • • • •

....... ...

The reaction mixture was charged to an evaporation vessel via an in-line filter to retain the 6APA precipitate. Phytosol D is then recirculated through the reaction vessel (to wash the 6-APA product) and the in-line filter is deflected back to the storage cylinder 30 minutes after the Phytosol D has passed. The 6-APA precipitate is then isolated from the in-line filter. When all of the Phytosol has evaporated, the remaining aqueous solution contains PAA as an oil suspension.

(iv) isolation of 6-APA (alternative method)

The enzymatic liquid containing 6-APA and PAA is mixed with Phytosol A or Phytosol D or with an alternative solvent mixture containing 1,1,1,2-tetrafluoroethane and another solvent such as an aliphatic alcohol, ketone or ether. An inorganic acid such as 1 to 4M nitric acid or sulfuric acid is added slowly until the pH drops below

2.5. The pH is suitably adjusted to a value of 1.5 to 2.0. The temperature is lowered to between 2 and 20 ° C during the acid reaction. The two immiscible layers were separated by standing the mixture and the bottom layer containing the PAA solution in the solvent was drained. The top layer containing 6-APA is reacted with stirring at reduced temperature with an aqueous base solution such as sodium hydroxide or potassium hydroxide until a pH of 3.8 is reached at which the 6-APA free acid precipitates. The precipitated 6-APA is isolated by filtration.

(v) PAA isolation

The aqueous solution containing the oily PAA suspension obtained after step (iii) is extracted into a Phytosol solvent and the immiscible Phytosol layer containing PAA in solution is separated from the aqueous layer. Isolation of PAA is accomplished by evaporation of Phytosol. Alternatively, PAA can be isolated after step (iv) by evaporating the solvent from the isolated PAA solution.

15 Dec • 0000 00 0 0 «00 ·· · · 0 0 0 0 0 • • '0 0 0 0 0 0 0 0 • 000 000000 «00 0 • · 0 00 0 0 00 0 000 00 00 0 0 Example C2 - Preparation of 6-APA The method described in Example C1 is followed getting the foundation for comparison with other examples. At way with Pen-G (30 g) reacts with immobilized enzyme and final the product (6-APA) was dried, weighed and tested using high pressure liquid chromatography (HPLC). The results are as follows: Weight of Pen-G used = 30.0 g Weight of immobilized enzyme = 38.0 g Volume of water used = 4,880 ml Volume of 5% NaOH used = 65 ml The volume of enzyme liquid obtained = 550 ml Enzyme Liquid Test · = 31 100 gg / yr i- ko-6-APA The yield of the conversion step = 96% Weight obtained by 6-APA = 13.23 g Total yield from Pen-G = 74%

Example 2

The suspension was prepared in a reaction vessel containing Pen-G (40 g), water (50 mL) and Phytosol (2 kg). A 5% sodium hydroxide solution was added over 2 hours maintaining the pH at 8.0. The total volume of sodium hydroxide solution used to convert completely was 85 ml. After completion of the reaction, the enzyme resin is filtered off and the phytosol is evaporated back to the storage tank.

Enzyme liquid volume = 160 ml

Enzyme Liquid Test - 142,300 gg / ml

Weight obtained 6-APA = 22.76 g

Conversion yield = 98.2%.

80 ml of enzyme liquid is precipitated and 6-APA is isolated according to the method described in Example 1 (iii).

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Weight obtained 6-APA = 9.6 g

Total Pen-G yield - 84.4% • 00 0 «» 0 0 «« 0 0

0 · ·

0 0 0

0 «0

0,000 «« 0

0 0 · 0 0

The product obtained is dry and no further vacuum drying is required.

Example C3

To obtain a direct comparison with the 6-APA isolation step of Example 2, a second 80 ml portion of the enzyme liquid was precipitated using MIBK, washed with acetone, and dried under vacuum for 20 hours. Weight obtained 6-APA = 9.45 g

Isolation step yield = 83.0%.

Example 3 - Effect of pH on PAA extraction

This assay is performed to evaluate the efficacy and selectivity of Phytosol D in extracting PAA from the enzyme liquid at various pH values.

The enzyme liquid was prepared in the same manner as described in Example 1 using the resin with the enzyme of Example C2.

The 20 ml liquid samples are extracted with 40 ml of Phytosol D at various pH values using a hand held apparatus as follows:

The apparatus consists of a 100 ml volumetric glass tube fitted with a filter assembly and a collar fitted with a needle valve. The substance or solution to be extracted is filled into a tube. The filter is then assembled by means of a sealing ring, then by a sleeve which ensures that a tight connection is achieved. Phytosol gas is introduced into the glass tube from the aerosol can via a needle valve. The contents of the tube are mixed by vigorous shaking, then the tube is inverted and allowed to stand until the layers are separated. Phytosol is then

/ «··· * · '· 00 0 0 · · 0 0 0 0 · 00 0 00 0 • 0 0000 0 0 · 0 0 • · · · 0 0 0 00 0 00 00 released through a needle valve into an evaporation vessel taking care not to extrude the aqueous layer. The results are shown in the table below.

PH Obtained 6-APA (total% by weight) Obtained by PAA (total% by weight) 8, 0 0 0 .6,5 0 <2 5.3 0 6.5 3.8 <1 > 99 1.4 <1 > 99

Example 4 - Effect of Phytosol A presence on enzyme yield

The experiment is carried out in order to determine the effect of the presence of Phytosol A on the enzymatic yield - i.e. (6-APA obtained by the enzyme) - (theoretical yield of 6-APA). In the experiment, Pen-G (30 g) was dissolved in water (300 mL) and deacylation was carried out according to the procedure described in Example 1.

Volume of 2.5% NaOH used = 134 ml

Volume of the obtained enzyme liquid = 450 ml

Enzyme Fluid Assay = 38,120 pg / ml as 6-APA

Enzyme yield = 98.2% of theory

It should be noted that the yield is higher in the presence of Phytosol A (compared to Example 4 and Example C2).

Example 5 - Effect of using a more concentrated Pen-G solution

This experiment involves conversion of a more concentrated PenG solution to omit the further concentration step prior to 6-APA precipitation and the isolation step of Example 1 (iii). In the experiment, Pen-G (30 g) was dissolved in water (150 ml) and Phytosol A (2 kg) was used and the method was carried out as described above.

Final volume of enzyme liquid = 350 ml ····

Enzyme Fluid Test Conversion yield = 4800 µ9 / τυ1 as 6-APA = 96%

6-APA was isolated as described in Example 1 (iii) by adding enzyme liquid to Phytosol D (2 kg) and acidifying to pH 3.8 using 1N nitric acid.

Weight of isolated product 6-APA - 5.6 g

It should be noted that the concentration of the enzyme liquid is preferably as high as possible, thereby minimizing the losses required by 6-APA in the mother liquors.

Example 6 - Isolation of PAA

The above aqueous solution (300 ml) containing PAA (about 14 g) in a fox oil suspension was charged to the reaction vessel. Phytosol D was charged to the flask and stirred for 30 minutes. The two layers were allowed to settle for 15 minutes, then separated using a viewing window at the bottom of the vessel. Phytosol D is then evaporated and returned to the supply cylinder. PAA is obtained from the vessel as a whitish crystalline solid.

Weight of PAA obtained = 14.2 g

Although it is difficult to accurately determine the PAA content of the starting solution, it has been shown that a yield close to the theoretical yield can be achieved.

Example 7 - Use of sulfuric acid for pH adjustment

The enzymatic reaction was carried out according to the procedure generally described in Example 1 (ii) using Pen-G (40 g), enzyme resin (61 g), water (50 ml), 5 µl ^. % sodium hydroxide solution to maintain pH at 8.0 and 37 ° C.

0000 • 0 · 00 00 • 0 0 000 000 0 • * 0 * 00 0000 0 · 0 0 0 0000 0 0 0 0 «000 00 0 0 00 0 000 00 00 00 00 ··

The results are as follows: Volume of Enzyme Liquid Concentration

Enzyme Step Yield = 145 mL = 13.2% (HPLC) = 83%

Precipitation and isolation were carried out as described in Example 1 (iii) using a mixed solvent containing Phytosol (2 kg) and isopropanol (30 mL). 6-APA is precipitated by the addition of 1M. sulfuric acid to pH 3.8 at 4 ° C.

6-APA is isolated as a white solid.

Product weight = 14.8 g

HPLC analysis showed that PAA is only present as a very small trace.

Preferred embodiments of the present invention have the following advantages:

The enzyme reaction can be carried out in a solution with a higher Pen-G concentration than hitherto, thereby eliminating or reducing the need for a potentially expensive thickening step.

A more efficient enzyme reaction is achieved, which is faster and / or has a higher yield. The conversion yield turned out to be 98% or higher.

The enzyme activity is not impaired by said solvent system.

It eliminates or reduces the need for a large amount of organic solvents, thereby eliminating problems associated with such solvents as storage, recovery and processing prior to spillage.

Phytosol has been shown to have good removal efficiency

PAA, while the solubility of 6-APA in the solvent is negligible.

• ···· 44 4 • · 4 .4 4 4 4 21 · * · ·· 4 44 44 4 4 4 4 4 4 • • 44 4 44 4 44 4444444 44 5 in * 4 4 »» 4 9 44 44 Crystal forms se. can change for use another solvents during precipitation of 6-APA, which may be advantageous in the

co-current processing.

Dry 6-APA is obtained directly without the need for further drying of the product.

Overall, the process is faster and cheaper than established industrial processes.

The present invention is not limited to penicillin and cephalosporin enzymes in a broad sense, but is also suitable for other similar enzymes including acylases, amidases, proteases, and esterases. The solvents described in the present invention can be used to significantly increase the reaction rates and facilitate product recovery.

Readers should be reminded of all articles and documents filed simultaneously or prior to this application in connection with this application and which are accessible to the public for review together with this application and whose content is incorporated herein by reference.

All embodiments included in the present application (including the appended claims, annotation and figures) and / or all steps of any method or process herein may be combined in any combination except combinations where at least some of these embodiments and / or steps are mutually exclusive.

Unless intended otherwise, all embodiments of the present application (including the appended claims, annotations, and drawings) may be replaced by alternative embodiments serving the same, equivalent, or similar purposes. Thus, unless stated otherwise, each embodiment of the present invention is only one example of a general series of equivalent or similar embodiments.

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The invention is not limited to the details of the preceding embodiments. The invention includes any new embodiments or novel combinations of embodiments described herein (including the appended claims, annotation and figures) or any new steps or novel combinations of steps of any method of the present invention.

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Claims (14)

, PATENT N A process for preparing a second compound by catalytic conversion of a first compound, comprising contacting said first compound with a catalyst in a solvent mixture comprising water and a first non-aqueous solvent. 2. The process of claim 1 wherein said catalyst is an enzyme. The method of claim 2, wherein said enzyme is sensitive to said second compound such that relatively high concentrations of said second compound reduce the ability of the enzyme to effect conversion. The method of any one of claims 2 or 3, wherein said enzyme is capable of catalyzing a deacylation reaction. The method of any one of:. wherein R @ 1 HQ is an alkylcarbonyl group and a cyclic group. of claims 1 to 4, said first compound having R ¹ being optionally substituted Q is optionally substituted The method of any one of the first solvents to the solvents is at least 2 of claims 1 to 5, wherein the weight ratio of said weight of water in said mixture is indicated. A method according to any one of claims 1 to 6, wherein the ratio of said first compound to water present in said mixture, in% w / v, is at least 10. »· · · · · · · · · · · · · · · · · · · - · 0 · 0 0000 0 0. · 0 4 0 0 00 0 0 0 0 4 • · * ·· 0 00 00 8. The process of any one of claims 1 to 7, wherein said first non-aqueous solvent has a boiling point at atmospheric pressure of less than 80 ° C and greater than -90 ° C. The process of any one of claims 1 to 8, wherein said first solvent is. organic. A process according to any one of claims 1 to 9, wherein said first solvent comprises an optionally substituted C 1 -C 4 alkane. Process according to any one of claims 1 to 10, characterized in that said first solvent is halogenated. The process according to any one of claims 1 to 11, wherein said first solvent is non-chlorinated. 13 . The method of any one of claims 1 to 12, wherein said first solvent is tetrafluoroethane. The method of any one of claims 1 to 13, comprising the step of isolating the second compound from the other compounds / solvents by precipitating the second compound and subsequently recovering the precipitate. The method of any one of claims 1 to 13, comprising adjusting the pH in the presence of the second solvent to a value at which the second compound is substantially soluble in the aqueous phase of the solvent mixture, while the reaction by-products are substantially soluble in the immiscible said second phase of the solvent mixture. 9 * 99 • 9 · · · · · · · · · 9 9 ♦ 9 9 9 Method according to any one of claims 1 to 16, characterized by: characterized in that said first compound is natural penicillin or a biosynthetic penicillin prepared by adding a precursor to the penicillin fermentation broth, or. cephalosporin and said second compound is 6-aminopenicillanic acid or 7-aminodesacetoxycephalosporanic acid. 17. A method of removing a third compound, for example an acid or acid salt, especially phenylacetic acid, from a mass. a compound-containing material comprising: a) contacting the mass of material with the solvent by saturating the solvent with said third compound; and b) separating the saturated solvent from the remainder of said mass of material. . .