This invention relates to the method of enzymatic resolution of serine racemic derivative.

The method used thus far has involved producing a racemic mixture of a-methylserine enantiomers which is converted into a mixture of secondary amine diastereomers. This requires using a costly enantiomerically pure reagent.

The method of enzymatic resolution of the racemic derivative of a-methylserine involves acylation of N-blocked-a-substituted serine ester in a homogeneous system with the use of an ancyl group donor in a solvent selected from among polar solvents. The N-blocked-a- substituted serine ester is preferably N-benzoyl-a-methylserine ethyl ester or N-benzoyl-a-ethylserine ethyl ester or N-benzoyl-a-(4- benzamidobutylo) serine ethyl ester. In a preferred embodiment of the invention, solvents are selected from among ether-like solvents, more specifically hexane/THF, methyl tert-butyl ether, 1,2- dichloroethane. In the most preferred embodiment of the invention, hexane/THF is used and the optimum composition of the solvent is 50-70 % of hexane, kerosene fraction, and 30-50 % of THF.

In a preferred embodiment of the invention, the enzyme used as a catalyst is a lipase immobilised using any method and selected from among Lipozyme RM IM, i.e. Mucor miehei lipase immobilised on ion-exchange resin, Lipozyme TL IM, i.e. Thermomyces lanuginosus lipase immobilised on acrylic resin, and Novozym 435, i.e. Candida antarctica Lipase B immobilised on acrylic resin. Lipozyme RM IM, Lipozyme TL IM, Novozym 435 are trade names owned by Novozymes A/S from Danemark. The optimum catalyst for N-benzoyl-a-methyl serine ethyl ester is Novozym 435, for N-benzoyl-a-ethylserine ethyl ester is Lipozyme TL IM , and for N-benzoyl-a-(4-benzamidobutylo) serine ethyl ester is Lipozyme RM IM . The acyl group is donated by the ester selected from among enol esters, preferably vinyl acetate and/or isopropenyl acetate. The optimum donor of acyl group is isopropenyl acetate for N-benzoyl-a-methylserine ethyl ester and vinyl acetate for N- benzoyl-a-ethylserine ethyl ester and/or N-benzoyl-a-(4- benzamidobutylo) serine ethyl ester .

In a preferred embodiment of the invention, for N-benzoyl-a- methylserine ethyl ester the acylation is carried out at the molar ratio of the substrate to the acyl group donor in the range of 1 to 2 up to 1 to 4, and the optimum substrate/acyl group donor molar ratio is 1 to 2. There is 20 to 100 mg of the enzyme and 2 to 12 ml of the solvent per 1 mmol of the substrate, preferably 40 mg of the enzyme and 4 ml of the solvent per 1 mmol of the substrate. The acylation is carried out at a temperature of 5 to 50 degrees Celsius, preferably 30 degrees Celsius.

In a preferred embodiment of the invention, for N-benzoyl-a- ethylserine ethyl ester the acylation is carried out at the molar ratio of the substrate to the acyl group donor in the range of 1 to 2 up to 1 to 6, and the optimum substrate/acyl group donor molar ratio is 1 to 4. There is 100 to 400 mg of the enzyme and 2 to 12 ml of the solvent per 1 mmol of the substrate, preferably 400 mg of the enzyme and 6 ml of the solvent per 1 mmol of the substrate. The acylation is carried out at a temperature of 5 to 50 degrees Celsius, preferably 30 degrees Celsius.

In a preferred embodiment of the invention, for N-benzoyl-a- (4-benzamidobutylo) serine ethyl ester the acylation is carried out at the molar ratio of the substrate to the acyl group donor in the range of 1 to 2 up to 1 to 6, and the optimum substrate/acyl group donor molar ratio is 1 to 4. There is 100 to 400 mg of the enzyme and 2 to 12 ml of the solvent per 1 mmol of the substrate, preferably 400 mg of the enzyme and 8,4 ml of the solvent per 1 mmol of the substrate. The acylation is carried out at a temperature of 5 to 50 degrees Celsius, preferably 30 degrees Celsius.

An advantage of the method according to the invention consists in reducing the costs of obtaining an enantiomerically pure product, using an environmentally-friendly catalyst and limiting emissions to the environment. The process causes practically no losses and enables full recovery of solvents and reagents as well as multiple use of the catalyst.

The subject of the invention is shown by way of examples of the embodiment.

Example I

A 500 ml conical flask was filled with 20 mmol of N-benzoyl- a-methylserine ethyl ester in the form of light straw-coloured oil and 80 ml of a mixture composed of 70 per cent of hexane, kerosene fraction, and 30 per cent of THF. The flask was closed with a ground glass stopper and secured with a clip, and then stirred for approximately 10 minutes until the ester was dissolved. The flask was opened and 40 mmol of isopropenyl acetate and 800 mg of Novozym 435 were placed inside. The flask was closed with a ground glass stopper and secured with a clip, and put on an orbital shaker thermostated at 30 degrees Celsius. The shaker speed was set at 120 rpm. The reaction was carried out for 16 hours, controlled with HPLC, using a chiral stationary phase (Chiralcell OD column, 250 x 4.6 mm), mobile phase of hexane and isopropanol at 95:5, flow rate at 0.5 ml/min, column temperature at 25 degrees Celsius, 250 nm UV detector. After 16 hours, the enzyme was filtered off under reduced pressure. The filtrate was evaporated on a rotary evaporator, and the obtained N-benzoyl-a-methylserine ethyl ester acetate was separated from unreacted substrate by column chromatography on silica gel 60, mobile phase of hexane/ethyl acetate at 85: 15.

Approximately 2 g of enantiomerically pure (S)-benzoyl-a- methylserine ethyl ester and approximately 3.5 g of enriched (R)- benzoyl-a-methylserine ester acetate were obtained. The enriched ester acetate was subjected to alcoholysis to eliminate the acyl group and re-used in the enzymatic reaction. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis, followed by purification on ion-exchange resin to obtain enantiomerically pure amino acid. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis to obtain enantiomerically pure amino acid. The N-benzoyl amino acid resulting from the basic hydrolysis was dissolved in methanol and tested for rotation using a polarimeter at 589 nm, and then compared with the values in the literature to confirm the enantiomeric purity of the product. The purity of the product of basic hydrolysis was also confirmed based on the melting point and comparison with the values in the literature.

Example II

A 750 ml round-bottomed flask was filled with 20 mmol of N- benzoyl-a-methylserine ethyl ester in the form of light straw- coloured oil and 80 ml of a mixture composed of 70 per cent of hexane, kerosene fraction, and 30 per cent of THF. The flask was closed with a ground glass stopper and secured with a clip, and then stirred for approximately 10 minutes until the ester was dissolved. The flask was opened and 40 mmol of isopropenyl acetate and 800 mg of Novozym 435 were placed inside. The flask was closed with a ground glass stopper and secured with a clip, and put on reciprocal shaker thermostated at 30 degrees Celsius. The shaker speed was set at 100 rpm. The reaction was carried out for 16 hours, controlled with HPLC, using a chiral stationary phase (Chiralcell OD column, 250 x 4.6 mm), mobile phase of hexane and isopropanol at 95:5, flow rate at 0.5 ml/min, column temperature at 25 degrees Celsius, 250 nm UV detector. After 16 hours, the enzyme was filtered off under reduced pressure. The filtrate was evaporated on a rotary evaporator, and the obtained N-benzoyl-a-methylserine ethyl ester acetate was separated from the unreacted substrate by column chromatography on silica gel 60, mobile phase of hexane/ethyl acetate at 85: 15. Approximately 1.9 g of enantiomerically pure (S)-benzoyl-a- methylserine ethyl ester and approximately 3.6 g of enriched (R)- benzoyl-a-methylserine ester acetate were obtained. The enriched ester acetate was subjected to alcoholysis to eliminate the acyl group and re-used in the enzymatic reaction. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis, followed by purification on ion-exchange resin to obtain enantiomerically pure amino acid. The N-benzoyl amino acid resulting from the basic hydrolysis was dissolved in methanol and tested for rotation using a polarimeter at 589 nm, and then compared with the values in the literature to confirm the enantiomeric purity of the product. The purity of the product of basic hydrolysis was also confirmed based on the melting point and comparison with the values in the literature.

Example III

A 100 ml conical flask was filled with 4 mmol of N-benzoyl-a- (4-benzamidobutylo) serine ethyl ester in the form of colorless crystals and 33,6 ml of a mixture composed of 50 % of hexane, kerosene fraction, and 50 % of THF. The flask was closed with a ground glass stopper and secured with a clip, and then stirred for approximately 10 minutes until the ester was partially dissolved. The flask was opened and 16 mmol of vinyl acetate and 1600 mg of Lipozyme TL IM were placed inside. The flask was closed with a ground glass stopper and secured with a clip, and put on an orbital shaker thermostated at 30 degrees Celsius. The shaker speed was set at 180 rpm. The reaction was carried out for 384 hours, but after 338 800mg of Lipozyme TL IM was added. The reaction was controlled with HPLC, using a chiral stationary phase (Chiralcell OD column, 250 x 4.6 mm), mobile phase of hexane and isopropanol at 90: 10, flow rate at 1 ml/min, column temperature at 25 degrees Celsius, 225 nm UV detector. After 384 hours, the enzyme was filtered off under reduced pressure. The filtrate was evaporated on a rotary evaporator, and the obtained N-benzoyl-a-(4-benzamidobutylo) serine ethyl ester acetate was separated from unreacted substrate by column chromatography on silica gel 60, mobile phase of hexane/ethyl acetate at 50:50.

Approximately 600mg of enantiomerically pure (S)-benzoyl-a- (4-benzamidobutylo) serine ethyl ester and approximately 1.1 g of enriched ( )-benzoyl-a-(4-benzamidobutylo) serine ester acetate were obtained. The enriched ester acetate was subjected to alcoholysis to eliminate the acyl group and re-used in the enzymatic reaction. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis, followed by purification on ion-exchange resin to obtain enantiomerically pure amino acid. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis to obtain enantiomerically pure amino acid. After the alkaline hydrolysis step, the resulting N- benzoylamino acid was recrystallized from wet ethyl acetate and the absolute configuration was confirmed in a X-ray study of the crystal.

Example IV

A 250 ml conical flask was filled with 8 mmol of N-benzoyl-a- (4-benzamidobutylo) serine ethyl ester in the form of of colorless crystals and 67,2 ml of a mixture composed of 50 per cent of hexane, kerosene fraction, and 50 per cent of THF. The flask was closed with a ground glass stopper and secured with a clip, and then stirred for approximately 10 minutes until the ester was partially dissolved. The flask was opened and 32 mmol of vinyl acetate and 3200 mg of Lipozyme TL IM were placed inside. The flask was closed with a ground glass stopper and secured with a clip, and put on reciprocal shaker thermostated at 30 degrees Celsius. The shaker speed was set at 100 rpm. The reaction was carried out for 384 hours, but after 338 800mg of Lipozyme TL IM was added. The reaction was controlled with HPLC, using a chiral stationary phase (Chiralcell OD column, 250 x 4.6 mm), mobile phase of hexane and isopropanol at 90: 10, flow rate at 1 ml/min, column temperature at 25 degrees Celsius, 225 nm UV detector. After 384 hours, the enzyme was filtered off under reduced pressure. The filtrate was evaporated on a rotary evaporator, and the obtained N-benzoyl-a-(4-benzamidobutylo) serine ethyl ester acetate was separated from unreacted substrate by column chromatography on silica gel 60, mobile phase of hexane/ethyl acetate at 50:50.

Approximately 1.3 g of enantiomerically pure (S)-benzoyl-a-(4- benzamidobutylo) serine ethyl ester and approximately 2 g of enriched ( )-benzoyl-a-(4-benzamidobutylo) serine ester acetate were obtained. The enriched ester acetate was subjected to alcoholysis to eliminate the acyl group and re-used in the enzymatic reaction. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis, followed by purification on ion-exchange resin to obtain enantiomerically pure amino acid. Po etapie hydrolizy zasadowej otrzymany N-benzoiloaminokwas rekrystalizowano z mokrego octanu etylu. After the alkaline hydrolysis step, the resulting N-benzoylamino acid was recrystallized from wet ethyl acetale.

Example V

A 250 ml conical flask was filled with 8 mmol of N-benzoyl-a- ethylserine ethyl ester in the form of yellow oil and 48 ml of a mixture composed of 70 per cent of hexane, kerosene fraction, and 30 per cent of THF. The flask was closed with a ground glass stopper and secured with a clip, and then stirred for approximately 10 minutes until the ester was dissolved. The flask was opened and 32 mmol of vinyl acetate and 3200 mg of Lipozyme RM IM were placed inside. The flask was closed with a ground glass stopper and secured with a clip, and put on an orbital shaker thermostated at 30 degrees Celsius. The shaker speed was set at 180 rpm. The reaction was carried out for 161 hours, controlled with HPLC, using a chiral stationary phase (Chiralcell OD column, 250 x 4.6 mm), mobile phase of hexane and isopropanol at 95:5, flow rate at 0.5 ml/min, column temperature at 25 degrees Celsius, 225 nm UV detector. After 161 hours, the enzyme was filtered off under reduced pressure. The filtrate was evaporated on a rotary evaporator, and the obtained N-benzoyl-a-ethylserine ethyl ester acetate was separated from unreacted substrate by column chromatography on silica gel 60, mobile phase of hexane/ethyl acetate at 85: 15.

Approximately 1 g of enantiomerically pure (S)-benzoyl-a- ethylserine ethyl ester and approximately 1.5 g of enriched (R)- benzoyl-a-ethylserine ester acetate were obtained. The enriched ester acetate was subjected to alcoholysis to eliminate the acyl group and reused in the enzymatic reaction. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis, followed by purification on ion-exchange resin to obtain enantiomerically pure amino acid. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis to obtain enantiomerically pure amino acid. After the alkaline hydrolysis step, the resulting N- benzoylamino acid was recrystallized from wet ethyl acetate and the absolute configuration was confirmed in a X-ray study of the crystal.

Example VI

A 250 ml flat-bottomed flask was filled with 12 mmol of N- benzoyl-a-ethylserine ethyl ester in the form of yellow oil and 72 ml of a mixture composed of 70 per cent of hexane, kerosene fraction, and 30 per cent of THF. The flask was closed with a ground glass stopper and secured with a clip, and then stirred for approximately 10 minutes until the ester was dissolved. The flask was opened and 48 mmol of vinyl acetate and 800 mg of Lipozyme RM IM were placed inside. The flask was closed with a ground glass stopper and secured with a clip, and put on reciprocal shaker thermostated at 30 degrees Celsius. The shaker speed was set at 120 rpm. The reaction was carried out for 158 hours, controlled with HPLC, using a chiral stationary phase (Chiralcell OD column, 250 x 4.6 mm), mobile phase of hexane and isopropanol at 95:5, flow rate at 0.5 ml/min, column temperature at 25 degrees Celsius, 225 nm UV detector. After 158 hours, the enzyme was filtered off under reduced pressure. The filtrate was evaporated on a rotary evaporator, and the obtained N-benzoyl-a-ethylserine ethyl ester acetate was separated from the unreacted substrate by column chromatography on silica gel 60, mobile phase of hexane/ethyl acetate at 85: 15.

Approximately 1.3 g of enantiomerically pure (S)-benzoyl-a- ethylserine ethyl ester and approximately 2 g of enriched (R)- benzoyl-a-ethylserine ester acetate were obtained. The enriched ester acetate was subjected to alcoholysis to eliminate the acyl group and re-used in the enzymatic reaction. The resulting enantiomer (S) of the ester was dissolved in methanol, and the absolute configuration was confirmed by comparing the specific optical rotation with the reaction product of Example V. The resulting (S) enantiomer of the ester was subjected to two-stage acidic and basic hydrolysis, followed by purification on ion-exchange resin to obtain enantiomerically pure amino acid. Po etapie hydrolizy zasadowej otrzymany N-benzoiloaminokwas rekrystalizowano z mokrego octanu etylu. After the alkaline hydrolysis step, the resulting N- benzoylamino acid was recrystallized from wet ethyl acetale.

The products obtained by the method according to the invention can be used in the synthesis of atypical oligopeptides, peptoids and peptidomimetics as well as a chiral building block in organic synthesis and manufacture of pharmaceuticals.