ethyl (3S)-3-hydroxy-4-iodobutanoate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R).

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 72 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R).

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 72 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography

Conditions: temp:30.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: The recombinant E. coli HB101(pNTS1G) obtained in Example 10 was inoculated in 100 ml of a 2×YT medium sterilized in a 500 ml Sakaguchi flask, and cultured with agitation at 37° C. for 13 hours. Glucose, 0.5 g, 3.2 mg of NADP, and then 0.5 g of ethyl 4-iodoacetoacetate were added to 50 ml of the resultant culture. The culture was stirred at 30° C. while being adjusted at pH 6.5 with a 5 M sodium hydroxide solution to allow for reaction for 72 hours. After the reaction, the reaction solution was subjected to extraction using ethyl acetate, the solvent was removed under decompression, and the concentrate was purified by silica gel column chromatography, to obtain 900 mg of ethyl (S)-4-iodo-3-hydroxybutyrate. The optical purity of the ethyl (S)-4-iodo-3-hydroxybutyrate was analyzed as follows and found to be 91.6% e.e. That is, the sample was heated together with sodium cyanide in dimethyl sulfoxide to obtain ethyl 4-cyano-3-hydroxybutyrate, which was then changed to a benzoic ester using benzoyl chloride under the presence of pyridine. The optical purity of the benzoic ester was measured by an HPLC method. NMR(CDC13)δ(ppm): 1.28(3H,t), 2.65(2H,d), 3.31(3H,m), 4.00(1H,m), 4.20(2H,q); Column: Chiralpak AS (0.46×25 cm) manufactured by Daicel Chemical Industries, Co., Ltd.; Column temperature: 25° C.; Eluent: n-hexane/ethanol of 95/5; Flow rate: 1 ml/min.; Detection: 254 nm; Elution time: 19.6 minutes for (S), 21.3 minutes for (R). coli HB101(pNTS1G) obtained in Example 10 to allow for reaction for 72 hours After the reaction the reaction solution was subjected to extraction the solvent was removed under decompression the concentrate was purified by silica gel column chromatography

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of imidazole (252.3 g, 7.36 mol) in dry DMF (7.6 L) are added ethyl 3(S)-hydroxy-4-iodobutyrate (2) (949.6 g, 3.68 mol, GC purity 85.9%) and anhydrous NaI (1106 g, 7.36 mol) under argon atmosphere at ambient temperature. The suspension is cooled to 0° C. and tert-butyl(chloro)dimethylsilane (838 g, 5.56 mol) is added portionwise. The reaction mixture is stirred at 0° C. for 1.5 hours followed by stirring for 15.5 hours from 0° C. to ambient temperature. Then the mixture is cooled to 0° C. and H2O (4.2 L) is added. After 2 hours of stirring additional amount of H2O (6.2 L) and saturated Na2S2O3 solution (0.5 L) are added. The product is extracted with t-BuMeO. The combined organic layers are washed with water and dried (MgSO4). Ether is removed completely under the reduced pressure (20 mbar) at 60° C. to produce a yellow oily residue. This residue is purified further by vacuum distillation (80-89° C. at 0.150-0.310 mbar) to give 1193.5 g (97%) of ethyl 3(S)-(tert-butyldimethylsilyloxy)-4-iodobutyrate (3) as a pale yellow oil (GC purity 96.1%). 1H NMR (300 MHz, CDCl3): δ 4.13 (qd, J=7.2 and 2.1 Hz, 2H), 4.01 (m, 1H), 3.28 (dd, J=10.2 and 4.2 Hz, 1H), 3.24 (dd, J=10.2 and 6.0 Hz, 1H), 2.66 (dd, J=15.3 and 4.8 Hz, 1H), 2.51 (dd, J=15.3 and 7.2 Hz, 1H), 1.26 (t, J=7.2 Hz, 3H), 0.87 (s, 9H), 0.10 (s, 3H), 0.05 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 170.8, 68.3, 60.5, 42.5, 25.6, 17.9, 14.1, 12.9, −4.6, −5.0. by stirring for 15.5 hours from 0° C. to ambient temperature Then the mixture is cooled to 0° C. are added The product is extracted with t-BuMeO The combined organic layers are washed with water dried (MgSO4) Ether is removed completely under the reduced pressure (20 mbar) at 60° C. to produce a yellow oily residue This residue is purified further by vacuum distillation (80-89° C. at 0.150-0.310 mbar)

Conditions: temp:0.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of imidazole (252.3 g, 7.36 mol) in dry DMF (7.6 L) are added ethyl 3(S)-hydroxy-4-iodobutyrate (2) (949.6 g, 3.68 mol, GC purity 85.9%) and anhydrous NaI (1106 g, 7.36 mol) under argon atmosphere at ambient temperature. The suspension is cooled to 0° C. and tert-butyl(chloro)dimethylsilane (838 g, 5.56 mol) is added portionwise. The reaction mixture is stirred at 0° C. for 1.5 hours followed by stirring for 15.5 hours from 0° C. to ambient temperature. Then the mixture is cooled to 0° C. and H2O (4.2 L) is added. After 2 hours of stirring additional amount of H2O (6.2 L) and saturated Na2S2O3 solution (0.5 L) are added. The product is extracted with t-BuMeO. The combined organic layers are washed with water and dried (MgSO4). Ether is removed completely under the reduced pressure (20 mbar) at 60° C. to produce a yellow oily residue. This residue is purified further by vacuum distillation (80-89° C. at 0.150-0.310 mbar) to give 1193.5 g (97%) of ethyl 3(S)-(tert-butyldimethylsilyloxy)-4-iodobutyrate (3) as a pale yellow oil (GC purity 96.1%). 1H NMR (300 MHz, CDCl3): δ 4.13 (qd, J=7.2 and 2.1 Hz, 2H), 4.01 (m, 1H), 3.28 (dd, J=10.2 and 4.2 Hz, 1H), 3.24 (dd, J=10.2 and 6.0 Hz, 1H), 2.66 (dd, J=15.3 and 4.8 Hz, 1H), 2.51 (dd, J=15.3 and 7.2 Hz, 1H), 1.26 (t, J=7.2 Hz, 3H), 0.87 (s, 9H), 0.10 (s, 3H), 0.05 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 170.8, 68.3, 60.5, 42.5, 25.6, 17.9, 14.1, 12.9, −4.6, −5.0. by stirring for 15.5 hours from 0° C. to ambient temperature Then the mixture is cooled to 0° C. are added The product is extracted with t-BuMeO The combined organic layers are washed with water dried (MgSO4) Ether is removed completely under the reduced pressure (20 mbar) at 60° C. to produce a yellow oily residue This residue is purified further by vacuum distillation (80-89° C. at 0.150-0.310 mbar)

Conditions: temp:59.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of ethyl 3(S)-hydroxy-chlorobutyrate (1) (639.0 g, 3.84 mol) in dry acetone (7.7 L) is added anhydrous NaI (2300 g, 15.34 mol). The mixture is stirred vigorously at 58-60° C. for 120 hours under argon atmosphere. The acetone is distilled off under the reduced pressure at 40-60° C. The residue is diluted with water (5.75 L) followed by the addition of saturated Na2S2O3 solution (1.53 L) and t-BuMeO (2.3 L). The mixture is stirred vigorously at ambient temperature for 30 minutes. Then the organic layer is separated and the water phase is extracted additionally with t-BuMeO (2×1.15 L). The combined organic layers are washed with water (800 mL) and dried (MgSO4). Evaporation of the solvent under the reduced pressure (20 mbar) at 40° C. affords 949.9 g (96%) of ethyl 3(S)-hydroxy-4-iodobutyrate (2) as yellow oil (GC purity 85.9%). A product with GC purity of 98-99% can be obtained by vacuum distillation (73-89° C. at 0.180-0.330 mbar) of the crude product. 1H NMR (300 MHz, CDCl3): δ 4.17 (q, J=7.2 Hz, 2H), 3.99 (m, 1H), 3.34 (dd, J=10.3 and 5.2 Hz, 1H), 3.28 (dd, J=10.3 and 5.7 Hz, 1H), 3.21 (br s, 1H), 2.67 (dd, J=16.5 and 4.3 Hz, 1H), 2.58 (dd, J=16.5 and 7.9 Hz, 1H), 1.27 (t, J=7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 171.7, 67.4, 61.0, 40.7, 14.1, 12.0. The acetone is distilled off under the reduced pressure at 40-60° C The residue is diluted with water (5.75 L) followed by the addition of saturated Na2S2O3 solution (1.53 L) and t-BuMeO (2.3 L) The mixture is stirred vigorously at ambient temperature for 30 minutes Then the organic layer is separated the water phase is extracted additionally with t-BuMeO (2×1.15 L) The combined organic layers are washed with water (800 mL) dried (MgSO4) Evaporation of the solvent under the reduced pressure (20 mbar) at 40° C.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: In accordance with Scheme I, (S)-3-hydroxy-4-[[(4-methylphenyl)sulfonyl]oxy]butanoic acid methyl ester 2, (S)-3-hydroxy-4-iodobutanoic acid ethyl ester 3 and (S)-oxiraneacetic acid ethyl ester 4 are prepared from (S)-malic acid 1 using previously described methods [S. Saito et. al. Chem. Lett. 1389, (1984) and M. Larcheveque et. al. Tetrahedron Lett. 1781, 28 (1987)]. The esters 2, 3 and 4 are individually reacted with propargylamine in an alcohol solvent at temperatures ranging from room temperature to the reflux temperature of the solvent, preferably in the presence of an alkali metal carbonate, to produce (S)-4-hydroxy-1-(2-propynyl)-2-pyrrolidinone 5. Compound 5 is then reacted with an aryl or a (C1 -C6) carboxylic acid anhydride such as acetic anhydride in the presence of pyridine and preferably dimethylaminopyridine in an ether or a chlorohydrocarbon solvent, such as dichloromethane, to give the intermediate of general formula 6, for example, (S)-4-(acetyloxy)-1-(2-propynyl)-2 -pyrrolidinone.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: In accordance with Scheme I, (S)-3-hydroxy-4-[[(4-methylphenyl)sulfonyl]oxy]butanoic acid methyl ester 2, (S)-3-hydroxy-4-iodobutanoic acid ethyl ester 3 and (S)-oxiraneacetic acid ethyl ester 4 are prepared from (S)-malic acid 1 using previously described methods [S. Saito et. al. Chem. Lett. 1389, (1984) and M. Larcheveque et. al. Tetrahedron Lett. 1781, 28 (1987)]. The esters 2, 3 and 4 are individually reacted with propargylamine in an alcohol solvent at temperatures ranging from room temperature to the reflux temperature of the solvent, preferably in the presence of an alkali metal carbonate, to produce (S)-4-hyroxy-1-(2-propynyl)-2-pyrrolidinone 5. Compound 5 is then reacted with an aryl or a (C1 -C6) carboxylic acid anhydride such as acetic anhydride in the presence of pyridine and preferably dimethylaminopyridine in an ether or a chlorohydrocarbon solvent, such as dichloromethane, to give the intermediate of general formula 6, for example, (S)-4-(acetyloxy)-1-(2-propynyl)-2 -pyrrolidinone.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: In accordance with Scheme I, (S)-3-hydroxy-4-[[(4-methylphenyl)sulfonyl]oxy]butanoic acid methyl ester 2, (S)-3-hydroxy-4-iodobutanoic acid ethyl ester 3 and (S)=oxiraneacetic acid ethyl ester 4 are prepared from (S)-malic acid 1 using previously described methods [S. Saito et. al. Chem. Lett. 1389, (1984) and M. Larcheveque et. al. Tetrahedron Lett. 1781, 28 (1987)]. The esters 2, 3 and 4 are individually reacted with propargylamine in an alcohol solvent at temperatures ranging from room temperature to the reflux temperature of the solvent, preferably in the presence of an alkali metal carbonate, to produce (S)-4-hydroxy-1-(2-propynyl)-2-pyrrolidinone 5. Compound 5 is then reacted with an aryl or a (C1 -C6) carboxylic acid anhydride such as acetic anhydride in the presence of pyridine and preferably dimethylaminopyridine in an ether or a chlorohydrocarbon solvent, such as dichloromethane, to give the intermediate of general formula 6, for example, (S)-4-(acetyloxy)-1-(2-propynyl)- 2-pyrrolidinone.