methyl (2R)-2-hydroxy-2-phenylacetate

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

Procedure: A suspension of 2-methanesulfinylpyrimidine-5-carboxylic acid (4-fluorophenyl)amide (100 mg, 0.36 mmol) and (R)-(−)-methyl mandelate (86 mg, 0.52 mmol) in 3.6 mL of THF was treated dropwise with DBU (80 μL, 0.54 mmol) at ambient temperature. The reaction mixture clarified and turned yellow over the course of 5 min. The reaction was quenched after 24 hours by the addition of 5 mL of 1 M HCl. The mixture was extracted with 2×25 mL portions of ethyl acetate. The organic extracts were combined, dried using Na2SO4 and concentrated in vacuo. The crude material was dissolved in 30% (v/v) ethyl acetate/hexanes and filtered though a 10 g plug of silica gel. The eluent was concentrated to dryness and triturated with 3×5 mL portions of hexane followed by 3×10 mL portions of 10% (v/v) portions of ethyl acetate/hexanes. The residue was dried to yield 47 mg (34%) of the titled compound as a white solid. ESI-MS m/z 382 (MH+), 380 (M−H−). turned yellow over the course of 5 min The reaction was quenched after 24 hours by the addition of 5 mL of 1 M HCl The mixture was extracted with 2×25 mL portions of ethyl acetate dried concentrated in vacuo The crude material was dissolved in 30% (v/v) ethyl acetate/hexanes filtered though a 10 g plug of silica gel The eluent was concentrated to dryness triturated with 3×5 mL portions of hexane The residue was dried

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

Procedure: To a stirred solution of D-(-)-mandelic acid (20 g) in methanol (100 ml) was added concentrated sulfuric acid (6 g) and the mixture was refluxed for 2.5 hours. After cooling to room temperature it was concentrated to about 50 ml and the residue was dissolved in methylene chloride (about 300 ml). The solution was the washed with water, saturated sodium bicarbonte and water, dried (magnesium sulfate) and evaporated to give a clear oil, which crystallized upon cooling to give 21.7 g of methyl D-(-)-mandelate, a compound of Formula 6, wherein R5 is phenyl. the mixture was refluxed for 2.5 hours was concentrated to about 50 ml the residue was dissolved in methylene chloride (about 300 ml) washed with water, saturated sodium bicarbonte and water dried (magnesium sulfate) evaporated to give a clear oil, which crystallized upon cooling

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

Procedure: Into methanol (20 ml) were added (R)-mandelic acid (5.0 g) and p-toluenesulfonic acid (0.1 g), and the resulting mixture was heated under reflux for 2 hours. The reaction mixture was cooled, then neutralized, and concentrated. Ethyl acetate and water were added to the resulting residue, and the organic layer was collected by layer separation. The organic layer was washed with a saturated aqueous sodium chloride solution and dried with anhydrous magnesium sulfate. The desiccant was removed by filtration, and the solvent was distilled off from the filtrate to obtain a colorless oily compound mentioned above (5.35 g, 97.9%). the resulting mixture was heated under reflux for 2 hours The reaction mixture was cooled concentrated Ethyl acetate and water were added to the resulting residue the organic layer was collected by layer separation The organic layer was washed with a saturated aqueous sodium chloride solution dried with anhydrous magnesium sulfate The desiccant was removed by filtration the solvent was distilled off from the filtrate to obtain a colorless oily compound

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

Procedure: A suspension of 2-methanesulfinylpyrimidine-5-carboxylic acid (4-fluorophenyl)amide (100 mg, 0.36 mmol) and (R)-(−)-methyl mandelate (86 mg, 0.52 mmol) in 3.6 mL of THF was treated dropwise with DBU (80 μL, 0.54 mmol) at ambient temperature. The reaction mixture clarified and turned yellow over the course of 5 min. The reaction was quenched after 24 hours by the addition of 5 mL of 1 M HCl. The mixture was extracted with 2×25 mL portions of ethyl acetate. The organic extracts were combined, dried using Na2SO4 and concentrated in vacuo. The crude material was dissolved in 30% (v/v) ethyl acetate/hexanes and filtered though a 10 g plug of silica gel. The eluent was concentrated to dryness and triturated with 3×5 mL portions of hexane followed by 3×10 mL portions of 10% (v/v) portions of ethyl acetate/hexanes. The residue was dried to yield 47 mg (34%) of the titled compound as a white solid. ESI-MS m/z 382 (MH+), 380 (M−H−). turned yellow over the course of 5 min The reaction was quenched after 24 hours by the addition of 5 mL of 1 M HCl The mixture was extracted with 2×25 mL portions of ethyl acetate dried concentrated in vacuo The crude material was dissolved in 30% (v/v) ethyl acetate/hexanes filtered though a 10 g plug of silica gel The eluent was concentrated to dryness triturated with 3×5 mL portions of hexane The residue was dried

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

Procedure: To a solution of 10.0 g (40 mmol) (E)-4-methyl-6-(2,6,6-trimethyl-cyclohex-1-enyl)-hex-3-enoic acid [H. S. Corey, J. R. D. Cormick, W. E. Swensen, J. Am. Chem. Soc. 1964, 86, 1884] in 100 ml MTBE were added at r.t. 6.64 g (40 mmol) (R)-(−)-mandelic acid methyl ester [α]D=−144.9 (c=1.04, MeOH) and 1.0 g DMAP. Then a solution of 8.24 g (40 mmol) DCC in 100 ml MTBE was added and the reaction mixture stirred for 3 h, filtered and the residue washed with MTBE. The combined organic phases were concentrated in vacuo and the residue (17.6 g) was purified by flash chromatography (250 g silicagel, hexane/MTBE 9:1) affording 12.2 g (88%) 4-methyl-6-(2,6,6-trimethyl-cyclohex-1-enyl)-hex-3-enoic acid (R)-methoxycarbonyl-phenyl-methyl ester (12) in form of a colorless oil. filtered the residue washed with MTBE The combined organic phases were concentrated in vacuo the residue (17.6 g) was purified by flash chromatography (250 g silicagel, hexane/MTBE 9:1)

Conditions: temp:28.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of 12.5 g (50 mmol) (E,E)-4,8,12-trimethyl-trideca-3,7,11-trienoic acid (isomeric purity of 95%) [K. Ishihara et al., J. Am. Chem. Soc. 2002, 124, 3647-3655] in 100 ml methyl tert-butyl ether (MTBE) were added at r.t. 8.35 g (50 mmol) (R)-(−)-mandelic acid methyl ester, [α]D=−147.3 (c=1.0, MeOH), and 1.5 g 4-dimethylaminopyridine (DMAP). After stirring for 15 min at 28° C., 10.5 g (51 mmol) N,N′-dicyclohexylcarbodiimide (DCC) were added, during which the temperature raised to 35° C. The reaction mixture was stirred for 1.5 h at 38-40° C., cooled to r.t. and filtered. The resulting solution was concentrated in vacuo. The residue (23.1 g) was purified over 100 g silicagel, elution of the product with hexane/MTBE 1:1. Concentration of the combined fractions in vacuo and drying of the product in high vacuo (50° C./0.08 Torr) afforded 19.5 g (97%) of (E,E)-4,8,12-trimethyl-trideca-3,7,11-trienoic acid (R)-methoxycarbonyl-phenyl-methyl ester (1) in form of a colorless oil. raised to 35° C The reaction mixture was stirred for 1.5 h at 38-40° C. cooled to r.t. filtered The resulting solution was concentrated in vacuo The residue (23.1 g) was purified over 100 g silicagel, elution of the product with hexane/MTBE 1:1 Concentration of the combined fractions in vacuo and drying of the product in high vacuo (50° C./0.08 Torr)

Conditions: temp:28.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a solution of 12.5 g (50 mmol) (E,E)-4,8,12-trimethyl-trideca-3,7,11-trienoic acid (isomeric purity of 95%) [K. Ishihara et al., J. Am. Chem. Soc. 2002, 124, 3647-3655] in 100 ml methyl tert-butyl ether (MTBE) were added at r.t. 8.35 g (50 mmol) (R)-(−)-mandelic acid methyl ester, [α]D=−147.3 (c=1.0, MeOH), and 1.5 g 4-dimethylaminopyridine (DMAP). After stirring for 15 min at 28° C., 10.5 g (51 mmol) N,N′-dicyclohexylcarbodiimide (DCC) were added, during which the temperature raised to 35° C. The reaction mixture was stirred for 1.5 h at 38-40° C., cooled to r.t. and filtered. The resulting solution was concentrated in vacuo. The residue (23.1 g) was purified over 100 g silicagel, elution of the product with hexane/MTBE 1:1. Concentration of the combined fractions in vacuo and drying of the product in high vacuo (50° C./0.08 Torr) afforded 19.5 g (97%) of (E,E)-4,8,12-trimethyl-trideca-3,7,11-trienoic acid (R)-methoxycarbonyl-phenyl-methyl ester (1) in form of a colorless oil. raised to 35° C The reaction mixture was stirred for 1.5 h at 38-40° C. cooled to r.t. filtered The resulting solution was concentrated in vacuo The residue (23.1 g) was purified over 100 g silicagel, elution of the product with hexane/MTBE 1:1 Concentration of the combined fractions in vacuo and drying of the product in high vacuo (50° C./0.08 Torr)

Conditions: temp:70.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: To a dispersion of 1.0 g (26.3 mmol) of sodium borohydride in THF (80 ml), there was added 3.95 g (26.3 mmol) of L-(+)-tartaric acid, and the mixture was heated under reflux for four hours in an oil bath at 70° C. with stirring. The resulting mixture was cooled in a cryostat at -20° C., a solution of 1.0 g (6.6 mmol) of phenylglyoxylic acid in THF (5 ml) was added dropwise with stirring over a period of ten minutes, and stirring was continued for 67 hours. The reaction mixture was cooled in an ice bath, 30 ml of 1N HCl was added, and stirring was further continued for 30 minutes. After distilling off the solvent under reduced pressure, the residue was extracted twice with 20 ml ether, and the combined extract was washed with 50 ml of a saturated aqueous solution of sodium chloride and then dried over anhydrous magnesium sulfate. The dried solution was concentrated to about 30 ml, and an ethereal solution of diazomethane was added to the concentrate to esterify the reaction product. After distilling off the solvent under reduced pressure, the residue was purified by silica gel column chromatography, giving 1.03 g of methyl (R)-(-)-2-hydroxy-2-phenylacetate as a fraction in ethyl acetate:n-hexane (1:5). Its specific rotation [α]D21 was -100.0° (cl. 09, MeOH). The synthesis yield was 93% and the optical yield was 65% e.e. (see Reference 3). the mixture was heated The resulting mixture was cooled in a cryostat at -20° C. with stirring over a period of ten minutes stirring The reaction mixture was cooled in an ice bath stirring was further continued for 30 minutes After distilling off the solvent under reduced pressure the residue was extracted twice with 20 ml ether the combined extract was washed with 50 ml of a saturated aqueous solution of sodium chloride dried over anhydrous magnesium sulfate The dried solution was concentrated to about 30 ml an ethereal solution of diazomethane was added to the concentrate After distilling off the solvent under reduced pressure the residue was purified by silica gel column chromatography

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

Procedure: (R)-Mandelic acid (2.568 g, 16.87 mmol) and 2,2-dimethoxypropane (5.1 mL, 41.48 mmol) were dissolved in MeOH (100 mL) and 12M HCl (100 mL) was added. The resulting solution was stirred for 20 hr under a CaCl2 tube and evaporated to dryness under reduced pressure. EtOAc (100 mL) and saturated aqueous NaHCO3 (100 mL) were added, and the aqueous phase was extracted with EtOAc (3×100 mL). The organic fractions were dried with MgSO4, and evaporated under reduced pressure to give (R)-methyl mandelate, (R)-4, quantitatively (2.78 g) as a white solid which was of sufficient purity for the next step. evaporated to dryness under reduced pressure EtOAc (100 mL) and saturated aqueous NaHCO3 (100 mL) were added the aqueous phase was extracted with EtOAc (3×100 mL) The organic fractions were dried with MgSO4 evaporated under reduced pressure

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

Procedure: A reaction mixture was formed by admixing (i) 50 mg NAD, (ii) 4 ml of 2-propanol and (iii) 5 mmoles of methyl benzoylformate, Compound 4, in a liquid medium containing 1 gm of lyophilized PED alcohol dehydrogenase preparation, 75 ml of 50 mM phosphate buffer, pH 7.1, and 25 ml of hexane. The pH of the reaction was maintained constant by addition of 1N NaOH. The reaction mixture was maintained at room temperature until product formation stopped. When product formation stopped, (R)-methyl mandelate, Compound 4a, was isolated by removal of the hexane layer and extraction of the aqueous layer with ethyl ether (3×75 ml). A reaction mixture was formed in a liquid medium containing

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

Procedure: The transformed strain XL10-Gold (pTrcFadLPL) prepared in Example 3 was inoculated again in 250 mL Erlenmeyer flask including 100 mL of LB liquid medium containing 50 μg/L of ampicillin, and then cultured at 37° C. Gene expression was induced by adding 0.1 mM of IPTG, when absorbance was 0.5 at 600 nm wavelength by spectrophotometer. 5 hours after inducing the expression, culture broth was centrifuged at 4° C., 600 rpm for 5 minutes to collect cells, and then, the collected cells were washed with 50 mM Tris-HCl solution (pH 8.0), followed by resuspending in 3 mL of buffer. Said re-suspension was put into a 10 mL flask, to which 30 mg of racemic mandelic acid methyl ester was added and allowed to react for 48 hours with stirring. Then, the suspension was centrifuged at 4° C. and 6000 rpm for 5 minutes to remove cells. The supernatant was collected and adjusted to a pH of 3.0. After extraction of the supernatant with 10 mL of ethyl acetate and hexane, the extract was analyzed by liquid chromatography. The analysis results showed that (S)-mandelic acid and (R)-mandelic acid methyl ester were obtained. cultured at 37° C 5 hours was centrifuged at 4° C. 600 rpm for 5 minutes to collect cells the collected cells were washed with 50 mM Tris-HCl solution (pH 8.0) was put into a 10 mL flask, to which 30 mg of racemic mandelic acid methyl ester was added to react for 48 hours was centrifuged at 4° C. 6000 rpm for 5 minutes to remove cells The supernatant was collected After extraction of the supernatant with 10 mL of ethyl acetate and hexane The analysis results

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

Procedure: (R)-Mandelic acid (2.568 g, 16.87 mmol) and 2,2-dimethoxypropane (5.1 mL, 41.48 mmol) were dissolved in MeOH (100 mL) and 12M HCl (100 mL) was added. The resulting solution was stirred for 20 hr under a CaCl2 tube and evaporated to dryness under reduced pressure. EtOAc (100 mL) and saturated aqueous NaHCO3 (100 mL) were added, and the aqueous phase was extracted with EtOAc (3×100 mL). The organic fractions were dried with MgSO4, and evaporated under reduced pressure to give (R)-methyl mandelate, (R)-4, quantitatively (2.78 g) as a white solid which was of sufficient purity for the next step. evaporated to dryness under reduced pressure EtOAc (100 mL) and saturated aqueous NaHCO3 (100 mL) were added the aqueous phase was extracted with EtOAc (3×100 mL) The organic fractions were dried with MgSO4 evaporated under reduced pressure