Title of Invention

"A PROCESS FOR OPTICAL RESOLUTION OF RACEMIC 1-ARYLETHYL ACETATES"

Abstract

The present invention provides a process for the enantioselective resolution of racemic 1-arylethyl acetates with an indigenous soil microbe strain Pseudomonas fluorescens RRLJ 134 isolated from the top soil of a tea plantation of north-east India. The strain, maintained in nutrient agar slant and grown in King's B medium, was inoculated with the substrate 1-aryl acetates in succinate medium. The resolution of R/S-1-arylethyl acetate with Pseudomonas fluorescens RRLJ 134 was highly enhanced in presence of surfactant Tween-80 (0.01%) to afford S(-)-1-arylethyl acetate and R(+)-l-arylethanol in high enantiomeric excess.

Full Text

The present invention relates to a process for optical resolution of racemic 1- arylethyl acetates using soil bacteria strain Pseudomonas fluorescens RRLJ 134 in presence of surfactant. The fluorescent Pseudomonads are well known as bacterial biocontrol agent and have been utilized for a diverse range of soil borne diseases. The Pseudomonas fluorescens RRLJ 134 is an organism isolated from the top soil of a tea plantation in north-east India and identified through various morphological, physiological and biochemical tests. The strain showed in vitro antibiosis (fungal pathogen control) against Fusarium and Fomes spp (B S Dileep Kumar. I Berggren and A M Martensson. Plant and Soil. 2001. 229. 25). In addition P. fluorescens RRLJ 134 organism showed growth promoting activity by enhancing shoot length, root length, seed germination and yield of the crop (B S Dileep Kumar and B Bezbantah, Indian J Microbiology. 1996. 36, 45). Further, it exhibited bio-control of plant diseases by controlling the fusarial wilt of pea. However the fluorescent Pseudomonads, despite their enormous potential have received scant attention in bio-transformation reactions. Reference may be made to M Torimura, H Yoshida, K Kano, T Ikeda, T Nagasawar. & T Ueda. Chem Lett.. 1998. 295 wherein Pseudomonas specis has been used for hydroxylation of nicotinic acid. Another reference may be made to / Kumar. K Manju & R S Jolly, Tetrahedron:Asymmetry. 2001. 12. 1431 wherein Pseudomonas fluorescens cell free extract has been employed as a new bio-catalyst for preparation of enantiomerically pure 2-arylpropionic acids. Still another reference may be made to Y Soeda. K Toshima & S Matsumura. Chem. Lett. 2001. 76. wherein Pseudomonas fluorescens lipase is utilized as enzyme based polymerization of glycidol. Still another reference may be made to A Ghanem & V Schuris. Tetrahedron:Asvmmetrv. 2001. 12. 276L wherein peracelylated p-cyclodextrin has been employed as an additive to enhance the reaction rate and enantiomeric ratio, E, in Pseudomonas capacia lipase catylased lipase triesterification of racemic l-(2- furyl)etahnol with isopentyl acetate. Still another reference may be made to H Idogaki, N Kasa, M Tekeuchi, M Hatada, & T Suzuki, Tetrahedron: Asymmetry, 2001, 12, 369, wherein the hydrolytic activity of Pseudomonas sp DS-K-19 was increased 20 folds compared to additive free medium by addition of 0.2% v/v 4-chloro-3-acetoxybutyronitrile. The main objective of the present invention is to employ Pseudomonas fluorescens RRLJ 134 strain for optical resolution of racemic 1-aryl ethylacetate. Another objective of the present invention is to study the effect of surfactant in enantioselective resolution compared to surfactant free medium, by addition of surfactant. Accordingly, the present invention provides a process for the optical resolution of racemic 1-aryl ethylacetate into an enantiomerically pure S-(-)-l-arylethylacetates in presence of surfactant Tween-80 [polyoxyethylene(20) sorbitan monooleate], (Formula Removed) wherein Ar is Ph or p-tolyl; which comprises : a bio-organic transformation reaction of R/S-1-arylethyl acetate characterized in that with indigenous soil microbe Pseudomonas fluorescens RRLJ 134 in succinate media in presence of surfactant Tween-80 in shaking incubation for 36 h at 28-30°C to afford S(-)-l-arylethyl acetate and R(+)-1-arylethanol in high enantiomeric excess. The process involves the use of Pseudomonas florescens RRLJ 134 strain, grown in King's B medium broth, to inoculate in succinate media containing substrate and biosurfactant and then keeping it in a shaking incubator at an ambient temperature at a constant rotation. The starin is obtained from the top soil of a tea plantation in Doars region of West Bengal, India. The biological material is deposited at MTCC Division of IMT, Chandigarh on 29-11-2005. In the first step of the resolution process, Pseudomonas fluorescens RRLJ 134 strain, maintained in nutrient agar slants (Plant Pathologist's Book, 1968, Commonwealth Mycological Institute, Surrey, KEW, England, p-267) was grown in King's B medium broth (E.O. King, M K Wood & D E Raney. J Lab Clin Med, 1954, 44, 301). In 15 sets of 250 ml conical flask, 100 ml of succinate media (J M Meyer & M A Abdallah, J Gen Microbiol, 1978,107,319) was taken in each flask to which 2 ml (0.7 mmol) melhanolic solution of (RS) -phenylethyl acetate and 0.5 ml of 18h old inoculum of Pseudomonas fluorescens RRU 134 strain grown in King's B medium, were added. The reaction was kept in shaking incubation temperature (28-30°C) for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate to obtain the products which were isolated by preparative silica gel TLC and identified as S(-)phenylethyl acetate and R(+)phenyl ethanol. The optical rotation and enantiomeric excess (ee) of the products showed for R(+)phenylethyl acetate [a]D 2s +10° and ee 30 % and S(-)phenylethanol [cc]D 25 -8° and ee 20 %. In the second step, the resolution process of (RS)-phenylethylacetate was carried in presence of 10 rag of surfactant Tween-80. In 15 sets of 250 ml conical flask, 100 ml of succinate media was taken in each flask to which 2 ml (0.7 mmol) methanolic solution phenylethyl acetate, 10 mg of Tween-80 and 0.5 ml of the of 18h old inoculum of Pseudomonas fluorescens RRLJ 134 strain grown in King's B medium, were added. The reaction was kept in shaking incubation temperature (28-30°C) for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate and solvent removed to obtain the products which were isolated by preparative silica gel TLC and identified as S(-)phenylethyl acetate and R(+)phenyl ethanol. The optical rotation and enantiomeric excess (ee) of the products showed for S(-)phenylethyl acetate [a]D2s -86° and ee 92% and R(+)phenylethanol [a]D 25 +19° and ee 77%. In the third step, the resolution process of (RS)-phenylethylacetate was carried in presence of 20 mg of surfectant Tween-80. In 15 sets of 250 ml conical flask, 100 ml of succinate media was taken in each flask to which 2 ml (0.7 mmol) methanolic solution phenylethyl acetate, 20 mg of Tween-80 and 0.5 ml of the 18 h old strain grown in King's B medium, were added. The reaction was kept in shaking incubation temperature (28- 30°C) for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate and solvent removed to obtain the products which were isolated by preparative silica gel TLC and identified as S(-) phenylethyl acetate and R(+)phenyl ethanoL The optical rotation and enantiomeric excess (ee) of the products showed for S(-)phenylethyl acetate [a]D 25 -64° and ee 80% and R(+) phenylethanol [a]D 25 +17°andee68%. In an embodiment of the present invention, the Pseudomonas fluorescens RRLJ 134 strain maintained in nutrient Agar slants was further grown hi King's B medium having the following compositions per liter at pH 7. Peptone 20.0 g Potasium monohydrogen phostate 2.5 g Magnesium sulfate heptahydrate 6.0 g Glycerol 15.0g AgarAgar 15.0g In another embodiment of the present invention, the enantioselective resolution of 1-arylethyl acetates were carried using Pseudomonas fluorescens RRLJ 134 strain in succinate medium having the following composition per liter at pH 7. Succinic acid 4.0 g Ammonium sulfate 1.0 g Potasium dihydrogen phostate 3.0 g Potasium monohydrogen phostate 0.1 g Magnesium sulfate heptahydrate 0.2 g Still in another embodiment of the present invention, the solvent used for preparation of substrate solution may be protic solvents such as methanol and ethanol. Still in another embodiment of the process, the whole cell Pseudomonas fluorescens RRLJ 134 strain showed the enantioselective resolution of (R/S)-l-phenyl ethyl acetate to a smaller degree. Still in another embodiment of the process, Pseudomonas fluorescens RRLJ 134 strain showed enhanced enantioselective resolution in presence of surfactant Tween-80 with inversion of rotation. Still in another embodiment of the present invention, the surfactant Tween-80 afforded optimum enantioselective resolution of (R/S)-1-arylethyl acetates when the concentration of the surfactant is 0.01% in the inoculum broth. The following examples are given by illustrations and should not contrued the scope of the invention. Example -1 Preparation of stock solution A stock solution of 1.14 g of (R/S)-l-phenylethyl acetate in methanol (20, ml) is prepared and kept in a 50 ml Erlenmeyer flask. Preparation of King's B medium In 100 ml distilled water in a 250 ml Erlenmeyer flask was added peptone (20.0 g), peptone (20.0 g), potasium monohydrogen phosphate (2.5 g), magnesium sulfate heptahydrate (6.0 g), glycerol (15.0 g) and agar agar (15.0 g) and stirred well to afford a clean solution. Preparation of succinate medium In 100 ml distilled water in a 250 ml Erlenmeyer flask was added succinic acid (4.0 g), ammonium sulfate (1.0 g), potasium dihydrogen phostate (3.0 g), potasium monohydrogen phostate (0.1 g), magnesium sulfate heptahydrate (0.2 g) and stirred well to afford a clean solution at pH 7.0. Enantioselective resolution of (R/SVl-phenvlethyl acetate with Pseudomonas fluorescens RRLJ 134 strain To 100 ml of succinate media taken in 250 ml Erlenmeyer flask were added 2 ml of t he stock solution of (R/S)-l-phenylethyl acetate and 0.5 ml of the Pseudomonas fluorescens RRLJ 134 strain grown in King's B medium. The reaction was kept in shaking incubation temperature (28-30°C) at rotation 120 rpm for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate and solvent removed to obtain the products in 38% overall yield, which were isolated by preparative silica gel TLC and identified as R(+)-l-phenylethyl acetate and S(-)-l-phenyl ethanol. R(+)-l-phenylethyl acetate: !HNMR 6 6.8-7.1 (m, 5H, aromatic protons), 5.58 (q, 1H, 7.0 Hz, methine proton), 1.75 (s, 3H, methyl protons), 1.28 (4 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 25 +10° (1.5, CHC13); Enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 30 %. S(-)-l-phenylethanol: !HNMR 8 6.7-7.0 (m, 5H, aromatic protons), 4.40 (q, 1H, 6.5 Hz, methine proton), 3.5 (bs, 1H, hydroxyl proton), 1.18 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [ 25 -8° and ee 20%. Example - II Enantioselective resolution of (R/SVl-phenvlethvl acetate with Pseudomonas fluorescens RRLJ 134 strain in presence of surfactant Tween-80 (0.01%^: Preparation of stock solution of the substrate, King's B medium and succinate medium were carried similar to the procedure as described in example-I. To 100 ml of succinate media taken in 250 ml Erlenmeyer flask were added 2 ml of the stock solution of (R/S)-l-phenylethyl acetate, Tween-80 (10 mg, 0.01%) and 0.5 ml of the Pseudomonas fluorescens RRLJ 134 strain grown in King's B medium. The reaction was kept in shaking incubation temperature (28-30°C) at rotation 120 rpm for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate and solvent removed to obtain the products in 45% overall yield, which were isolated by preparative silica gel TLC and identified as S(-)-lphenylethyl acetate and R(+)-l-phenyl ethanol. S(-)-l-phenylethyl acetate: 'HNMR 8 6.8-7.1 (m, 5H, aromatic protons), 5.58 (q, 1H, 7.0 Hz, methine proton), 1.75 (s, 3H, methyl protons), 1.28.(d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 2s -86° (1.5, CHC13); Enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 92 %. R(+)-l-phenylethanol: 'HNMR 8 6.7-7.0 (m, 5H, aromatic protons), 4.40 (q, 1H, 6.5 Hz, methine proton), 3.5 (bs, 1H, hydroxyl proton), 1.18 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 25 +19° (1.5, CHCb); enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 77%. Example -III Enantioselective resolution of (R/SVl-phenylethyl acetate with Pseudomonas fluorescens RRLJ 134 strain in presence of surfactant Tween-80 (0.02%) : Preparation of stock solution of the substrate, King's B medium and succinate medium were carried similar to the procedure as described in example-I. To 100 ml of succinate media taken in 250 ml Erlenmeyer flask were added 2 ml of the stock solution of (R/S)-l-phenylethyl acetate, tween-80 (20 mg, 0.02%) and 0.5 ml of the Pseudomonas fluorescens RRLJ 134 strain grown in King's B medium. The reaction was kept in shaking incubation temperature (28-30°C) at rotation 120 rpm for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate and solvent removed to obtain the products in 42% overall yield, which were isolated by preparative silica gel TLC and identified as S(-)-lphenylethyl acetate and R(+)-l-phenyl ethanol. S(->l-phenylethyl acetate: 1HNMR 8 6.8-7.1 (m, 5H, aromatic protons), 5.58 (q, 1H, 7.0 Hz, methine proton), 1.75 (s, 3H, methyl protons), 1.28 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 2s -64° (1.5, CHC13); Enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 80 %. R(+)-l-phenylethanol: 1HNMR 8 6.7-7.0 (m, 5H, aromatic protons), 4.40 (q, 1H, 6.5 Hz, methine proton), 3.5 (bs, 1H, hydroxyl proton), 1.18 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 25 +17° (1.5, CHC13); enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 68 %. Example - IV Knantioselective resolution of (R/SVl-(p-tolvn-ethvl acetate with Pseudomonas fluorescens RRLJ 134 strain in presence of surfactant Tween-80 (0.01%'): Preparation of stock solution of the substrate, King's B medium and succinate medium were carried similar to the procedure as described in example-I. To 100 ml of succinate media taken in 250 ml Erlenmeyer flask were added 2 ml of the stock solution of (R/S)-l-(p-tolyl)-ethyl acetate, Tween-80 (10 mg, 0.01%) and 0.5 ml of the Pseudomonas fluorescens RRLJ 134 strain grown in King's B medium. The reaction was kept in shaking incubation temperature (28-30°C) at rotation 120 rpm for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium suifate and solvent removed to obtain the products in 47% overall yield, which were isolated by preparative silica gel TLC and identified as S(-)-l-(p-tolyl)- ethyl acetate and R(+)-l-(p-tolyl)-ethanol. S(-)-l-(p-tolyl)-ethyl acetate: 'HNMR 8 6.8-7.2 (m, 4H, aromatic protons), 5.68 (q, 1H, 7.0 Hz, methine proton), 2.20 (s, 3H, methyl protons), 1.90 (s, 3H, methyl protons), 1.40 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 25 -49° (1.5, CHCls); Enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 99 %. R(+)-l-(p-tolyl)-ethanol: 'HNMR 8 6.7-7.0 (m, 5H, aromatic protons), 4.45 (q, 1H, 6.5 Hz, methine proton), 3.60 (bs, 1H, hydroxyl proton), 1.80 (s, 3H, methyl protons), 1.30 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 25 +19° (1.5, CHC13); enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 77 %. Example - V Enantioselective resolution of rR/SVp-tolylethyl acetate with Pseudomonas Preparation of stock solution of the sub Uiate, King's B medium and succinate medium were carried similar to the procedure as described in example-I. To 100 ml of succinate media taken in 250 ml Erlenmeyer flask were added 2 ml of the stock solution of (R/S)-l-tolylethyl acetate, tween-80 (20 mg, 0.02%) and 0.5 ml of the Pseudomonas fluorescens RRLJ 134 strain grown in King's B medium. The reaction was kept in shaking incubation temperature (28-30°C) at rotation 120 rpm for 36 hr. The reaction broth were extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate and solvent removed to obtain the products in 44% overall yield, which were isolated by preparative silica gel TLC and identified as S(-)-l-tolylethyl acetate and R(+)-l-tolylethanol. S(-)-l-(p-tolyl)-ethyl acetate: 'HNMR 8 6.8-7.2 (m, 4H, aromatic protons), 5.68 (q, 1H, 7.0 Hz, methine proton), 2.20 (s, 3H, methyl protons), 1.90 (s, 3H, methyl protons), 1.40 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 25 -42° (1.5, CHC13); Enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 90 %. R(+)-l 6.5 Hz, methine proton), 3.60 (bs, 1H, hydroxyl proton), 1.80 (s, 3H, methyl protons), 1.30 (d, 3H, J= 7.0 Hz, methyl protons); Optical rotation [a]D 25 +15° (1.5, CHC13); enantiomeric excess (ee) determined by HPLC in chiral cell OD column ee 72 %. The main advantages of the present inventions are : 1. The organism Pseudomonas fluorescens RRLJ 134 could be isolated from the top soil of tea plantation of north-east India and maintained in nutrient Agar. 2. The strain could be further grown in King's B medium broth. 3. The inoculation of the Pseudomonas fluorescens RRLJ 134 can be carried in succinate media along with the substrate. 4. The enantiomeric resolution of the substrate (RS)-arylethyl acetate can be carried in high enantiomeric excess of the S(-)-aryl ethyl acetate in presence of surfactant. 5. The enantiomeric excess of the R(+)-arylethyl alcohol could also be achieved in optimum resolution. 6. Protic solvents like methanol and ethanol can be employed for the preparation of substrate solution. We claim: 1. A process for the preparation of enantiomerically pure S-(-)-l-arylethylacetates which comprises : a bio-organic transformation reaction of R/S-1-arylethyl acetate characterized in that with indigenous soil microbe Pseudomonasfluorescens RRLJ 134 in succinate media in presence of surfactant Tween-80 (polyoxyethylene(20) sorbitan monooleate), incubating for 36 hour at 28-30°C to obtain S(-)-l-arylethyl acetate. 2. A process as claimed in claim 1, wherein succinate media composition per litre at Ph 7 contains, succinic acid 4.0 gm, ammonium sulfate 1.0 gram„potassium dihydrogen phosphate 3.0 gram, potassium monohydrogen phosphate 0.1 gram, ammagnesium sulfate heptahydrate 02 gram. 3. A process for the preparation of enantiomerically pure S-(-)-l-arylethylacetates substantially as herein described with reference to examples accompanying this specification.

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