Reportedly, conversion and enantioselectivity in lipase catalysed hydrolysis can be improved by the addition of a suitable cosolvent [45] . First, the reaction was carried out in the absence of a cosolvent and the product was obtained with 82% ee and 19% conversion. When the reaction was carried out with ethanol, isopropyl ether or acetone as a cosolvent, the conversion dropped from 19 to 8%. Similar observations have been reported by Szymanski et al. for the enzymatic hydrolysis of α-acetoxyamides when acetone was used as a cosolvent [46] . Dioxane and DMSO showed moderate conversion (25% - 37%). All long chain hydrophopic cosolvents (n-octane, isooctane and hexane) gave good conversion (up to 56%). This result is consistent with the literature reports that hydrophobic solvents favor the binding of hydrophobic substrates to lipase [27] . Among the cosolvents tested in this study, only hexane showed reasonable conversion (40%) and good ee (90%) (Figure 1).

Scheme 2. Enantioselective hydrolysis of (RS)-1a.

Further, the reaction time was varied from 12 to 60 h using hexane as cosolvent. It was found that 48 h showed improvement in both conversion (40%) and enantiomeric excess (89.6%) (Table 1). Further increase in the reaction time resulted in decrease in ee (82.2%). Hence 48 h was chosen as the optimum incubation time for further experiments.

To study the effect of temperature on conversion and ee of enantioselective hydrolysis, the reaction was performed at various temperatures from 25˚C - 35˚C. The product 2a was obtained with good conversion (40%) and ee (89%) at 25˚C (Table 2). With increasing temperature (30˚C & 35˚C), the conversion also increased (66 & 68% respectively) but the enantiomeric excess reduced sharply (61 & 57% respectively) (Table 2). This Candida grows best at 25˚C [36] , which supports the observation that maximum conversion is obtained at 25˚C. All further experiments were carried out at 25˚C.

In whole cell biocatalysis, conversion and enantioselectivity can be controlled by optimizing the substrate concentration due to the presence of multiple enzymes with opposite stereo preferences and different Km values for the same substrate [45] . The substrate 1a was added in varied concentrations (1 - 2.5 mM) keeping all the conditions identical (Figure 2). Notably, higher conversion (53%) was observed with low substrate concentration (1 mM), while the enantiomeric excess was moderate (75%). At a substrate concentration of 1.5 mM, the enanti-

omeric excess increased to 89% with good conversion (39%). Further increase in the substrate concentration showed a drop in conversion however the ee was maintained. Hence, 1.5 mM of substrate was chosen as the optimum substrate concentration.

Under the optimized reaction conditions, three substrates were used (Scheme 3). The experiment with substrate 1a yielded product (R)-1-(2-bromocyclohex-1-en-1-yl)but-3-en-1-ol 2a in 33% isolated yield and 89% ee (E = 31) (Table 3, entry 2a). To further explore the substrate scope of this biocatalyst, substrate 1b (Scheme 3) was subjected to hydrolysis using fermenting cells of C. parapsilosis ATCC 7330. Initially, under the optimized reaction time of 48 h, a moderate conversion (20%) was obtained with excellent ee (>99%). The conversion was further improved to 32% by extending the reaction time to 60 h and the product (R)-1-(2-bromocyclohept-1-en-1-yl) but-3-en-1-ol 2b was isolated with moderate yield (29%) and excellent ee (> 99%) (E => 200) (Table 3, entry 2b). The obtained high enantioselectivity suggests that the substrate with the 7-membered ring is seemingly better than the one with the 6-membered ring for the biocatalyst. Another substrate 1c (Scheme 3) with an alkyne chain was subjected for enantioselective hydrolysis and the product (R)-1-(2-bromocyclohex-1-en-1-yl) but-3- yn-1-ol 2c was obtained with excellent ee (>99%) in lesser reaction time (12h) compared to substrates 1a & 1b. Further increase in the reaction time resulted in decrease in ee. The product 2c was isolated in 42% yield and 99% ee (E => 500) (Table 3, entry 2c).

Scheme 3. Enantioselective hydrolysis of (RS)-1a-c.

^aAbsolute configuration was assigned by comparing specific rotation value with the analogue substrate [47] ; ^bConversion and ee was determined by GC using Chirasil Dex CB chiral column; ^cEnantiomeric excess of the unreacted acetates were determined after hydrolysis using 5% methanolic KOH; ^dEnantioselectivity ratios were calculated according to: E = ln[(eep(1 − ees)/( eep+ees)]/ln[(eep(1 + ees)/(eep + ees) [48] ; E values in the range 200 - 500 are mentioned as >200, more than 500 as >500.

¹H NMR (500 MHz,CDCl₃) δ:5.86 (dd, J = 8,5.5 Hz, 1H), 5.74 - 5.82 (m, 1H), 5.10 - 5.15 (ddd, J = 17, 3.5, 1.5 Hz, 1H), 5.06 - 5.09 (ddd, J = 9.5, 1.5, 0.5 Hz, 1H), 2.51 - 2.55 (m, 2H), 2.44 - 2.50 (m, 1H), 2.36 - 2.41 (m, 1H), 2.08 - 2.17 (m, 2H), 2.06 (brs, 3H), 1.66 - 1.72 (m, 3H), 1.61 - 1.64 (m, 1H); ¹³C NMR (125 MHz, CDCl₃) δ: 169.9, 133.7, 133.3, 121.3, 117.6, 75.7, 36.7, 36.6, 25.9, 24.5, 21.9, 20.9; HRMS for C₁₂H₁₇O₂Br (Cal: 295.0310 [M + Na]⁺, Found: 295.0310); ee = 59%, retention times 15.0 (S, major), 15.5 min (R, minor); [α] D²⁵ + 1.1 (c 1, CHCl₃).

¹H NMR (500 MHz, CDCl₃) δ: 5.74 - 5.79 (m, 2H), 5.06 - 5.11 (m, 1H), 5.03 - 5.04 (m, 1H), 2.72 - 2.79 (m, 2H), 2.37 - 2.43 (m, 1H), 2.27 - 2.35(m, 1H), 2.19 - 2.26 (dd, J = 6.5, 5 Hz, 2H), 2.02 (brs, 3H), 1.70 - 1.74 (m, 2H), 1.53 - 1.56 (m, 3H), 1.38 - 1.52 (m, 1H); ¹³C NMR (125 MHz, CDCl₃) δ: 169.9, 138.7, 133.2, 125.0, 117.7, 76.7, 41.5, 36.7, 31.6, 27.9, 25.9, 25.1, 20.9; HRMS forC₁₃H₁₉O₂Br (Cal: 309.0466 [M + Na]⁺, Found: 309.0466); ee = 46%, retention times 20.3 (S, major), 21.5 min (R, minor); [α] D²⁵ + 1.7 (c 1, CHCl₃).

¹H NMR (500 MHz, CDCl3) δ: 5.87 - 5.89 (m, 1H), 2.56 - 2.66 (m, 2H), 2.52 - 2.54 (m, 2H), 2.18 - 2.28 (m, 1H), 2.08 (s, 3H), 1.97 - 1.98 (t, J = 2.5 Hz, 1H), 1.62 - 1.75 (m, 4H), 1.59 (brs, 1H); ¹³CNMR (125 MHz, CDCl₃) δ: 169.7, 133.1, 122.1, 79.4, 74.0, 70.2, 36.7, 26.0, 24.4, 22.4, 21.8, 20.8; HRMS for C₁₂H₁₅O₂Br (Cal: 293.0153 [M + Na]⁺, Found: 293.0154); ee => 99%, retention times19.4 (R, minor), 19.8 min (S, major); [α] D²⁵ + 3.2 (c 0.5, CHCl₃).

¹H NMR (500 MHz,CDCl₃) δ: 5.78 - 5.86 (m, 1H), 5.12 - 5.18 (m, 2H), 4.78 - 4.81 (dd = t, J = 6.5 Hz, 1H), 2.50 - 2.52 (m, 2H), 2.28 - 2.34 (m, 3H), 2.04 - 2.10 (m, 1H), 1.63 - 1.74 (m, 5H); ¹³C NMR (125 MHz, CDCl₃) δ:136.9, 134.3, 120.3, 118.0, 73.2, 38.9, 36.8, 25.4, 24.7, 22.1; ee = 89%; retention times 15.0 (S, minor), 15.5 min (R, major); [α]D²⁵ + 25.4 (c 0.5, CHCl₃).

Lipase PS (from Burkholderia cepacia) and Amano lipase AK (from Pseudomonas fluorescence) were obtained from Amano Pharmaceuticals Co., Nagoya, Japan. CAL B (Candida antarctica lipase B, Novozym 435) was purchased from Sigma. Candida parapsilosis ATCC 7330 was obtained from ATCC (Manassas, VA) and maintained at 4˚C in yeast malt agar medium that contained 5 g/L peptic digest of animal tissue, 3 g/L malt extract, 3 g/L yeast extract, 10 g/L dextrose and 20 g/L agar. All chemicals used were of analytical grade and distilled prior to use. TLC was carried out on Kieselgel 60 F254 aluminium sheets (Merck1.05554). ¹H and ¹³C NMR spectra were recorded in CDCl₃ solution on a Bruker AVANCE III 500 MHz spectrometer. Chemical shifts are expressed in ppm values using TMS as an internal standard. The enantiomeric excess was determined by gas chromatography using Perkin Elmer Clarus 600 gas chromatograph and Shimadzu GC 2014 gas chromatograph fitted with flame ionisation detector using VARIAN Chirasil Dex CB chiral column (0.25 μm × 25 mm × 30 m). The injector and detector were set at 220˚C, respectively. Nitrogen was used as the carrier gas. Optical rotations were determined using Autopol-IV automatic polarimeter.

Tin powder (1.5 mmol) was suspended in a mixture of water (2 mL) and diethyl ether (2mL). To this mixture allyl bromide (3 mmol) was added and treated with few drops of 48% hydrobromic acid and the mixture was stirred well for 5 min at RT. To this reaction mixture bromoaldehyde (1 mmol) was added and the resultant mixture was stirred for 6 h at RT (reaction was monitored by TLC). After the complete conversion, the reaction was quenched with water (5 ml) and extracted with 20% dichloromethane/hexane mixture (10 mL × 3). The organic layers were combined and dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude reaction mixture was purified by silica gel column chromatography using hexane/ethyl acetate (9:1) as a mobile phase (Yield: 60% - 80%). The respective bromo alcohol (1 mmol) was stirred with (2 mL) of acetic anhydride containing catalytic amount of sodium acetate for 24 h at room temperature. The progress of the reaction was monitored by TLC. After the completion of reaction, the reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over sodiumsulphate and concentrated under reduced pressure. The crude reaction mixture was purified by column chromatography over silica gel (100 - 200 mesh) using hexane/ethyl acetate mixture (9:1) as a mobile phase to obtain the pure product (Yield: 70% - 80%).

Scheme 4. Radical cyclization of 2a.

Candida parapsilosis ATCC 7330 was pre-cultured for 12 h at 25˚C with shaking at 200rpm in yeast malt broth medium that contained 5 g/L peptic digest of animal tissue, 3 g/L malt extract, 3 g/L yeast extract and 10 g/L dextrose. The pre-cultured broth, 4 mL [4% (v/v)] was transferred to a 500 mL Erlenmeyer flask that contained 96 mL of yeast malt broth. The culture was grown on rotatory shaker at 25˚C and 200 rpm for 12 h.

To the 500 mL Erlenmeyer flask that contained 12 h cultivated cells (100 mL culture), the substrate (racemic acetate, 1.5 mM) in 500 μL of hexane as co-solvent was added and the incubation was continued at 25˚C and 200 rpm. After getting the maximum conversion and enantiomeric excess of the product, the cells were centrifuged at 6000 rpm for 10 min. The formed product and the remaining substrate were extracted from the supernatant using ethylacetate. The organic layer was dried over anhydrous sodium sulphate. The solvent was removed by evaporation and the crude extract was purified by silica gel column chromatography using hexane/ ethyl acetate (95:05) as a mobile phase eluent.

The respective enantiomerically enriched (R)-1-(2-bromocyclohex-1-en-1-yl)but-3-en-1-ol 2a (1 mmol) was dis- solved in ethanol (25 mL). To this bis-tri-n-butyltinoxide (5 mol%), sodium borohydride (1 mol) and catalytic amount of azobisisobutyro nitrile (AIBN) were added. The reaction mixture was refluxed for 3 h at 80˚C under N₂ atmosphere and the reaction was monitored by TLC. After the completion of reaction, the reaction mixture was concentrated under reduced pressure. The products were purified by column chromatography over silica gel (100 - 200 mesh) using hexane/ethyl acetate mixture (9:1) as a mobile phase to obtain the cyclized product 3a and 3b (Yield: 70% - 80%).