chiral gas chromatography ( gc ) analyses were performed by using a heptakis(2,3dioacetyl6otbutyldimethylsilyl)cyclodextrincolumn ( machereynagel , dren , germany ) on a gc14a gas chromatograph ( shimadzu , tokyo , japan ) and by using a heptakis(2,6odiomethyl3opentyl)cyclodextrincolumn ( machereynagel , dren , germany ) on a gc2010 gas chromatograph ( shimadzu , tokyo , japan ) equipped with an aoc20i autoinjector ( shimadzu , tokyo , japan ) .
chiral hplc analyses were performed by using the column odh ( daicel chemical industries , osaka , japan ) with nhexane and 2propanol as solvent on a hitachi elite lachrom device ( hitachi high technologies america , san jose , usa ) .
ag ( zwingenberg , germany ) and used as glycerolstabilized crude cell extracts or as lyophilisates ( brsehaber et  al . , 2008 ) .
plasmids containing the genes for the esterases a3 ( elend et  al . , 2006 ) , ce ( elend et  al . , 2006 ) and cl1 ( schmeisser et  al . , 2003 ) were cloned as described .
one equivalent ( 18  mmol of 1a or 3a , and 20  mmol of 2a ) of the phenylalkyl carboxylic acid was stirred with 3  eq . of ethanol at 110c in toluene as solvent with some drops of concentrated sulfuric acid as catalyst .
next , the solvent was evaporated and the product was purified by silica gel column chromatography ( npentane / ethyl acetate , 10:1 ) . using this procedure ,
1b was obtained as light yellow oil ( 1.8  g , 9.3  mmol , 52% yield ) , 2b was obtained as colourless oil ( 2.6  g , 14.6  mmol , 73% yield ) and 3b was obtained as colourless oil ( 2.5  g , 13  mmol , 72% yield ) .
the structures were confirmed by nmr spectroscopy and matched literature data ( yang et  al . , 1999 ) .
the test for hydrolytic activity towards the target substrates was based on a ph assay ( baumann et  al . , 2000 ) .
wells were filled with 20  l of a substrate stock solution ( 200  mm in dmso ) , 5  l of a solution of the ph indicator bromothymol blue ( btb , 16  mm in 16% ethanol and 84% water ) , 30  l of a glycerol stock solution of the different esterases and 145  l of sodium phosphate buffer ( 5  mm , ph 7.4 ) to get a final volume of 200  l .
positive controls contained 5  l of btb solution , 175  l of sodium phosphate buffer ( 5  mm , ph 7.4 ) and 20  l of dmso containing the correspondent acids 1a3a at final concentrations of 120  mm .
each mtp was then sealed with adhesive foil , incubated at 37c for 72  h and stirred at 400  r.p.m . in a thermoshaker ( eppendorf , hamburg , germany ) .
active esterases were then identified by visible colour changes and used in analytical scale experiments . to a stirred solution of the ethyl esters 1b3b ( 0.076  mmol of 1b or 2b , or 0.15  mmol of 3b ) in 1.8  ml of sodium phosphate buffer ( 50  mm , ph 7.4 ) and 10% dmso if necessary ,
200  l of esterase solution was added to a total volume of 2  ml .
the reaction mixture was stirred at 1000  r.p.m . in a thermoshaker at 20c or 30c .
samples ( 400  l ) were taken after 2 , 7 and 24  h. these samples were acidified with 20  l of 1  n hcl and subsequently extracted three times with 400  l diethyl ether .
the combined organic layers were dried over anhydrous sodium sulfate and afterwards the organic solvent was evaporated in a speed vac ( thermo fisher scientific , langenselbold , germany ) .
the mixture was then treated with tms diazomethane ( hashimoto et  al . , 1981 ) in order to convert the carboxylic acid ( hydrolysis product ) in its methyl ester to allow analysis of substrate and product concentration and optical purity in one run by gc .
conversion and e values were then calculated from the enantiomeric excesses from substrate and product following the equations developed by chen and colleagues ( 1982 ) . to a 40  mm solution of 1b3b ( 1 
g ) in sodium phosphate buffer ( 50  mm , ph 7.4 ) containing 10% ( v / v ) dmso , esterase ( lyophilisate dissolved in buffer , units as given in table  2 ) was added and hydrolysis was performed at 20c or 30c .
after tlc analysis indicated 50% conversion , the reaction was stopped by addition volume of 1  n hcl , and substrate and product were extracted three times with diethyl ether . after drying the organic phase over anhydrous na2so4 ,
the solvent was removed by evaporation under reduced pressure and the product was purified by column chromatography ( npentane / ethyl acetate ) .
the purity of the biocatalysis products was confirmed by nmr spectroscopy and was in all cases >  99% . using this protocol , ( r)()1b was obtained as a light yellow oil ( 411  mg , 21  mmol , 41.4% yield ) .
( s)(+)1a was obtained as colourless oil ( 230  mg , 14  mmol , 27.7% yield , 72%ee ) ; ( s)(+)1b was obtained as light yellow oil ( 420  mg , 2.2  mmol , 42% yield ) .
enantiomeric excess was determined after hydrolysis to 1a as 87%ee ; ( r)()1a was obtained as light yellow oil ( 330  mg , 2  mmol , 38.5% yield , 98%ee ) ; ( r)()2b was obtained as colourless oil ( 164  mg , 9.2  mmol , 16.4% yield ) .
enantiomeric excess was determined after hydrolysis to 2a as 82%ee ; ( s)(+)2a was obtained as a white powder ( 181  mg , 12  mmol , 21.5% yield , 97%ee ) ; ( s)(+)2b was obtained as a colourless oil ( 218  mg , 12.2  mmol , 28.3% yield ) .
enantiomeric excess was determined after hydrolysis to 2a as 92%ee ; ( r)()2a was obtained as a white powder ( 180  mg , 12  mmol , 21.3% , 56%ee ) ; ( r)()3b was obtained as colourless oil ( 380  mg , 1.99  mmol , 38% yield ) .
enantiomeric excess was determined after hydrolysis to 3a as 69%ee ; ( s)(+)3a was obtained as yellow oil ( 360  mg , 2.18  mmol , 41.9% yield , >  99%ee ) ; ( s)(+)3b was obtained as colourless oil ( 150  mg , 0.78  mmol , 15% yield ) .
enantiomeric excess was determined after hydrolysis to 3a as 97%ee ; ( r)()3a was obtained after column chromatography ( npentane / ethyl acetate ) as yellow oil ( 330  mg , 2  mmol , 39% yield , 95%ee ) .
chiral gas chromatography ( gc ) analyses were performed by using a heptakis(2,3dioacetyl6otbutyldimethylsilyl)cyclodextrincolumn ( machereynagel , dren , germany ) on a gc14a gas chromatograph ( shimadzu , tokyo , japan ) and by using a heptakis(2,6odiomethyl3opentyl)cyclodextrincolumn ( machereynagel , dren , germany ) on a gc2010 gas chromatograph ( shimadzu , tokyo , japan ) equipped with an aoc20i autoinjector ( shimadzu , tokyo , japan ) .
chiral hplc analyses were performed by using the column odh ( daicel chemical industries , osaka , japan ) with nhexane and 2propanol as solvent on a hitachi elite lachrom device ( hitachi high technologies america , san jose , usa ) .
ag ( zwingenberg , germany ) and used as glycerolstabilized crude cell extracts or as lyophilisates ( brsehaber et  al . , 2008 ) .
plasmids containing the genes for the esterases a3 ( elend et  al . , 2006 ) , ce ( elend et  al . , 2006 ) and cl1 ( schmeisser et  al . , 2003 ) were cloned as described .
one equivalent ( 18  mmol of 1a or 3a , and 20  mmol of 2a ) of the phenylalkyl carboxylic acid was stirred with 3  eq . of ethanol at 110c in toluene as solvent with some drops of concentrated sulfuric acid as catalyst .
next , the solvent was evaporated and the product was purified by silica gel column chromatography ( npentane / ethyl acetate , 10:1 ) . using this procedure ,
1b was obtained as light yellow oil ( 1.8  g , 9.3  mmol , 52% yield ) , 2b was obtained as colourless oil ( 2.6  g , 14.6  mmol , 73% yield ) and 3b was obtained as colourless oil ( 2.5  g , 13  mmol , 72% yield ) .
the structures were confirmed by nmr spectroscopy and matched literature data ( yang et  al . , 1999 ) .
the test for hydrolytic activity towards the target substrates was based on a ph assay ( baumann et  al . , 2000 ) .
wells were filled with 20  l of a substrate stock solution ( 200  mm in dmso ) , 5  l of a solution of the ph indicator bromothymol blue ( btb , 16  mm in 16% ethanol and 84% water ) , 30  l of a glycerol stock solution of the different esterases and 145  l of sodium phosphate buffer ( 5  mm , ph 7.4 ) to get a final volume of 200  l .
positive controls contained 5  l of btb solution , 175  l of sodium phosphate buffer ( 5  mm , ph 7.4 ) and 20  l of dmso containing the correspondent acids 1a3a at final concentrations of 120  mm .
each mtp was then sealed with adhesive foil , incubated at 37c for 72  h and stirred at 400  r.p.m . in a thermoshaker ( eppendorf , hamburg , germany ) .
active esterases were then identified by visible colour changes and used in analytical scale experiments .
to a stirred solution of the ethyl esters 1b3b ( 0.076  mmol of 1b or 2b , or 0.15  mmol of 3b ) in 1.8  ml of sodium phosphate buffer ( 50  mm , ph 7.4 ) and 10% dmso if necessary , 200  l of esterase solution was added to a total volume of 2  ml .
the reaction mixture was stirred at 1000  r.p.m . in a thermoshaker at 20c or 30c .
samples ( 400  l ) were taken after 2 , 7 and 24  h. these samples were acidified with 20  l of 1  n hcl and subsequently extracted three times with 400  l diethyl ether .
the combined organic layers were dried over anhydrous sodium sulfate and afterwards the organic solvent was evaporated in a speed vac ( thermo fisher scientific , langenselbold , germany ) .
the mixture was then treated with tms diazomethane ( hashimoto et  al . , 1981 ) in order to convert the carboxylic acid ( hydrolysis product ) in its methyl ester to allow analysis of substrate and product concentration and optical purity in one run by gc .
conversion and e values were then calculated from the enantiomeric excesses from substrate and product following the equations developed by chen and colleagues ( 1982 ) .
to a 40  mm solution of 1b3b ( 1  g ) in sodium phosphate buffer ( 50  mm , ph 7.4 ) containing 10% ( v / v ) dmso , esterase ( lyophilisate dissolved in buffer , units as given in table  2 ) was added and hydrolysis was performed at 20c or 30c .
after tlc analysis indicated 50% conversion , the reaction was stopped by addition volume of 1  n hcl , and substrate and product were extracted three times with diethyl ether . after drying the organic phase over anhydrous na2so4 ,
the solvent was removed by evaporation under reduced pressure and the product was purified by column chromatography ( npentane / ethyl acetate ) .
the purity of the biocatalysis products was confirmed by nmr spectroscopy and was in all cases >  99% . using this protocol , ( r)()1b was obtained as a light yellow oil ( 411  mg , 21  mmol , 41.4% yield ) .
( s)(+)1a was obtained as colourless oil ( 230  mg , 14  mmol , 27.7% yield , 72%ee ) ; ( s)(+)1b was obtained as light yellow oil ( 420  mg , 2.2  mmol , 42% yield ) .
enantiomeric excess was determined after hydrolysis to 1a as 87%ee ; ( r)()1a was obtained as light yellow oil ( 330  mg , 2  mmol , 38.5% yield , 98%ee ) ; ( r)()2b was obtained as colourless oil ( 164  mg , 9.2  mmol , 16.4% yield ) .
enantiomeric excess was determined after hydrolysis to 2a as 82%ee ; ( s)(+)2a was obtained as a white powder ( 181  mg , 12  mmol , 21.5% yield , 97%ee ) ; ( s)(+)2b was obtained as a colourless oil ( 218  mg , 12.2  mmol , 28.3% yield ) .
enantiomeric excess was determined after hydrolysis to 2a as 92%ee ; ( r)()2a was obtained as a white powder ( 180  mg , 12  mmol , 21.3% , 56%ee ) ; ( r)()3b was obtained as colourless oil ( 380  mg , 1.99  mmol , 38% yield ) .
enantiomeric excess was determined after hydrolysis to 3a as 69%ee ; ( s)(+)3a was obtained as yellow oil ( 360  mg , 2.18  mmol , 41.9% yield , >  99%ee ) ; ( s)(+)3b was obtained as colourless oil ( 150  mg , 0.78  mmol , 15% yield ) .
enantiomeric excess was determined after hydrolysis to 3a as 97%ee ; ( r)()3a was obtained after column chromatography ( npentane / ethyl acetate ) as yellow oil ( 330  mg , 2  mmol , 39% yield , 95%ee ) .
 summaryenantiomerically pure arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities . 
 a library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter platebased screening using a phindicator assay . out of these , 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity . 
 the most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions . 
 thus , both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields . 
Experimental procedures
Materials
General procedure for the synthesis of the phenylalkyl carboxylic acid ethylesters
Highthroughput screening
General procedure for esterasecatalysed analytic scale kinetic resolutions
Esterasecatalysed preparativescale kinetic resolution of 1b3b