in recent years , ene - reductases became 
 prominent for providing 
 a green alternative for the highly selective production of chiral 
 molecules .
the enzymes are members of 
 the flavin - dependent old yellow enzyme ( oye ) family and catalyze the 
 asymmetric reduction of a broad variety of activated alkenes at the 
 expense of a nicotinamide cofactor .
ene - reductases 
 have been utilized for the synthesis of diverse enantiomerically pure 
 products , including asymmetric building blocks such as chiral acyloins and the  roche - ester  .
further examples encompass enantiopure - 
 and -amino acid derivatives , molecules for 
 pharma applications , and compounds of 
 commercial importance , such as the fragrances lilial or lysmeral .
pregabalin [ ( s)-3-aminomethyl-5-methylhexanoic 
 acid ] , the active ingredient in lyrica , is a -substituted -amino 
 acid belonging to the class of drugs known as gaba analogues . the 
 manufacture of pregabalin begins with the enzymatic resolution of 
 a -cyanodiester followed by decarboxylation 
 to give the penultimate intermediate , ( s)-ethyl 3-cyano-5-methylhexanoate 
 ( s)-4b .
reduction of the nitrile moiety 
 gives pregabalin . while this chemo - enzymatic 
 process achieved a large increase in efficiency over the initial manufacturing 
 protocol , we were interested in evaluating 
 stereoselective approaches independent of kinetic resolution protocols 
 with the objective of further increasing process efficiency .
we postulated 
 that asymmetric bioreduction of a -cyanoacrylate ester by an 
 ene - reductase could provide access to ( s)-4b , thus enabling a novel route to pregabalin , and set out to evaluate 
 the feasibility of this potential strategy ( scheme 1 ) .
this approach could also be useful for the stereoselective 
 synthesis of related -substituted -amino acids ( baclofen ) 
 or derivatives ( brivaracetam ) .
-cyanoacrylate esters 
 ( e)-1a5a and ( z)-1a5a were prepared 
 from the appropriate -keto ester via 
 enol triflate precursors using the procedure described by frantz et 
 al .
( scheme 2 ) . 
 the selectivity for ( z)-enol triflates was excellent 
 with no trace of ( e)-isomers present at the end of 
 the reaction , and ( z)-enol triflates were isolated 
 in 8092% yield after chromatography . the conditions to generate 
 ( e)-analogues gave poorer selectivity ( > 50:1 to 
 5:1 ) , 
 as the steric bulk of the r group increased from me to i - bu .
furthermore , lower isolated yields ( 5471% ) 
 were observed for the ( e)-enol triflates , which was 
 due to less effective extraction of the product from the me4noh / water mixture going in hand with 
 competing hydrolysis of triflic anhydride .
treatment of ( e)- or ( z)-enol triflates with zn(cn)2 in the presence of a palladium(0 ) catalyst using the procedure 
 of tebben et al . generated the desired 
 -cyanoacrylate esters in 5186% yield .
substrates ( z)-6a and ( e)-7a were obtained by transesterification from ( z)-3a and ( e)-5a , respectively .
since the ( e / z)-configuration 
 of alkenes has been 
 shown to have a dramatic impact on the stereochemical outcome of ene - reductions , stereochemically pure substrates 1a4a were used . in addition , the size of the alcohol moiety of the ( activating ) 
 ester group was varied in order to test its influence on the substrate - docking 
 mode of substrates 5a7a within the 
 active site .
all 
 substrates were tested using a set of native ene - reductases , which 
 have shown to possess a broad substrate spectrum on acrylic ester 
 derivatives ( scheme 1 , table 1 ) .
we envisaged that 
 the appropriate combination of substrate- and 
 enzyme - engineering would enable us to access both stereoisomers of 
 gaba - precursors with high conversion and perfect stereoselectivity . 

interestingly , in line with previous reports , ( e)--cyanoacrylate 
 esters generally were converted to the ( s)-products 
 while the bioreduction of ( z)-analogues gave the 
 enantiomeric ( r)--cyano esters .
accordingly , 
 ( s)-1b was obtained from ( e)-1a in perfect conversion and stereoselectivity using 
 oye2 , oye3 , and ebp1 , while ( r)-1b was 
 formed from ( z)-1a using ncr ( ee > 
 99 , 
 c 94% ) .
the same enzymes showed similar activities toward ( e)-2a and ( z)-2a : oyes 13 and ncr produced ( s)-2b from ( e)-2a ( ee > 99 , c > 99% ) 
 and 
 ( r)-2b could be obtained with perfect 
 ee from ( z)-2a using opr3 , xenb , and 
 ncr . in analogy ,
( s)-3b could be produced 
 from ( e)-3a ( ee > 99 , c > 99% ) 
 and the 
 reduction of ( e)-4a afforded ( s)-4b with perfect stereoselectivity in up 
 to 48% conversion using oye1 , oye2 , and ncr . however , with increasing 
 steric demand of the substrates , the conversion of ( z)--cyanoacrylate esters dropped , resulting in low reactivity 
 toward ( z)-3a and no activity toward 
 ( z)-4a at all .
in contrast , decreasing 
 the size of the ester moiety from ethyl to methyl had a strongly positive 
 impact on reaction rates : by applying methyl ( e)--cyanoacrylate 
 ( z)-6a instead of the ethyl ester ( z)-3a , the activity of ebp1 forming the ( r)-enantiomer could be improved from 5% conversion ( 85% 
 ee ) to 64% conversion and perfect stereoselectivity .
in the same way , 
 ethyl ester ( z)-4a was not accepted 
 by any of the enzymes , but the corresponding methyl ester ( z)-5a was converted with modest rates ( oye3 , 
 c 29% ) . for the bulkier substrates 4a , 5a , and 7a bearing the isobutyl pharmacophor side chain 
 of pregabalin , the stereorecognition was not as homogeneously conserved 
 as for the smaller analogues . whereas the less bulky e - configured substrates strictly yielded s - products 
 with the majority of enzymes in up to quantitative conversion , the 
 stereopreference of oye3 switched to r ( c > 99 , 
 ee 
 > 99 ) for ( e)-5a . this exceptional 
 behavior 
 of oye3 has been observed before and depends 
 on the steric demand of the ester group , going from absolute r - selectivity for the methyl ester ( e)-5a to moderate s - preference for the ethyl 
 ester ( e)-4a ( ee 38% ) .
in order to push 
 the incomplete s - selectivity , the size of the ester 
 group was increased to n - propyl [ ( e)-7a ] , which successfully resulted in ( s)-7b as the sole product .
overall , opr3 , xena , and yqjm 
 showed poor activities toward all tested substrates and nera and xenb 
 accepted only the smaller substrates 1a3a .
using oye2 in combination with a recently developed adh - based 
 nadh - cofactor recycling system , ( s)-1b3b could be produced 
 on preparative scale ( > 1 g ) with perfect ee and good isolated yields 
 ( up to 83% ) , which nicely demonstrates the applicability of this bioreduction 
 protocol to asymmetric syntheses .
double enzyme and half substrate 
 amount : > 99% conversion , > 99 e.e .
mutant 
 variants were constructed . a basic sequence alignment 
 of opr1 identified many potential sites for mutation of which positions 
 cys20 , ile287 , and his245 were selected for investigation .
( neutral at ph 7.5 ) was mutated to a negatively charged asp to enhance 
 the polarity around the active site entrance for improved substrate 
 binding .
the sequence alignment showed a high variability of 14 amino 
 acids at position ile287 , which is located in a flexible loop close 
 to the active site entrance extending from arg279 to thr290 . in order 
 to make this region more rigid
cys20 located at the 
 surface opposite to the active site was exchanged by a tyr to probe 
 whether such a distant mutation had any effects on the chiral recognition 
 process .
results from bioreduction 
 screens on opr1 variants ( table 2 ) showed improved 
 conversions for variant cys20tyr 
 with ( e)-4a and ( e)-5a compared to wild - type opr1 , while ile287his did not give 
 improved conversions for any of the substrates .
however , cys20tyr 
 and ile287his both gave greatly enhanced stereoselectivities in the 
 bioreduction of ( e)-5a and slight improvements 
 for ( e)-2a and ( z)-2a .
variant his245asp gave the highest increase in selectivity 
 for the bioreduction of ( e)-5a and also 
 showed the highest improvements in conversion for several of the substrates .
the asymmetric bioreduction of -cyanoacrylate 
 esters using 
 ene - reductases provides an elegant alternative to metal - dependent 
 hydrogenation protocols for the asymmetric synthesis of precursors 
 for gaba analogues , such as pregabalin .
this concept was successfully 
 applied to the production of a library of chiral -cyano esters . 

the stereochemical outcome could be switched via substrate - engineering 
 through size - variation of the alkoxy moiety and by employing stereochemically 
 pure ( e)- or ( z)-isomers , which 
 allowed to access both enantiomers of each product with up to quantitative 
 conversion in enantiomerically pure form . in addition , stereoselectivities 
 and conversions could be improved by mutant variants of opr1 . finally , 
 the utility of the system for preparative - scale applications was demonstrated 
 by the isolation of gram - amounts of -cyano esters with perfect 
 ee . upscaling to industrial production
should be 
 feasible as the procedure utilized inexpensive cosubstrate to recycle 
 the nicotinamide cofactor , improvement of the volumetric efficiency 
 would be feasible by development of more active enzymes via enzyme 
 engineering and/or screening natural diversity .
further process efficiency 
 could be achieved by the implementation of a hydrolase - reductase cascade 
 process : in the case of pregabalin synthesis , 
 the addition of a hydrolase to the bioreduction of ( e)-4a could result in a one - pot process to ( s)-3-cyano-5-methylhexanoic acid , the penultimate precursor to pregabalin , thus eliminating an additional step for hydrolysis 
 of ( s)-4b . in conclusion , this study 
 demonstrates the applicability of ene - reductases as a general synthetic 
 tool by meeting the demand for green and highly stereoselective synthetic 
 methods .
gc analyses were carried out using 
 a chiraldex g - ta column ( 30 m  0.25 mm i d , 0.12 m film ) 
 or a hydrodex--tbdac capillary column ( 250 m  0.25 mm 
 i d ) .
nmr spectra were obtained on a 400 mhz or a 300 mhz nmr spectrometer . 

chemical shifts are reported relative to tms (  0.00 ) , and coupling 
 constants ( j ) are given in hz .
high resolution mass 
 spectra were obtained either on a oa - tof mass spectrometer with direct 
 insertion and electron - impact ionization ( 70 ev ) or on a ion trap 
 ft - ms using electrospray ionization in positive mode or apci ionization 
 in negative mode .
tlc plates were run on silica gel , and compounds 
 were visualized by spraying with phosphomolybdic acid stain or by 
 uv ( 254 nm ) .
12-oxophytodienoate reductase isoenzymes 
 opr1 and opr3 from lycopersicon esculentum and the 
 oye homologue yqjm from bacillus subtilis were overexpressed 
 and purified as reported .
the cloning , purification , and 
 characterization of oye isoenzymes from yeast ( oye1 from saccharomyces 
 pastorianus , oye2 and oye3 from saccharomyces cerevisiae ) and nicotinamide - dependent cyclohexenone reductase ( ncr ) from zymomonas mobilis were performed according to literature 
 methods .
xenobiotic reductases xena and xenb from pseudomonas putida and p. fluorescens , 
 respectively , and glycerol trinitrate reductase nera from agrobacterium radiobacter were obtained as recently published.lycopersicon esculentum opr1 variants cys20tyr , 
 ile287his , and his245asp were constructed in pet26b using quikchange 
 lightning site - directed mutagenesis kits ( stratagene , la jolla , ca ) 
 and were expressed as his - tagged proteins in e. coli bl21 cells .
the cloning and purification of epb1 from candida 
 albicans and of lycopersicon esculentum opr1 
 variants cys20tyr , ile287his , and his245asp can be found in the supporting information . an aliquot 
 of enzyme ( opr1-wt , opr3 , oye1 - 3 , xena , xenb , yqjm , ebp1 , nera , or 
 ncr ; protein concentration in biotransformations : 75125 g / ml ) 
 was added into a 1.5 ml microcentrifuge tube containing a tris - hcl 
 buffer solution ( 0.8 ml , 50 mm , ph 7.5 ) , the substrate ( 10 mm ) , and 
 the cofactor ( nadh , 15 mm ) .
after 24 h , the products were extracted with ethyl acetate 
 ( 0.6 ml ) .
the organic phase was dried over na2so4 and analyzed on gc to determine enantiomeric excess and conversion . 
 for every screening a control experiment without
absolute configurations of 1b , 2b , and 3b were determined by 
 preparation of ( s)-1b , ( s)-2b , and ( s)-3b via bioreductions 
 of ( e)-1a , ( e)-2a , and ( e)-3a at preparative 
 scale and subsequent conversions to suitable derivatives and comparison 
 of specific rotations of the latter with literature values . for determination 
 of the absolute configuration of 4b , both reference materials 
 ( r)- and ( s)-4b were 
 prepared . for
the assignment of the absolute configurations of 5b , 6b , and 7b , enantiomerically 
 pure ( s)-isomers were prepared by transesterification 
 of the corresponding ethyl esters ( s)-4a and ( s)-3a .
the ketone 
 ( 1.0 equiv ) was added slowly , over 15 min , to a stirred suspension 
 of nah ( 60% in mineral oil , 1.2 equiv ) in dry thf ( 500 ml ) at 0 c . 
 after the addition was complete , the cooling bath was removed , and 
 the resulting mixture was stirred at rt for 40 min .
the reaction was 
 cooled to 0 c , and the carbonate ester ( 1.5 equiv ) was added 
 slowly over 1 h. after the addition was complete , the reaction was 
 allowed to slowly warm to rt with stirring ( 18 h ) .
the reaction was 
 quenched and acidified to ph 6 by careful addition of acetic 
 acid ( 60 ml ) .
water ( 400 ml ) was added , and the resulting mixture 
 was extracted with et2o .
the organic extracts were washed 
 with brine , dried over na2so4 , and concentrated 
 under reduced pressure to give the crude -ketoester . using general procedure a , 2-butanone ( 51.6 ml , 41.5 g , 576 mmol ) , 
 nah ( 60% in mineral oil , 27.6 g , 691 mmol ) in dry thf ( 500 ml ) , and 
 diethyl carbonate ( 105 ml , 102 g , 864 mmol ) gave the crude product 
 as a clear yellow oil , which was distilled twice under reduced pressure 
 ( 60 c , 4 mbar ) resulting in a 2:1 mixture of the desired product 
 and the regioisomer ethyl 2-methyl-3-oxobutanoate as a clear colorless 
 oil ( 31.9 g , 38% ) : h nmr ( 300 mhz , cdcl3 )  
 1.09 ( t , j = 7.5 hz , 3h ) , 1.28 ( t , j = 7.0 hz , 3h ) , 2.56 ( q , j = 7.5 hz , 2h ) , 3.43 ( s , 
 1h ) , 4.19 ( q , j = 7.0 hz , 2h ) .
ethyl 2-methyl-3-oxobutanoate : h nmr ( 300 mhz , cdcl3 )  1.28 ( t , j = 7.0 hz , 3h ) , 1.34 ( d , j = 7.0 hz , 3h ) , 
 2.24 ( s , 3h ) , 3.49 ( q , j = 7.0 hz , 1h ) , 4.19 ( q , j = 7.0 hz , 2h ) . using 
 general procedure
a , 2-pentanone ( 61.5 ml , 49.6 g , 576 mmol ) , nah 
 ( 60% in mineral oil , 27.6 g , 691 mmol ) in dry thf ( 500 ml ) , and diethyl 
 carbonate ( 104.9 ml , 102.1 g , 864 mmol ) gave the crude product as 
 a clear yellow oil , which was distilled under reduced pressure ( 60 
 c , 4 mbar ) yielding the desired product as a clear colorless 
 oil ( 39.9 g , 44% ) : h nmr ( 300 mhz , cdcl3 )  
 0.93 ( t , j = 7.5 hz , 3h ) , 1.28 ( t , j = 7.0 hz , 3h ) , 1.561.70 ( m , 2h ) , 2.51 ( t , j = 7.0 hz , 2h ) , 3.42 ( s , 2h ) , 4.19 ( q , j = 7.0 hz , 
 2h ) . using general procedure a , 4-methyl-2-pentanone ( 608.0 ml , 487.0 
 g , 4.86 mol ) , nah ( 60% in mineral oil , 256.7 g , 1.32 equiv ) in dry 
 thf ( 4.87 l ) , and diethyl carbonate ( 884.0 ml , 1.5 equiv ) gave the 
 crude product as an orange oil .
the crude product was distilled under 
 reduced pressure ( 6482 c , 710 mbar ) the desired 
 product as a pale yellow oil ( 661.2 g , 79% isolated yield ) : h nmr ( 300 mhz , cdcl3 )  0.86 ( d , j = 6.4 hz , 6h ) , 1.21 ( t , j = 7.2 hz , 3h ) , 2.09 ( j = 6.7 hz , 1h ) , 2.35 ( d , j = 6.9 hz , 2h ) , 
 3.34 ( s , 2h ) , 4.12 ( q , j = 7.2 hz , 2h ) . using general procedure
a , 4-methyl-2-pentanone ( 72.1 ml , 57.7 
 g , 576 mmol ) , nah ( 60% in mineral oil , 27.6 g , 691 mmol ) in dry thf 
 ( 500 ml ) , and dimethyl carbonate ( 72.8 ml , 77.8 g , 864 mmol ) gave 
 the crude product as a cloudy yellow oil .
the crude product was distilled 
 under reduced pressure ( 60 c , 4 mbar ) to give the desired product 
 as a clear colorless oil ( 61.9 g , 68% ) : h nmr ( 300 mhz , 
 cdcl3 )  0.93 ( d , j = 6.5 hz , 6h ) , 
 2.092.22 ( m , 1h ) , 2.41 ( d , j = 7.0 hz , 2h ) , 
 3.42 ( s , 2h ) , 3.73 ( s , 3h ) .
lithium triflate ( 2.0 equiv ) was added to a stirred solution of 
 the keto ester ( 1.0 equiv ) in dry ch2cl2 ( 150 
 ml ) .
the resulting mixture was cooled to 0 c , and diisopropylethylamine 
 ( 1.1 equiv ) was added dropwise over 10 min . after the addition was 
 complete the mixture was stirred at 0 c for 20 min before triflic 
 anhydride ( 1.1 equiv ) was added slowly over 30 min .
the resulting 
 mixture was stirred at 0 c for 1 h before carefully being quenched 
 by addition of saturated aqueous nh4cl ( 60 ml ) .
the reaction 
 was allowed to warm to rt and an additional portion of ch2cl2 ( 30 ml ) was added .
the layers were separated , and 
 the organic layer was washed with aqueous hcl ( 1 m , 2  30 ml ) , 
 water ( 30 ml ) , and brine ( 30 ml ) .
et2o ( 60 ml ) was added to the resulting semisolid , and 
 the mixture was filtered .
the solids were extracted with more et2o ( 2  20 ml ) , and the combined ether extracts were concentrated 
 under reduced pressure to give the crude product . using general procedure b , lithium 
 triflate ( 24.0 g , 154 mmol ) , ethyl acetoacetate ( 9.7 ml , 10.0 g , 76.8 
 mmol ) in dry ch2cl2 ( 150 ml ) , diisopropylethylamine 
 ( 14.7 ml , 10.9 g , 84.5 mmol ) and triflic anhydride ( 14.22 ml , 23.9 
 g , 84.5 mmol ) gave the crude product as a clear yellow oil ( 18.6 g , 
 92% ) which was used without further purification : h nmr 
 ( 300 mhz , cdcl3 )  1.30 ( t , j = 
 7.0 hz ) , 2.16 ( s , 3h ) , 4.24 ( q , j = 7.0 hz , 2h ) , 
 5.75 ( q , j = 1.0 hz , 1h ) ; c nmr ( 100 
 mhz , cdcl3 )  14.2 , 21.1 , 61.4 , 113.0 , 118.5 ( q , j = 319.8 hz , cf3 ) , 155.2 , 162.5 . using general procedure b , lithium 
 triflate ( 21.6 g , 139 mmol ) , ethyl 3-oxopentanoate ( 10.0 g , 69.4 mmol ) 
 in dry ch2cl2 ( 150 ml ) , diisopropylethylamine 
 ( 13.3 ml , 9.9 g , 76.3 mmol ) , and triflic anhydride ( 12.8 ml , 21.5 
 g , 76.3 mmol ) gave the crude product as a clear dark orange oil .
purification 
 by flash chromatography ( silica gel , cyclohexane / etoac , 9:1 ) afforded 
 the desired product as a clear pale yellow oil ( 17.7 g , 92% yield ) : h nmr ( 300 mhz , cdcl3 )  1.18 ( t , j = 7.5 hz , 3h ) , 1.31 ( t , j = 7.0 hz , 3h ) , 
 2.44 ( qd , j = 7.5 , 1.0 hz , 2h ) , 4.25 ( q , j = 7.0 hz , 2h ) , 5.75 ( t , j = 1.0 hz , 1h ) .
using general procedure b , lithium triflate ( 19.7 g , 126 mmol ) , 
 ethyl 3-oxohexanoate ( 10.0 g , 63.2 mmol ) in dry ch2cl2 ( 150 ml ) , diisopropylethylamine ( 12.1 ml , 9.0 g , 69.5 mmol ) , 
 and triflic anhydride ( 11.7 ml , 19.6 g , 69.5 mmol ) gave the crude 
 product as a clear dark red oil ( 17.0 g , 93% yield ) which was used 
 without further purification : h nmr ( 300 mhz , cdcl3 )  0.99 ( t , j = 7.5 hz , 3h ) , 1.30 
 ( t , j = 7.0 hz , 3h ) , 1.551.69 ( m , 2h ) , 2.37 
 ( t , j = 7.5 hz , 2h ) , 4.25 ( q , j = 
 7.0 hz , 2h ) , 5.74 ( t , j = 1.0 hz , 1h ) ; c nmr ( 100 mhz , cdcl3 )  13.3 , 14.2 , 19.5 , 36.5 , 
 61.5 , 112.1 , 118.6 ( q , j = 319.8 hz , cf3 ) , 158.9 , 162.7 ; 
 hrms ( ei ) m / z calcd 
 for c9h13f3o5s [ m ] 290.0439 , found 290.0436 .
a 20 l jacketed reactor was charged 
 with ch2cl2 ( 2.45 l ) , ethyl 3-oxo-5-methylhexanoate 
 ( 163.3 g ; 1 equiv ) , and lithium triflate ( 295.86 g ; 2 equiv ) under 
 an inert atmosphere . the resulting white suspension was stirred and 
 cooled to 0 c .
n , n - diisopropylethylamine 
 ( 134.8 g ; 182 ml ; 1.1 equiv ) was added over 10 min at < 10 c . 

the suspension was stirred for 20 min at 0 c , and then triflic 
 anhydride ( 294.28 g ; 175 ml ; 1.1 equiv ) was added slowly to the mixture 
 over 30 min at < 10 c .
the suspension was stirred at 0 c 
 for 1 h. the mixture then was allowed to warm from 0 to 25 c 
 over 1 h while quenching with satd nh4cl solution ( 980 
 ml ) .
dichloromethane ( 490 ml ) was added , and the mixture was stirred 
 for 5 min and then allowed to settle , and the layers were separated . 

the combined organic phases were then washed with 1 m hcl ( 2 
  490 ml ) , water ( 490 ml ) , and 20% brine ( 490 ml ) and then stirred 
 for 30 min with mgso4 ( 165 g ) for 1530 
 min .
the filtrate was concentrated under 
 vacuum at 40 c to give an orange / brown oil containing 
 crystalline solids .
the mixture was filtered and the solids were washed with mtbe 
 ( 2  165 ml ) .
the filtrate was concentrated under vacuum at 40 
 c to give the title product as an orange / brown oil : overall 
 yield 271 g ( 94% ; corrected for 2.1% w / w mtbe content ) ; 87.7% area 
 purity by gc ; h nmr ( 400 mhz , cdcl3 ) :  
 0.94 ( 6h , d , j = 6.7 hz ) , 1.24 ( 3h , t , j = 7.2 hz ) , 1.89 ( 1h , hept , j = 6.8 hz ) , 2.17 ( 2h , 
 d , j = 7.2 hz ) , 4.18 ( 2h , q , j = 
 7.2 hz ) , 5.67 ( 1h , s ) . using general procedure b , lithium triflate ( 19.7 g , 126 mmol ) , 
 methyl 5-methyl-3-oxohexanoate ( 10.0 g , 63.2 mmol ) in dry ch2cl2 ( 150 ml ) , diisopropylethylamine ( 12.1 ml ,
9.0 g , 69.5 
 mmol ) , and triflic anhydride ( 11.7 ml , 19.6 g , 69.5 mmol ) gave the 
 crude product as a clear orange oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac , 9:1 ) afforded the desired product 
 as a clear pale yellow oil ( 14.7 g , 80% ) : h nmr ( 300 mhz , 
 cdcl3 )  0.99 ( d , j = 6.6 hz , 6h ) , 
 1.98 ( dp , j = 13.6 , 6.8 , 1h ) , 2.27 ( d , j = 7.2 hz , 
 2h ) , 3.80 ( s , 3h ) , 5.76 ( s , 1h ) .
c nmr ( 75 mhz , cdcl3 )  21.9 , 25.8 , 43.7 , 51.9 , 112.4 , 118.4 ( q , j = 318.0 
 hz , cf3 ) , 158.3 , 162.8 ; hrms ( es ) m / z calcd for c9h14f3o5s [ m + h ] 291.0514 , found 291.0519 .
a mixture of me4noh ( 25% 
 in h2o , 5.0 equiv ) and water ( 72.5 ml ) was added dropwise 
 over 30 min to a vigorously stirred solution of the keto ester ( 1.0 
 equiv ) in heptane ( 300 ml ) at 0 c .
after the addition was complete , 
 the mixture was stirred at 0 c for 15 min before triflic anhydride 
 ( 2.5 equiv ) was added dropwise over 1 h. after the addition was complete , 
 the reaction was stirred for another 1 h at 0 c before being 
 quenched by careful addition of water ( 75 ml ) .
the mixture was allowed 
 to warm to rt , and the layers were separated .
the combined organic extracts 
 were washed with water ( 75 ml ) and brine ( 75 ml ) , dried over na2so4 , and concentrated under reduced pressure to 
 yield the crude product . using general procedure c , me4noh ( 25% in h2o , 137 ml , 

140 g , 384 mmol ) , water ( 72.5 ml ) , ethyl acetoacetate ( 9.7 ml , 10.0 
 g , 76.8 mmol ) in heptanes ( 300 ml ) , and triflic anhydride ( 32.3 ml , 
 54.2 g , 192.1 mmol ) gave the crude product as a clear colorless oil 
 ( 14.4 g , 71% ) , which was used without further purification : h nmr ( 400 mhz , cdcl3 )  1.31 ( t , j = 7.0 hz , 3h ) , 2.51 ( s , 3h ) , 4.22 ( q , j = 7.0 hz , 
 2h ) , 5.95 ( s , 1h ) . using general procedure c , me4noh ( 25% in h2o , 124 ml , 
 26.4 g , 347 mmol ) , water ( 72.5 ml ) , ethyl 3-oxopentanoate ( 10.0 g , 
 69.4 mmol ) in heptanes ( 300 ml ) , and triflic anhydride ( 29.2 ml , 48.9 
 g , 173.4 mmol ) gave the crude product as a clear pale yellow oil . 

purification by flash chromatography ( silica gel , cyclohexane / etoac , 
 19:1 ) afforded the desired product as a clear colorless oil ( 10.6 
 g , 55% ) : h nmr ( 400 mhz , cdcl3 )  1.21 
 ( t , j = 7.5 hz , 3h ) , 1.31 ( t , j = 
 7.0 hz , 3h ) , 2.94 ( q , j = 7.5 hz , 2h ) , 4.22 ( q , j = 7.0 hz , 2h ) , 5.92 ( s , 1h ) .
using general procedure c , me4noh ( 25% in h2o , 113 ml , 
 115 g , 316.1 mmol ) , water ( 72.5 ml ) , ethyl 3-oxohexanoate ( 10.0 g , 
 63.2 mmol ) in heptanes ( 300 ml ) , and triflic anhydride ( 26.6 ml , 44.6 
 g , 158.0 mmol ) gave the crude product as a clear yellow oil ( 13.8 
 g , 75% yield ) , which was used without further purification : h nmr ( 300 mhz , cdcl3 )  0.99 ( t , j = 7.5 hz , 3h ) , 1.30 ( t , j = 7.0 hz , 3h ) , 1.591.73 
 ( m , 2h ) , 2.90 ( t , j = 7.5 hz , 2h ) , 4.22 ( q , j = 7.0 hz , 2h ) , 5.95 ( s , 1h ) . a 20 l jacketed reactor was charged 
 with hexane ( 7.83 l ) and ethyl 3-oxo-5-methylhexanoate ( 261.0 g , 1 
 equiv ) under an inert atmosphere .
the solution was cooled to 5 c 
 and stirred vigorously while a mixture of 25% aq tetramethylammonium 
 hydroxide ( 2.76 kg , 2.76 l , 5 equiv ) and water ( 1.89 l ) was added 
 over 30 min at 15 c .
the biphasic mixture was stirred vigorously 
 for 10 min at 510 c , and then triflic anhydride ( 1.07 
 kg , 637 ml , 2.5 equiv ) was added dropwise to the mixture over 1.5 
 h at < 10 c .
the mixture was stirred vigorously at 5 c 
 for a further 1 h , agitation was stopped , and the layers were allowed 
 to settle before the upper organic layer was sampled .
the mixture 
 was then allowed to warm from 5 to 25 c over 1 h while being 
 quenched with water ( 1.97 l ) and then stirred for 5 min .
the combined organic layers were 
 washed with water ( 1.97 l ) and then 20% brine ( 1.97 l ) , stirred with 
 mgso4 ( 300 g ) for 1530 min , and then filtered . 

the residue was washed with etoac ( 260 ml ) , and the filtrate was concentrated 
 under vacuum at 40 c to give the e - vinyl 
 triflate as a brown oil ( overall yield of 378 g , 82% ; corrected for 
 8.4% w / w etoac content , 75.5% area purity by gc ) .
nmr analysis indicated 
 that the isomeric ratio was 6:1 : h nmr ( 400 mhz , cdcl3 ) :  0.92 ( 6h , d , j = 6.7 hz ) , 1.24 
 ( 3h , t , j = 7.2 hz ) , 1.95 ( 1h , hept , j = 6.8 hz ) , 2.17 ( 0.33h , d , j = 7.2 hz ) , 2.76 ( 2h , 
 d , j = 7.3 hz ) , 4.14 ( 2h , q , j = 
 7.2 hz ) , 4.18 ( 0.28h , q , j = 7.2 hz ) , 5.66 ( 0.14h , 
 s ) , 5.91 ( 1h , s ) . using general procedure c , me4noh ( 25% in h2o , 113 ml ,
115 g , 316 mmol ) , water ( 72.5 ml ) , 
 ethyl 3-oxo-5-methylhexanoate ( 10.0 g , 63.2 mmol ) in heptane ( 300 
 ml ) , and triflic anhydride ( 26.6 ml , 44.6 g , 158 mmol ) gave the crude 
 product as a clear pale yellow oil .
etoac , 19:1 ) afforded the desired 
 product as a clear colorless oil ( 10.0 g , 54% ) : h nmr 
 ( 400 mhz , cdcl3 )  0.99 ( d , j = 
 6.5 hz , 6h ) , 2.02 ( m , 1h ) , 2.83 ( d , j = 7.5 hz , 2h ) , 
 3.76 ( s , 3h ) , 5.99 ( s , 1h ) ; c nmr ( 125 mhz , cdcl3 )  21.8 , 22.1 , 26.5 , 39.8 , 51.9 , 112.9 , 118.4 ( q , j 
 = 319.8 hz , cf3 ) , 164.5 , 165.1 ; hrms ( es ) m / z calcd for c9h14f3o5s [ m + h ] 291.0514 , found 291.0516 .
the triflate ( 1.0 equiv ) and zn(cn)2 ( 0.6 equiv ) were 
 added to dry dmf ( 75 ml ) .
the mixture was purged with n2 for 15 min before pd(pph3)4 ( 0.01 equiv ) was 
 added , and the mixture was heated at 70 c for 15 h. the reaction 
 mixture was allowed to cool to rt , and toluene ( 125 ml ) was added . 

the mixture was washed with aqueous ammonium hydroxide ( 20% , 2  
 50 ml ) , and the aqueous washings were extracted with toluene ( 50 ml ) . 

the combined organic extracts were washed with brine ( 50 ml ) , dried 
 over na2so4 , and concentrated under reduced 
 pressure to give the crude product . using general 
 procedure d , ( z)-ethyl 3-(trifluoromethylsulfonyloxy)but-2-enoate 
 ( 12.3 g , 46.8 mmol ) , zn(cn)2 ( 3.30 g , 28.1 mmol ) in dmf 
 ( 75 ml ) , and pd(pph3)4 ( 0.54 g , 0.47 mmol ) gave 
 the crude product as a clear yellow oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac 9:1 ) afforded the desired product as 
 a clear pale yellow oil ( 4.23 g , 65% ) : h nmr ( 400 mhz , 
 cdcl3 )  1.33 ( t , j = 7.0 hz , 3h ) , 
 2.16 ( s , 3h ) , 4.28 ( q , j = 7.0 hz , 2h ) , 6.32 ( s , 
 1h ) ; c nmr ( 100 mhz , cdcl3 )  14.0 , 
 22.0 , 61.5 , 116.4 , 122.8 , 133.1 , 162.7 . using general 
 procedure d ,
( e)-ethyl 3-(trifluoromethylsulfonyloxy)but-2-enoate 
 ( 12.3 g , 46.8 mmol ) , zn(cn)2 ( 3.30 g , 28.1 mmol ) in dry 
 dmf ( 75 ml ) , and pd(pph3)4 ( 0.54 g , 0.47 mmol ) 
 gave the crude product as a clear red oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac , 9:1 ) afforded the desired product 
 as a clear pale yellow oil ( 4.52 g , 69% ) : h nmr ( 400 mhz , 
 cdcl3 ) :  1.31 ( t , j = 7.0 hz , 3h ) , 
 2.35 ( s , 3h ) , 4.23 ( q , j = 7.0 hz , 2h ) , 6.41 ( s , 
 1h ) ; c nmr ( 100 mhz , cdcl3 ) :  14.1 , 
 17.3 , 61.2 , 118.8 , 125.9 , 132.7 , 163.8 . using general procedure d , ( z)-ethyl 3-(trifluoromethylsulfonyloxy)pent-2-enoate ( 17.66 
 g , 63.9 mmol ) , zn(cn)2 ( 4.50 g , 38.4 mmol ) in dry dmf ( 75 
 ml ) , and pd(pph3)4 ( 0.74 g , 0.64 mmol ) gave 
 the crude product as a clear yellow oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac 9:1 afforded the desired product as 
 a clear pale yellow oil ( 7.27 g , 74% ) : h nmr ( 400 mhz , 
 cdcl3 )  1.24 ( t , j = 7.5 hz , 3h ) , 
 1.34 ( t , j = 7.1 hz , 3h ) , 2.45 ( qd , j = 7.4 , 1.5 hz , 2h ) , 4.29 ( q , j = 7.1 hz , 2h ) , 6.33 
 ( t , j = 1.5 hz , 1h ) ; c nmr ( 100 mhz , 
 cdcl3 )  12.0 , 14.0 , 29.3 , 61.5 , 115.9 , 129.2 , 131.4 , 
 162.9 ; hrms ( esi ) m / z calcd for 
 c8h11no2na ( m + na ) 176.0682 , 
 found 176.0680 . using general procedure d , ( e)-ethyl 3-(trifluoromethyl sulfonyloxy)pent-2-enoate ( 10.62 g , 38.4 
 mmol ) , zn(cn)2 ( 2.71 g , 23.1 mmol ) in dry dmf ( 75 ml ) , 
 and pd(pph3)4 ( 0.44 g , 0.38 mmol ) gave the crude 
 product as a clear yellow oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac 9:1 ) afforded the desired product as 
 a clear pale yellow oil ( 4.9 g , 84% ) : h nmr ( 400 mhz , 
 cdcl3 )  1.23 ( t , j = 7.6 hz , 3h ) , 
 1.32 ( t , j = 7.1 hz , 3h ) , 2.81 ( qd , j = 7.6 , 1.2 hz , 2h ) , 4.24 ( q , j = 7.1 hz , 2h ) 6.39 
 ( t , j = 1.2 hz , 1h ) ; c nmr ( 100 mhz , 
 cdcl3 )  12.4 , 14.1 , 23.6 , 61.2 , 118.0 , 131.8 , 132.6 , 
 163.7 ; hrms ( ei ) m / z calcd for c8h11no2 ( m ) 153.0790 , found 
 153.0798 . using general procedure d , ( z)-ethyl 3-(trifluoromethylsulfonyloxy)hex-2-enoate ( 13.6 g , 46.8 
 mmol ) , zn(cn)2 ( 3.30 g , 28.1 mmol ) in dry dmf ( 75 ml ) , 
 and pd(pph3)4 ( 0.54 g , 0.47 mmol ) gave the crude 
 product as a clear yellow oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac 9:1 , product from first column was 
 repurified on a second silica column ) afforded the desired product 
 as a clear pale yellow oil ( 4.56 g , 58% ) : h nmr ( 300 mhz , 
 cdcl3 )  0.98 ( t , j = 7.4 hz , 3h ) , 
 1.34 ( t , j = 7.1 hz , 3h ) , 1.68 ( m , 2h ) , 2.37 ( td , j = 7.5 , 1.5 hz , 2h ) , 4.29 ( q , j = 7.1 
 hz , 2h ) , 6.32 ( t , j = 1.5 hz , 1h ) ; c 
 nmr ( 100 mhz , cdcl3 )  13.1 , 14.0 , 20.8 , 37.7 , 61.5 , 
 115.9 , 127.8 , 132.3 , 162.9 ; hrms ( esi ) m / z calcd for c9h13no2na 
 ( m + na ) 190.0839 , found 190.0838 . using general procedure d , ( e)-ethyl 3-(trifluoromethylsulfonyloxy)hex-2-enoate ( 13.6 g , 46.8 
 mmol ) , zn(cn)2 ( 3.30 g , 28.1 mmol ) in dry dmf ( 75 ml ) , 
 and pd(pph3)4 ( 0.54 g , 0.47 mmol ) gave the crude 
 product as a clear yellow oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac , 19:1 , product from first column was 
 repurified on second silica column ) afforded the desired product as 
 a clear pale yellow oil ( 4.02 g , 51% ) : h nmr ( 400 mhz , 
 cdcl3 )  1.00 ( t , j = 7.4 hz , 3h ) , 
 1.32 ( t , j = 7.1 hz , 3h ) , 1.67 ( dq , j = 14.8 , 7.4 2h ) , 2.77 ( td , j = 7.6 , 1.1 hz , 2h ) , 
 4.23 ( q , j = 7.0 hz , 2h ) , 6.42 ( s , 1h ) ; c nmr ( 100 mhz , cdcl3 )  13.3 , 14.1 , 21.3 , 31.8 , 
 61.2 , 118.2 , 131.2 , 132.4 , 163.7 ; hrms ( ei ) m / z calcd for c9h13no2 ( m ) 167.0946 , found 167.0952 . using general procedure d , ( z)-ethyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate 
 ( 271.38 g , 1 equiv ) ,
zn(cn)2 ( 62.8 g , 0.6 equiv ) in dry 
 dmf ( 1.63 l ) , and pd(pph3)4 ( 10.31 g , 0.01 equiv ) 
 gave the crude product as a yellow oil .
purification by chromatography 
 on a biotage apparatus ( 25.3 g per run using a 65i cartridge ( 350 g 
 silica ) and 16% etoac in cyclohexane ) gave the product as 
 a yellow oil ( 67 g , 60% yield ) : h nmr ( 400 mhz , cdcl3 )  0.90 ( d , j = 6.6 hz , 6h ) , 1.27 
 ( t , j = 7.2 hz , 3h ) , 1.99 ( hept , j = 6.8 hz , 1h ) , 2.19 ( dd , j = 7.2 hz , 1.2 hz , 2h ) , 
 4.22 ( q , j = 7.2 hz , 2h ) , 6.21 ( t , j = 1.2 hz , 1h ) .
using general procedure d , ( e)-ethyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate 
 ( 378 g , 1 equiv ) , zn(cn)2 ( 87.6 g , 0.6 equiv ) in dry dmf 
 ( 2.27 l ) , and pd(pph3)4 ( 14.4 g ; 0.01 equiv ) 
 gave the crude product as a red oil ( 244 g , 108% ) .
a portion of the 
 crude product ( 111 g ) was purified by chromatography on a biotage 
 apparatus using a 65i cartridge ( 350 g silica ) and 16% etoac 
 in cyclohexane to give the product as an yellow - orange oil ( 55 g , 
 53% ) : h nmr ( 400 mhz , cdcl3 )  0.91 ( d , j = 6.7 hz , 6h ) , 1.24 ( t , j = 7.1 hz , 3h ) , 
 1.95 ( hept , j = 6.8 hz , 1h ) , 2.61 ( dd , j = 7.3 , 1.1 hz , 2h ) , 4.15 ( q , j = 7.1 hz , 2h ) , 6.37 
 ( t , j = 1.1 hz , 1h ) . using general procedure d , ( z)-methyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate 
 ( 14.7 g , 50.8 mmol ) and zn(cn)2 ( 3.58 g , 30.5 mmol ) in 
 dry dmf
( 75 ml ) and pd(pph3)4 ( 0.59 g , 0.51 
 mmol ) gave the crude product as a clear yellow oil .
purification by 
 flash chromatography ( silica gel , cyclohexane / etoac 9:1 ) afforded 
 the desired product as a clear pale yellow oil ( 7.32 g , 86% ) : h nmr ( 400 mhz , cdcl3 )  0.96 ( d , j = 6.5 hz , 6h ) , 2.05 ( m , 1h ) , 2.20 ( dd , j = 7.0 , 1.0 hz , 2h ) , 3.82 ( s , 3h ) , 6.29 ( t , j = 
 1.0 hz , 1h ) ; c nmr ( 100 mhz , cdcl3 )  
 21.9 , 27.1 , 44.8 , 52.3 , 116.0 , 127.5 , 132.6 , 163.2 . using general procedure d , ( e)-methyl 5-methyl-3-(trifluoromethylsulfonyloxy)hex-2-enoate 
 ( 9.96 g , 34.3 mmol ) , zn(cn)2 ( 2.42 g , 20.6 mmol ) in dry 
 dmf ( 75 ml ) and pd(pph3)4 ( 0.40 g , 0.34 mmol ) 
 gave the crude product as a clear red oil .
purification by flash chromatography 
 ( silica gel , cyclohexane / etoac 9:1 , product from first column was 
 repurified on a second silica column ) afforded the desired product 
 as a clear pale yellow oil ( 4.24 g , 74% ) : h nmr ( 400 mhz , 
 cdcl3 )  0.98 ( d , j = 6.5 hz , 6h ) , 
 2.02 ( m , 1h ) , 2.69 ( dd , j = 7.5 , 1.0 hz , 2h ) , 3.77 
 ( s , 3h ) , 6.45 ( t , j = 1.0 hz , 1h ) ; c 
 nmr ( 100 mhz , cdcl3 )  22.0 , 28.0 , 38.5 , 52.0 , 118.3 , 
 131.1 , 132.5 , 164.2 . to a solution of starting ester ( 1 mmol ) in alcohol 
 ( methanol or n - propanol , 25 ml ) in a round - bottom 
 flask ( 100 ml ) equipped with a reflux condenser was added hydrochloric 
 acid ( conc , 0.5 ml ) .
the resulting mixture was stirred under reflux 
 until all starting material was consumed . after the reaction was complete , 
 a solution of nahco3 ( saturated , 15 ml )
the combined 
 organic phases were dried over na2so4 and yielded 
 the crude product after the solvent was removed under reduced pressure . using general procedure
e , ( z)-ethyl 3-cyanohex-2-enoate [ ( z)-3a ] ( 300 mg , 1.79 mmol ) in methanol ( 40 ml ) and hydrochloric acid ( conc , 
 0.5 ml ) gave the product after 5 days reaction time .
purification 
 by flash chromatography ( silica gel , petroleum ether / etoac 20:1 ) afforded 
 the product ( 173 mg , 63% ) : h nmr ( 300 mhz , cdcl3 )  6.33 ( s , 1h ) , 3.83 ( s , 3h ) , 2.38 ( dd , j = 10.8 , 4.1 , 2h ) , 1.841.60 ( m , 2h ) , 0.98 ( t , j = 7.4 , 3h ) ; c nmr ( 75 mhz , cdcl3 )  
 163.3 , 131.9 , 128.2 , 115.9 , 52.3 , 37.7 , 20.8 , 13.1 ; hrms ( ei ) m / z calcd for c8h11no2 ( m ) 153.0790 , found 153.0786 . using general procedure e , 
 ( e)-ethyl 3-cyano-5-methylhex-2-enoate
[ ( e)-5a ] ( 150 mg , 0.82 mmol ) in n - propanol ( 30 ml ) and hydrochloric acid ( conc . , 0.5 ml ) gave the 
 pure product ( 120 mg , 75% ) after 5 days reaction time : h nmr ( 300 mhz , cdcl3 )  6.47 ( s , 1h ) , 4.14 ( t , j = 6.7 , 2h ) , 2.70 ( dd , j = 7.3 , 1.0 , 2h ) , 
 2.171.94 ( m , 1h ) , 1.791.64 ( m , 2h ) , 1.00 ( d , j = 6.0 , 6h ) , 0.98 ( t , j = 9.0 , 3h ) ; c nmr ( 75 mhz , cdcl3 )  163.9 , 133.0 , 130.6 , 
 118.4 , 66.8 , 38.5 , 28.0 , 22.0 , 21.8 , 10.4 ; hrms ( ei ) m / z calcd for c11h17no2 ( m ) 195.1259 , found 195.1277 .
the 
 starting acrylic ester ( 0.5 mmol ) was dissolved in thf ( 5 ml ) and 
 hydrogenated under h2 at atmospheric pressure at room temperature 
 employing 10% pd / c ( 5 mg ) as catalyst by stirring overnight at room 
 temperature .
afterward , the reaction mixture was filtered through 
 celite and concentrated to yield the pure hydrogenated product in 
 quantitative conversion . using general 
 procedure
f , hydrogenation of ( z)-ethyl 3-cyanobut-2-enoate 
 [ ( z)-1a ] ( 51 mg , 0.39 mmol ) gave the 
 pure product ( 97% , 50.3 mg , 0.35 mmol ) : h nmr ( 300 mhz , 
 cdcl3 )  4.22 ( dq , j = 14.3 , 7.2 , 
 2h ) , 3.302.98 ( m , 1h ) , 2.63 ( ddd , j = 56.4 , 
 16.6 , 7.2 , 2h ) , 1.39 ( d , j = 7.1 , 3h ) , 1.29 ( t , j = 7.1 , 3h ) ; c nmr ( 75 mhz , cdcl3 )  169.6 , 121.8 , 61.3 , 38.2 , 21.7 , 17.7 , 14.1 . using general 
 procedure f , hydrogenation of ( z)-ethyl 3-cyanopent-2-enoate 
 [ ( z)-2a ]
( 50 mg , 0.33 mmol ) gave the 
 pure product ( 97% , 49.5 mg , 0.32 mmol ) : h nmr ( 300 mhz , 
 cdcl3 )  4.20 ( q , j = 7.1 , 2h ) , 
 3.142.86 ( m , 1h ) , 2.63 ( ddd , j = 23.3 , 16.5 , 
 7.2 , 2h ) , 1.841.57 ( m , 2h ) , 1.29 ( t , j = 
 7.1 , 3h ) , 1.12 ( t , j = 7.4 , 3h ) ; c nmr 
 ( 75 mhz , cdcl3 )  169.8 , 121.0 , 61.3 , 36.4 , 29.1 , 
 25.2 , 14.1 , 11.4 . using general 
 procedure f , hydrogenation of ( z)-ethyl 3-cyanohex-2-enoate 
 [ ( z)-3a ] ( 51 mg , 0.31 mmol ) gave the 
 pure product ( 80% , 42 mg , 0.25 mmol ) : h nmr ( 300 mhz , 
 cdcl3 )  4.21 ( q , j = 7.1 , 2h ) , 
 3.03 ( m , 1h ) , 2.822.46 ( m , 2h ) , 1.821.34 ( m , 4h ) , 
 1.34 ( t , j = 9 , 3h ) , 0.98 ( t , j = 
 6.8 , 3h ) ; c nmr ( 75 mhz , cdcl3 )  169.8 , 
 121.1 , 61.3 , 36.8 , 33.8 , 27.4 , 20.2 , 14.1 , 13.4 . using general procedure f ,
hydrogenation of ( z)-methyl 
 3-cyano-5-methylhex-2-enoate [ ( z)-5a ] ( 90 mg , 0.54 mmol ) gave the pure product ( 93% , 85 mg , 0.50 mmol ) .
h nmr ( 300 mhz , cdcl3 ) :  3.75 ( s , 3h ) , 3.162.96 
 ( m , 1h ) , 2.63 ( ddd , j = 23.3 , 16.6 , 7.1 , 2h ) , 1.981.76 
 ( m , 1h ) , 1.65 ( ddd , j = 13.5 , 10.7 , 4.8 , 1h ) , 1.401.28 
 ( m , 1h ) , 0.97 ( dd , j = 6.6 , 3.5 , 6h ) ; c nmr ( 75 mhz , cdcl3 ) :  170.2 , 121.1 , 52.3 , 40.7 , 
 36.9 , 26.1 , 25.8 , 22.9 , 21.2 . using general 
 procedure
f , hydrogenation of ( z)-methyl 3-cyano - hex-2-enoate 
 [ ( z)-6a ] ( 40 mg , 0.24 mmol ) gave the 
 pure product ( 92% , 38 mg , 0.22 mmol ) : h nmr ( 300 mhz , 
 cdcl3 )  3.76 ( s , 3h ) , 3.162.94 ( m , 1h ) , 
 2.66 ( ddd , j = 23.4 , 16.6 , 7.2 , 2h ) , 1.731.53 
 ( m , 4h ) , 0.99 ( t , j = 6.9 , 3h ) ; c nmr 
 ( 75 mhz , cdcl3 )  170.3 , 121.1 , 52.2 , 36.6 , 33.8 , 
 27.4 , 20.2 , 13.4 . a 1:1 mixture of ( r)- and ( s)-ethyl 3-cyano-5-methylhexanoate ( 4b ) was prepared . using general procedure 
 e , rac - ethyl 3-cyano-5-methylhexanoate ( rac-4b ) ( 30 mg , 0,17 mmol ) in n - propanol 
 ( 20 ml ) and hydrochloric acid ( conc , 0.2 ml ) gave the pure product 
 ( 88% , 29 mg , 0,15 mmol ) after 24 h reaction time : h nmr 
 ( 300 mhz , cdcl3 )  4.11 ( t , j = 
 6.7 , 2h ) , 3.07 ( dt , j = 12.1 , 7.0 , 1h ) , 2.63 ( ddd , j = 48.8 , 16.5 , 7.1 , 2h ) , 1.981.56 ( m , 4h ) , 1.411.28 
 ( m , 1h ) , 1.040.85 ( m , 9h ) ; c nmr ( 75 mhz , cdcl3 )  169.9 , 121.2 , 66.9 , 40.8 , 37.2 , 26.1 , 25.8 , 22.9 , 
 21.9 , 21.2 , 10.3 ; hrms ( ei ) m / z calcd 
 for c11h18no2 ( [ m  h ] ) 196.1338 , found 196.1329 .
biotransformations of 
 ( e)-1a , ( e)-2a , and ( e)-3a were carried out in 100 
 ml volumes at 30 c for 1925 h. reactions contained alkene 
 substrate ( 10 mmol ) , 2-propanol ( 26 mmol ) , lactobacillus brevis alcohol dehydrogenase ( 3200 u ) , nadp ( 1 mmol ) , oye2 
 ( 48 g wet e. coli cells ; oye2 was constructed 
 in pet28b and expressed in bl21 e. coli cells ) , and 
 100 mm potassium phosphate buffer ( ph 7 ) .
the reaction 
 mixtures were centrifuged and the resulting supernatants were extracted 
 with etoac .
the organic layers were dried over mgso4 and 
 concentrated under reduced pressure , and the resulting crude products 
 were purified by kugelrohr distillation .
bioreduction 
 of ( e)-1a afforded 1.17 g ( 83% ) of ( s)-1b as a clear colorless liquid ( 100% ) .
spectroscopic 
 data were in agreement with data for rac-1b ; [ ]d + 30.7 ( c 1.07 , 
 meoh ) ; ee > 99% determined by gc analysis ( g - ta , 135165 
 c 
 temperature gradient ) , tr 5.09 min .
for 
 determination of absolute configuration , ( s)-1b ( 0.73 g , 5.2 mmol ) was converted to the methyl ester by 
 transesterification with ti(oet)4 ( 0.5 g , 2.2 mmol ) and ethylene glycol ( 0.07 g , 1.1 mmol ) 
 in refluxing meoh ( 25 ml ) for 118 h. the reaction mixture was concentrated 
 ( 5 ml ) under reduced pressure , diluted with 10 ml of 4 n aqueous 
 hcl , and extracted with etoac .
the organic extract was washed with 
 water , dried over magnesium sulfate , concentrated under reduced pressure 
 and kugelrohr distilled to give 0.46 g ( 60% ) of ( s)-methyl 3-cyanobutanoate as a clear liquid ( 97% , contaminated with 
 3% ( s)-1b ) .
spectral data and 
 specific rotation for ( s)-methyl 3-cyanobutanoate 
 were consistent with literature values : [ ]d + 33.2 ( c 1.11 , 
 meoh ) ; ee > 99% determined by gc analysis ( -ta , 135165 
 c temperature gradient ) tr 4.79 
 min ; lit .
bioreduction 
 of ( e)-2a afforded 1.0 g ( 64% ) of ( s)-2b as a clear colorless liquid ( > 99% ) . 

[ ]d + 12.7 ( c 1.03 , meoh ) ; ee > 99% 
 determined by gc analysis ( g - ta 135165 c temperature 
 gradient ) , tr 6.16 min .
for determination 
 of absolute configuration , ( s)-2b ( 0.73 
 g , 4.7 mmol ) was converted to the methyl ester by transesterification 
 according to the procedure described for transesterification of ( s)-1b .
0.56 g ( 84% ) of ( s)-methyl 
 3-cyanopentanoate was obtained as a clear liquid ( 97% , contaminated 
 with 3% ( s)-2b ) .
spectral data 
 and specific rotation for ( s)-methyl 3-cyanopentanoate 
 were consistent with literature values : [ ]d + 14.9 ( c 1.06 , meoh ) ; ee > 99% 
 determined by gc analysis ( g - ta , 135165 c temperature 
 gradient ) tr 5.82 min [ lit .
bioreduction 
 of ( e)-3a afforded 1.05 g ( 60% ) of ( s)-3b as a clear colorless liquid ( 98% ) .
spectroscopic 
 data were in agreement with data for racemic 3b : [ ]d + 2.2 ( c 1.03 , meoh ) ; ee > 99% 
 determined by gc analysis ( g - ta , 135 c - 165 c temperature 
 gradient ) tr = 7.18 min .
for determination 
 of absolute configuration , ( s)-3b ( 0.66 
 g , 3.9 mmol ) was converted to the corresponding carboxylic acid by 
 hydrolysis with 50 wt % naoh ( 0.25 ml ) in meoh ( 2.5 ml ) at room temperature 
 for 3 h. the reaction mixture was acidified with 2 ml of 4 n hcl , 
 diluted with 10 ml of water and extracted with etoac .
the organic 
 extract was dried over na2so4 and concentrated 
 under reduced pressure to give 0.5 g ( 91% ) of ( s)-3-cyanohexanoic 
 acid as a viscous liquid .
spectral data for ( s)-3-cyanohexanoic 
 acid were consistent with literature values for ( r)-3-cyanohexanoic acid and specific rotation was consistent with 
 literature value in magnitude but opposite in direction : [ ]d 3.1 
 ( c 1.18 , chcl3 ) . for chiral gc analysis , 
 the acid ( 0.25 mg in 0.5 ml etoac ) was converted to the methyl ester 
 by treatment with tms - diazomethane ( 0.02 ml ) and meoh ( 0.02 ml ) for 
 30 min at room temperature ; ee > 99% determined by gc analysis ( g - ta , 
 135165 c temperature gradient ) tr 6.66 min [ lit .
[ ]d + 1.1 ( c 1.1 , chcl3 ) , 98% ee ] . reference material for ( s)-4b was independently prepared from diethyl 
 2-(1-cyano-3-methylbutyl)malonate according to the published procedure and purified by kugelrohr distillation for characterization : 
 98.3% ee determined by gc analysis ( g - ta , 135165 c temperature 
 gradient ) tr = 7.53 min ; [ ]d 10.9 ( c 1.09 , meoh ) . 
 spectral data for ( s)-4b were in agreement 
 with data for rac-4b and with previously 
 reported data .
preparation of ( s)-4b also gave ( r)-diethyl 2-(1-cyano-3-methylbutyl)malonate 
 as a byproduct of the enzymatic hydrolysis step .
for preparation of ( r)-4b , the published procedure was modified by running the enzymatic hydrolysis 
 step to > 50% conversion to obtain ( r)-diethyl 
 2-(1-cyano-3-methylbutyl)malonate 
 with higher enantiomeric purity .
partial hydrolysis of ( r)-diethyl 2-(1-cyano-3-methylbutyl)malonate was carried out with 
 koh in etoh and h2o .
aqueous koh ( 8.8 wt % , 13.6 g , 21.3 
 mmol ) was slowly added over 1 h to an ice - cold solution of ( r)-diethyl 2-(1-cyano-3-methylbutyl)malonate ( 5.0 g , kugelrohr 
 distilled , 19.6 mmol , > 99% ee ) in etoh ( 5.9 ml ) .
the ice bath was 
 removed and the reaction was stirred for 18 h at rt .
the reaction 
 mixture was extracted with toluene ( 3  10 ml ) and the toluene 
 layers discarded .
the remaining aqueous layer was stirred at 100 c 
 for 4 h. the reaction mixture was extracted with etoac , dried over 
 mgso4 and concentrated under reduced pressure to give the 
 crude product .
purification of the crude product by kugelrohr distillation 
 afforded ( r)-4b as a clear colorless 
 liquid ( > 99% by gc , 1.18 g , 32.8% ) ; 89.8% ee determined by gc analysis 
 ( g - ta , 135165 c temperature gradient ) tr 7.34 min ; [ ]d + 10.9 ( c 1.08 , meoh ) .
spectral data for ( r)-4b were in agreement with data for rac-4b . using general procedure e , 
 ( s)-ethyl 3-cyano-5-methylhexanoate [ ( s)-4b ] ( 52 mg , 0.28 mmol ) in methanol 
 ( 5 ml ) and hydrochloric acid ( conc , 0.2 ml ) gave the pure product 
 ( 73% , 35 mg , 0.21 mmol ) after 24 h reaction time .
spectral data for 
 ( s)-5b were in agreement with data for rac-5b . using general procedure e , ( s)-ethyl 3-cyanohexanoate [ ( s)-3b ] ( 11 mg , 0.07 mmol ) in methanol ( 5 ml ) and hydrochloric 
 acid ( conc , 0.1 ml ) gave the pure product ( 54% , 7 mg , 0.04 mmol ) after 
 24 h reaction time .
spectral data for ( s)-6b were in agreement with data for rac-6b . using general procedure e , 
 ( s)-ethyl 3-cyano-5-methylhexanoate
[ ( s)-4b ] ( 30 mg , 0.16 mmol ) in n - propanol 
 ( 20 ml ) and hydrochloric acid ( conc , 0.5 ml ) gave the pure product 
 ( 99% , 32 mg , 0.16 mmol ) after 24 h reaction time .
 the asymmetric bioreduction of a library of -cyanoacrylate 
 esters using ene - reductases was studied with the aim to provide a 
 biocatalytic route to precursors for gaba analogues , such as pregabalin . 
 
 the stereochemical outcome could be controlled by substrate - engineering 
 through size - variation of the ester moiety and by employing stereochemically 
 pure ( e)- or ( z)-isomers , which 
 allowed to access both enantiomers of each product in up to quantitative 
 conversion in enantiomerically pure form . 
 in addition , stereoselectivities 
 and conversions could be improved by mutant variants of opr1 , and 
 the utility of the system was demonstrated by preparative - scale applications . 
Introduction
Results and Discussion
Conclusion
Experimental Section