aminobutyric acid ( gaba ) is the primary inhibitory neurotransmitter widely distributed in the mammalian central nervous system to regulate neuronal excitation.1 gaba deficiency has been associated with several neurodegenerative disorders such as parkinson 's disease,2 huntington 's disease,[2b ] , 3 and alzheimer 's disease4 in addition to psychiatric disorders including depression,5 alcoholism,6 and anxiety.[5b ] , [ 5c ] , 7 over the past few decades , a number of gaba derivatives showing biological activities have been developed as potential drugs for the treatment of neurological disorders.8 , 9 it has been shown that the biological activities of these compounds are largely dependent on the configuration of the chiral center . for example , only the ( s ) enantiomer of vigabatrin,10 the ( r ) enantiomer of baclofen,11 and the ( s ) enantiomer of pregabalin12 are the active pharmaceutical ingredients . the asymmetric synthesis of gaba derivatives has therefore attracted considerable attention , and several methodologies involving the use of metal catalysts , organocatalysts , and biocatalysts , or their combinations , to construct these chiral molecules have been established.[8a ] we recently reported a biocatalytic approach for the asymmetric michaeltype addition of acetaldehyde to nitroalkenes to afford enantioenriched nitroaldehydes,13 which can be readily converted into chiral gaba derivatives by two simple chemical steps . this methodology utilizes the catalytic promiscuity of the enzyme 4oxalocrotonate tautomerase ( 4ot).14 in this study , we investigated the effects of different substituents at the aromatic ring of the michael acceptor on the catalytic efficiency and stereoselectivity of 4otcatalyzed michaeltype addition reactions . we report the asymmetric 4otcatalyzed michaeltype addition of acetaldehyde ( 1 ; scheme 1 ) to a series of monosubstituted nitrostyrene derivatives ( see compounds 2c  m ) to yield enantioenriched ( r) or ( s)nitroaldehydes ( see compounds 3c  m ) , as well as the 4otcatalyzed michaeltype addition of 1 to ( e)2hydroxynitrostyrene ( 2b ) , which , after cyclization , affords 4(nitromethyl)chroman2ol ( 5 ; table 1 ) . preparativescale asymmetric 4otcatalyzed michaeltype additions of acetaldehyde 1 ( 50 equiv . ) to nitrostyrene derivatives 2a  n ( 1.52.0 mm ) in nah2po4 buffer ( ph 5.5 ) to yield nitroaldehydes 3a  n or chroman2ol 5 
   yield of isolated product .   determined by hplc analysis with a chiral stationary phase .   absolute configurations of the major enantiomers of 3c  m were determined by optical rotation measurements and comparison with literature data ( see table s3 ) .   compared to nitrostyrene derivatives previously reported result ; reaction performed with 1 ( 50 mm ) , 2a ( 1.3 mm ) , 4ot ( 73 m ) in phosphate buffer ( ph 7.3).[13a ]   diastereomeric ratio ( dr ) was determined by h nmr spectroscopy and comparison with literature data;15 enantiomeric excess ( ee ) of 5 was not determined . previously reported result ; reaction performed with 1 ( 65 mm ) , 2 g ( 1.3 mm ) , 4ot ( 36 m ) in phosphate buffer ( ph 5.5).[13c ]   previously reported result ; reaction performed with 1 ( 50 mm ) , 2n ( 2 mm ) , 4ot ( 28 m ) in phosphate buffer ( ph 5.5).[13b ] the 4otcatalyzed reaction of donor 1 with nitrostyrene derivatives 2b  l as potential acceptors ( scheme 1 ) was examined . in separate analyticalscale experiments , 2b  l and 1 were incubated with 4ot in nah2po4 buffer containing an appropriate cosolvent ( table s1 in the supporting information ) . the reactions were monitored by recording the change in the absorbance at  
 max of 2b l was observed over the course of time , which indicated depletion of these nitro olefins ( figures s1s10 ) . l under the same conditions but in the absence of 4ot showed a negligible decrease in the absorbance at  
 max of 2b  l , which indicated that 4ot was responsible for the catalysis ( figures s1s10 ) . next , preparativescale experiments were performed to allow product identification by nmr spectroscopy and hence to confirm that the 4otcatalyzed additions of 1 to 2b in addition , the enzymatic preparation of 3 m from 2 m and 1 was investigated . in separate experiments , 2b m , 1 , and 4ot were incubated in nah2po4 buffer with an appropriate cosolvent ( table 1 , table s2 ) . the progress of each reaction was monitored by uv spectroscopy ( figures s11s14 ) . upon completion of the reaction , workup and purification procedures analysis of the purified products by nmr spectroscopy confirmed the formation of nitroaldehydes 3c  m ( figures s16s25 ) . intriguingly , the expected nitroaldehyde 3b ( from 1 and 2b ) was not observed . instead , the formation of 4(nitromethyl)chroman2ol ( 5 ) as a result of hemiacetalization of 3b was established by nmr spectroscopy ( figure s15 ) and comparison with literature data.15 4substituted chroman2ol 5 is a synthetically valuable intermediate that can be easily converted into chromans and their derivatives with antimicrobial and anticancer properties.16 good to excellent yields ( up to 96  % ) were achieved for products 3c  f , 3h  i , 3 m , and 5 ( table 1 ) . the lower yields of 3j  l ( 3137  % ) were caused by the low conversion rates with nitrostyrene derivatives 2j m for determination of their enantiomeric excess ( ee ) values by chiral stationary phase hplc . good to excellent ee values ( between 82 and 97  % ) were achieved for 3c  m , which indicated that 4ot was highly stereoselective during the catalytic process ( table 1 ; figures s46s55 ) . interestingly , comparison with literature data revealed that the meta and parasubstituted nitroaldehydes ( i.e. , compounds 3d , 3f , 3 g , 3i , and 3j ) had the ( s ) configuration , whereas orthosubstituted nitroaldehydes 3e and 3k had the ( r ) configuration ( table 1 ; table s3 ) . the observed negative optical rotation of metasubstituted nitroaldehydes 3c , 3l , and 3 m could not be compared with literature data , as , to the best of our knowledge , these compounds have so far not been reported . we assume that the major enantiomers of 3c , 3l , and 3 m have the ( s ) configuration , because they showed the same negative optical rotation and elution order in the hplc chromatograms as those observed with metasubstituted products 3f and 3i . similarly , the absolute configuration of the major enantiomer of 3h was tentatively assigned as ( r ) by comparing its optical rotation data with those of 3e and 3k . r = parafluoro ) were performed to elucidate the influence of substituents on the kinetic parameters . to address the influence of the cosolvent and ph on the kinetic parameters , the kinetic assays of the 4otcatalyzed reaction with ( e)nitrostyrene ( 2n ) were performed at different ph values and in different solvent systems ( table 2 , entries 14 ) . we previously reported that lowering of the ph of the reaction media from 7.3 to 5.5 gave an approximately fourfold increase in both the turnover rate ( k 
 cat ) and michaelis constant ( k 
 m ) , which resulted in an unchanged catalytic efficiency ( k 
 cat / k 
 m ) ( table 2 , entries 1 and 2 ) . interestingly , switching the cosolvent of the reaction medium from etoh ( 10  % , v / v ) to dmso ( 40  % , v / v ) also resulted in a slight increase ( up to twofold ) in both k 
 cat and k 
 m ( table 2 , compare entries 3 and 4 with entries 1 and 2 ) . the kinetic data obtained with 2a in two different solvent systems showed similar changes in the k 
 cat and k 
 m values ( table 2 , entries 5 and 6 ) , which suggests that this solvent effect on the kinetic parameters is consistent for the different nitro olefins . this effect may be caused by a small conformational change in the structure of 4ot induced by the dmso cosolvent.17 
  apparent kinetic parameters for the 4otcatalyzed michaeltype addition of acetaldehyde ( 1 ) to para , meta , and orthosubstituted nitrostyrenes 2a  o 
   previously reported data ; assays were performed in etoh / phosphate buffer ( 10:90 , v / v ) at ph 7.3.[13a ]   previously reported data ; assays were performed in etoh / phosphate buffer ( 10:90 , v / v ) at ph 5.5.[13c ]   assays were performed in dmso / phosphate buffer ( 40:60 , v / v ) at ph 7.3 . assays were performed in dmso / phosphate buffer ( 40:60 , v / v ) at ph 5.5 . v / v ) at ph 5.5 to prevent nonenzymatic hydration of nitrostyrene derivatives 2b , 2e , 2h , and 2k in the aqueous solvent system . the kinetic parameters of the 4otcatalyzed additions of 1 to 2a  o were determined and compared with those of 2n ( r = h ) measured under the same solvent and ph conditions ( table 2 ) . in the case of parasubstituted nitrostyrenes , an increase in k 
 cat for substrates carrying electrondonating groups ( e.g. , hydroxy and methoxy groups ) and a decrease in k 
 cat for the substrate with a strongly electronwithdrawing group ( e.g. , nitro group ) the k 
 cat value seemed not to be significantly affected by fluoro and chloro substituents . the obtained k 
 m values suggest that the affinity of 4ot for parasubstituted nitrostyrenes is affected by the hydrophilicity of the substituents . substituents with high hydrophilicity generally resulted in increased k 
 m values , which indicates a lower affinity of 4ot towards the substrate ( table 2 , compare entries 6 , 8 , and 9 with entry 3 ) . the highest k 
 m values were obtained with hydroxy and fluorosubstituted nitrostyrenes ( table 2 , entries 6 and 8) . the presence of electrondonating groups ( e.g. , hydroxy and methoxy groups ) enhanced the k 
 cat values , whereas the presence of a strongly electronwithdrawing group ( e.g. , nitro group ) showed the opposite effect . furthermore , the affinity of 4ot towards metasubstituted nitrostyrenes seemed to be influenced by the hydrophilicity of the substituents as well , as the bromo and chlorosubstituted nitrostyrenes showed the lowest k 
 m values ( table 2 , compare entries 13 and 14 with entry 3 ) , whereas a more hydrophilic substituent ( e.g. , hydroxy group ) at the meta position led to an increased k 
 m value ( table 2 , compare entry 11 with entry 3 ) . the k 
 m values obtained with metasubstituted nitrostyrenes were generally somewhat lower than those determined for the corresponding parasubstituted substrates , which suggests that nitrostyrene derivatives with a meta substitution fit better to the active site of 4ot . the relatively low k 
 m values obtained with metasubstituted nitrostyrenes also resulted in higher k 
 cat / k 
 m values than that obtained with 2n ( table 2 , compare entries 1115 with entry 3 ) . the results of the kinetic assays with orthosubstituted substrates show that introducing a methoxy group into the ortho position of nitrostyrene significantly increased the value of k 
 cat , whereas the presence of a hydroxy group at this position resulted in a significant decrease in k 
 cat ( table 2 , compare entries 16 and 17 to entry 4 ) . the different k 
 m values observed for the different orthosubstituted nitrostyrenes may be related to steric effects . 4ot was shown to catalyze the asymmetric michaeltype addition of acetaldehyde to various ortho , meta , and parasubstituted nitrostyrenes . for most of the nitroaldehydes , excellent yields ( up to 96  % ) and high optical purities ( ee values up to 97  % ) were achieved . enzymatic access to ( r)nitroaldehydes was achieved with orthosubstituted nitrostyrenes , whereas ( s)nitroaldehydes were obtained with para and metasubstituted nitrostyrenes . apparently , attaching substituents to the ortho position of the aromatic substrate induced steric effects , which caused either substrate repositioning in the active site of 4ot or a stereofacial shielding effect . introducing electrondonating groups at the meta and para positions of the nitrostyrene derivatives significantly enhanced the catalytic rates ( up to fivefold improvement in k 
 cat ) . together with the ongoing enzyme engineering studies in our group , this work is an important step towards our aim of developing novel  michaelases  for carbon  carbon bondforming michaeltype addition reactions with high efficiency and stereoselectivity . finally , the 4otcatalyzed reaction between 1 and 2b did not give nitroaldehyde 3b as the final product ; instead , it yielded 4(nitromethyl)chroman2ol ( 5 ) as a result of hemiacetalization between the aldehyde and hydroxy groups of presumed intermediate 3b . this michaeltype addition / cyclization cascade reaction provides exciting options for the enzymatic synthesis of precursors for chromans and derivatives , which are valuable scaffolds for preparing biologically active natural products . 
 supporting information ( see footnote on the first page of this article ) : experimental procedures and characterization of the compounds .
the enzyme 4oxalocrotonate tautomerase ( 4ot ) promiscuously catalyzes the michaeltype addition of acetaldehyde to nitrostyrene derivatives to yield chiral nitroaldehydes , which are important precursors for pharmaceutically active aminobutyric acids . in this study , we investigated the effect of different substituents at the aromatic ring of the michael acceptor on the catalytic efficiency and stereoselectivity of the 4otcatalyzed acetaldehyde addition reactions . 
 highly enantioenriched ( r) and ( s)nitroaldehydes and 4substituted chroman2ol could be obtained in good to excellent yields by applying different substituents at appropriate positions of the aromatic substrate . 
 stereochemical control of these enzymatic michaeltype additions by  substrate engineering  allowed the enantioselective synthesis of valuable aminobutyric acid precursors . 
 in addition , the results suggest a novel enzymatic synthesis route towards precursors for chromans and derivatives , which are valuable scaffolds for preparing biologically active natural products .