through the process 
 of natural selection , nature has evolved well - adapted 
 macromolecular structures that interact with biological small molecules . 

oxidoreductases , for example , rely on the nicotinamide coenzymes to 
 supply them with the redox equivalents required to sustain their catalytic 
 cycles .
two forms of natural coenzymes exist : the phosphorylated ( nadp / nadph ) and nonphosphorylated ( nad / nadh ) forms 
 ( figure 1a ) .
nicotinamide 
 coenzymes essentially contain two structural motifs , the nicotinamide 
 moiety conferring their electrochemical function ( i.e. , serving as 
 an electron source or sink in the form of a hydride ) and the adenosine 
 dinucleotide moiety conferring the separation between anabolic and 
 catabolic pathways .
nadp is involved in anabolic redox processes , 
 whereas nad is mostly found in processes dealing with energy metabolism . 
 while this separation between different metabolic pathways is essential 
 for cellular survival , it is irrelevant in chemical applications of 
 redox enzymes . therefore , there is a renewed interest in the design 
 of simple synthetic analogues of the natural nicotinamide coenzymes . 

the laboratory - based design and synthesis of small molecule biomimetics 
 that can functionally substitute ( or even outperform ) those available 
 in nature is a major challenge .
the development of biomimetics that 
 can be synthesized easily and exploited widely would be a game changer 
 in establishing new manufacturing technologies that would be too expensive 
 using natural biological molecules .
the natural coenzymes nad(p)h 
 are prohibitively expensive and chemically too unstable for stoichiometric 
 use in fine and specialty chemicals manufacture .
this has prevented 
 their general uptake as a source of reducing equivalents in biocatalytic 
 oxidoreductase - catalyzed reactions and has led to the development 
 of in situ regeneration systems to replenish nad(p)h ( e.g. , using 
 enzymatic , photochemical , and electrochemical approaches ) or the use of hydrogen - borrowing biocatalytic cascades . in turn
, the limited stability and expense of natural nicotinamide 
 coenzymes have driven a search for more stable synthetic nicotinamide 
 coenzyme analogues that can interface generally with biological oxidoreductase 
 catalysts .
ene reductases ( ers ) from the old 
 yellow enzyme family ( ec 1.3.1.31 ) are particularly attractive as 
 they are a group of broad specificity biocatalysts that catalyze the 
 asymmetric reduction of activated c = c bonds , forming up to 
 two new stereogenic centers at the expense of the natural nicotinamide 
 coenzyme nad(p)h as an electron source .
in particular , ,-unsaturated 
 carbonyl compounds ( e.g. , enals and enones ) and nitroalkenes are excellent 
 substrates . in general , ,-unsaturated diesters as well as
in contrast , 
 the efficient reduction of ,-unsaturated monoacids or 
 monoesters requires an additional electron - withdrawing group in the 
 - or -position in order to activate the alkene moiety . the ability to form new stereogenic centers and
the wide acceptance 
 of different substrate types are driving the exploitation of ers toward 
 novel applications in redox biocatalysis and implementation in key 
 industrial processes .
ers have been studied extensively over the past decade , and there 
 is detailed information known , such as their structure , reaction mechanism , 
 substrate scope , kinetic properties , and biocatalytic approaches .
the catalytic cycle of er - catalyzed 
 reactions can be divided into two separated half - reactions : in the 
 reductive half - reaction , a hydride is transferred from nad(p)h to 
 the enzyme - bound flavin ( flavin mononucleotide ; fmn ) .
after release 
 of oxidized nad(p ) , this hydride is then transferred in 
 the oxidative half - reaction from the flavin n5 position to the activated 
 alkene substrate ( figure 1b ) .
( a ) structure of nad(p)h and synthetic nicotinamide biomimetic 
 mnadhs ( 15 ) and ( b ) the catalytic 
 cycle of er - catalyzed reactions .
recently , nicotinamide coenzyme biomimetics ( mnadhs ) were 
 shown 
 to replace nad(p)h in er - catalyzed reactions .
a series of mnadhs ( 15 ) has been 
 synthesized to replace the natural coenzymes for various ers ( figure 1a ) .
these coenzyme 
 biomimetics show great promise and have sparked renewed interest in 
 redox biocatalysis .
in addition , reduction of c = c 
 double bonds and oxyfunctionalization 
 reactions such as epoxidation or hydroxylation using mnadhs have been reported . however ,
structural  mechanistic 
 understanding of biomimetic chemistry in er - catalyzed reactions is 
 lacking , preventing the successful exploitation of these biomimetics . 

biomimetic complexes and analysis of reaction cycle 
 kinetics , both presented here for the first time for a panel of structurally 
 diverse nicotinamide biomimetics in combination with several ers . 

these structural  mechanistic analyses show how selected biomimetics 
 perform as well and , in some cases , even outperform the natural coenzymes . 

the chemical stability of the biomimetics , coupled with their enhanced 
 enzymatic reactivity , gives rise to the rare situation where chemical 
 designs surpass those available in the natural world .
these biomimetics 
 provide an attractive means of performing redox biotransformations 
 with ers ( and mutatis mutandis other redox biocatalysts ) 
 under mild reaction conditions and at relatively low cost .
we set out to determine the structures of 
 selected complexes formed 
 between er biocatalysts and the biomimetics to ascertain if the biomimetic 
 design features replicate those seen for the natural enzyme 
the tetrahydro forms of the natural ( nadph4 ) 
 and biomimetic ( mnadh4s ) ligands were used to prevent hydride 
 transfer in the crystal complexes ( figure 2 ) .
crystal structures 
 were determined for xena in complex with nadph4 and tetrahydro - biomimetics 
 ( 1b , 2b , and 3b ) at 1.61.8 
  resolution .
clear electron density was observed for the bound 
 nadph4 extending from the nicotinamide moiety and becoming 
 disordered after the diphosphate moieties .
nadph4 binds 
 in a stacked arrangement sitting above the isoalloxazine ring of the 
 fmn cofactor , and his181 and his178 are located 2.8 and 2.9  , 
 respectively , from the nicotinamide oxygen , with cys25 some 3.6  
 from the nicotinamide n7 position .
each of the three bound biomimetics 
 occupies the same region of the active site as that observed for nadph4 ( figure 2 ) , 
 with only very minimal changes observed in the positions of surrounding 
 residues in the active site .
however , the reduced bulk of the biomimetics 
 when compared to the dinucleotide portion of nadph4 ( figure 2 ) results in trp302 
 adopting an alternative conformation that effectively reduces the 
 volume of the active site in this region . this feature is observed 
 with each of the three biomimetics , with the remainder of the active 
 site residues remaining static when compared to the nadph4 complex .
the determined structures reveal that the biomimetic designs 
 capture the design features of the natural coenzyme in that the nicotinamide 
 moiety is positioned optimally over the re - face of 
 the enzyme - bound fmn cofactor for hydride transfer from the c4 atom 
 of the biomimetic to the n5 atom of the flavin isoalloxazine .
structures cocrystallized in the presence of nadph4 and biomimetic compounds 1b , 2b , and 3b are shown , superimposed based upon secondary 
 structure elements using ssm ( coot ) .
residues 
 trp302 , his181 , and his178 are shown in stick representation colored 
 with white carbon atoms and blue nitrogen atoms .
the ordered portion of the 
 nadph4 is shown as semitransparent spheres in all - atom 
 color ( carbon , purple ; oxygen , red ; phosphorus , orange ) with its associated 
 underlying fmn cofactor shown as gray spheres .
three biomimetic compounds 
 are shown in stick representation with their respective trp302 residues 
 colored to match the biomimetic .
dual occupancies are present 
 for residue trp302 for each of the three mimics , indicating that occupancy 
 is less than 100% for these compounds . in ers ,
hydride 
 transfer from the natural coenzyme to the flavin mononucleotide cofactor 
 occurs by quantum mechanical tunnelling .
the probability of hydride 
 transfer by quantum mechanical tunnelling is affected by sub - angstrom 
 changes in donor  acceptor distance .
although the 
 nicotinamide synthetic analogues and the natural coenzyme bind to 
 the er active site with similar geometry , subtle changes in the binding 
 mode will influence the kinetics of hydride transfer .
we determined 
 the catalytic parameters for the reductive half - reaction ( figure 1b ) for four ers , 
 namely , petnr , toye , xena , and tsoye , using anaerobic 
 rapid mixing stopped - flow measurements , employing the natural coenzymes 
 nad(p)h and the mnadh biomimetics ( 15 ) ( figure 1a ) .
kred , reaction rates [ s ] ; kd , dissociation constants [ m ] ; 
 n.d . , not determined 
 due to poor conversion in biocatalytic reactions .
reaction conditions : [ er ] = 810 
 m , buffer 50 mm mops ( ph 7.0 ) + 5 mm cacl2 , at 30 
 c , under anaerobic conditions with constant n2 flow , 
 [ coenzyme ] = varied . not 
 possible to measure at lower 
 concentrations of the coenzyme due to non - pseudo - first - order conditions
. rate of reduction occurs within 
 the dead time of the stopped - flow instrument at higher concentrations 
 of coenzyme .
table 1 reports 
 the reaction rates ( kred ) and the apparent 
 dissociation constants ( kd ) for flavin 
 reduction .
most ers possess a higher affinity for the natural coenzyme 
 nadph compared to that for nadh .
this affinity is evident 
 from the values of the apparent dissociation constants ( kd ) calculated from stopped - flow studies that differ by 
 about an order of magnitude for all ers investigated .
the exception 
 is tsoye , with which the kd values are essentially the same with both coenzymes .
furthermore , 
 the values of the kinetic constants for the reductive half - reaction 
 ( i.e. , hydride transfer ; kred ) depend 
 on the type of natural coenzyme employed .
the kred values differed from 10- to 36-fold higher when using nadph 
 compared to that using nadh .
in particular , petnr shows significantly 
 higher kd values compared to those of 
 the other ers , and it is the only enzyme where kred / kd is 2 orders of magnitude 
 higher for nadph compared to that for nadh . with petnr ,
the faster 
 rates of flavin reduction with nadph compared to that with nadh have 
 been attributed to promoting vibrations that couple to the reaction 
 coordinate and modulate the donor  acceptor distance for hydride 
 tunnelling .
the rates of the reductive 
 half - reactions were elevated using the 
 nicotinamide biomimetic 2 compared to that using nadph 
 for each of the four ers investigated .
additionally , biomimetic 2 binds with apparent higher affinity to the active site of 
 the ers compared with that of nadph in two cases out of four , namely , 
 with petnr and xena ( kd 184 and 8 m , 
 respectively ) . as a consequence ,
the kred / kd values for the reductive half - reaction 
 of petnr and xena with the synthetic biomimetic 2 indicate 
 superior catalytic efficiency compared to that of reactions with nadph . 
 the highest kred / kd value of 48  500 mm s was obtained employing biomimetic 2 with xena , and 
 to the best of our knowledge
, this is the first report of a nicotinamide 
 biomimetic surpassing the efficiency of natural coenzymes .
biomimetic 4 harbors an acid group instead of the 
 amide group in the nicotinamide moiety .
the kred values were higher than 500 s with 
 xena and tsoye and reached a maximum of 770 s with toye .
further detailed analysis is reported 
 in table 1 , which indicates 
 that each biomimetic performed 
 better than nadh in reactions with petnr ( i.e. , higher reaction rates ; 
 apparent dissociation constants were similar for the biomimetic and 
 nadh ) .
a similar trend was observed in hydride transfer reactions 
 mediated by toye . in this case , the kred values were elevated for all of the biomimetics when compared to 
 those for both natural coenzymes . with that said ,
the ratio kred / kd was always 
 in favor of the natural coenzymes due to the modest binding affinity 
 of toye for the synthetic biomimetics .
xena stands apart from the 
 other ers : xena was the only enzyme capable of catalyzing the reduction 
 of an activated alkene using biomimetic 5 ( see enzyme - catalyzed redox reactions using coenzyme biomimetics ) .
biomimetic 5 differs from the others in that it possesses 
 a nitrile instead of carbonyl moiety , and kinetic parameters for the 
 reaction of biomimetic 5 with xena were determined .
the kd values for the reduction with 5 and nadph with xena are similar , whereas kred differs by 3 orders of magnitude ( i.e. , 0.08 and 67 s for 5 and nadph , respectively ) . in general , 
 the highest reaction rate with xena was obtained using biomimetic 4 , whereas three biomimetics ( 1 , 2 , and 3 ) showed a higher apparent affinity compared 
 with that of the natural coenzymes .
reactions with tsoye confirmed the general trend , i.e. , increased reaction rates with 
 the biomimetics but elevated kd values 
 for the enzyme 
ers catalyze 
 the reduction of 
 activated alkenes following a ping - pong bibi mechanism .
stopped - flow experiments demonstrated that reductive 
 rates are higher for a number of nicotinamide biomimetics than for 
 natural coenzymes , but in order to obtain better insight into the 
 overall catalytic cycle ( relevant to exploitation of any er with a 
 coenzyme biomimetic in biocatalysis ) , steady - state kinetic investigations 
 were undertaken studying three ers , namely , petnr , toye , and xena . 
 in each case , 2-cyclohexen-1-one was used as the second substrate 
 in reactions employing nad(p)h and the biomimetics .
for many ers the 
 oxidative half - reaction limits the overall kinetics of the complete 
 catalytic cycle , and the kapp values were similar when using nadph or the synthetic biomimetics . 

only the use of nadh led to a slight deviation from this trend ( table 2 ) . again , biomimetic 2 is of particular interest as it gave the highest ratio of kapp / km in two cases 
 out of three ( i.e. , petnr , xena ) , confirming that this synthetic analogue 
 has an improved catalytic efficiency compared to that with nadph .
reaction conditions : buffer 50 mm 
 mops ( ph 7.0 ) + 5 mm cacl2 , at 30 c , under anaerobic 
 conditions with constant n2 flow . [ coenzyme ] = varied , 
 [ er ] = 100250 nm ; (mnadhs ) , see the supporting information .
our previous study revealed that biomimetic coenzymes 15 are readily accepted by the ers from thermus scotoductus ( tsoye ) and bacillus subtilis ( yqjm ) . to evaluate
whether this 
 was generally the case for all of the members of the er family , we 
 investigated a more representative selection of ers .
we carried out 
 the asymmetric reduction of ketoisophorone 6 to levodione 
 ( r)-6a using an extended panel of 12 
 ers from the oye family ( petnr , toye , oye2 , oye3 , xena , xenb , leopr1 , 
 nera , mr , tsoye , droye , and rmoye ) and the five biomimetics ( 15 ) .
the results in table 3 show that the biomimetics were overall well - accepted , 
 leading up to quantitative conversions , with the exception of mnadh 5 . in fact , xena was the only enzyme that efficiently accepted 
 mnadh 5 as the hydride source .
this finding is in line 
 with the kinetic parameters shown in tables 1 and 2 .
as expected , 
 the source of hydride , whether from the natural or synthetic coenzyme , 
 did not have a significant influence on the enantiomeric excess ( table 
 s3 , supporting information ) , which is dependent 
 on the intrinsic stereoselectivity of the enzyme only .
the slight 
 deviations in the enantiomeric excess are due to the slow spontaneous 
 racemization of the product in aqueous medium .
reaction conditions :   = 10 mm , [ er ] = 3.05.0 
 m , [ coenzyme ] = 11 mm ( unless 
 otherwise specified ) , mops buffer ( 50 mm , ph 7.0 , 5 mm cacl2 ) , inert atmosphere , 30 c , 4 h ; the mnadhs were directly added 
 as solids .
the biocatalytic 
 reduction described in the previous section was carried out at the 
 expense of stoichiometric amounts of nicotinamide biomimetic as hydride 
 donor , which is a limitation in developing inexpensive biocatalytic 
 transformations .
the rhodium complex [ cp*rh(bpy)(h2o ) ] is capable of reducing biomimetics 1 and 3 using formate as a hydride source .
hence , to use the biomimetics in catalytic amounts , we implemented 
 a recycling system for the er - catalyzed reduction of 6 to ( r)-6a , as depicted in figure 3a .
the reduced form 
 of the rh - catalyst binds the carbonyl moiety of the oxidized form 
 of the biomimetic , followed by hydride transfer from the metal center 
 to the aromatic ring of the biomimetic .
after the release of the reduced 
 mimic from the metal center , the organometallic catalyst is reduced 
 back at the expense of formate , which is oxidized to co2 . after a preliminary optimization of the reaction conditions ( see 
 the supporting information for more details ) , the reduction of ketoisophorone 6 was performed with tsoye , oxidized biomimetic 1a , and the rhodium complex [ cp*rh(bpy)(h2o ) ] , using sodium formate as the hydride donor . under these 
 reaction conditions ( see the supporting information for more details ) , 6.6 mm product with an ee of 85% was obtained 
 starting with 1 mm 1a . therefore , an in situ recycling 
 method for mnad1a with an er was demonstrated .
( a ) investigated 
 recycling system of mnadhs with tsoye and the rhodium 
 complex [ cp*rh(bpy)(h2o ) ] .
reaction conditions :   = 10 mm , [ er ] = 3.0 m , [ oxidized coenzyme 1a ] = 1 mm , [ nahco2 ] = 60 mm , [ rh complex ] = 0.25 
 mm , mops buffer ( 50 mm , ph 7.0 , 5 mm cacl2 ) , inert atmosphere , 
 50 c , 4 h ; the mnadhs were directly added as solids .
( b ) proposed mechanism for the reduction of 
 nad(p ) by [ cp*rh(bpy)(h2o ) ] .
the table shows that 66% conversion could be 
 obtained after 4 h reaction time .
the structural , 
 kinetic , and biocatalytic studies of the biomimetics with a wide range 
 of er enzymes demonstrate that they faithfully capture , at the functional 
 level , design aspects inferred from the structures of the natural 
 coenzymes and the natural coenzyme  er protein complexes . that 
 the biomimetics are also stable and can be recycled in biocatalytic 
 transformations
the general utility of these synthetic biomimetics beyond the er family 
 is also important in adopting their use more widely in biocatalytic 
 manufacture .
enzymes were chosen to encompass some 
 of the most useful transformations for the manufacture of chemical 
 products such as the monooxygenation of unfunctionalized carbon 
hydrogen 
 bonds or of ketones to esters , the chemoselective reduction of carboxylic 
 acids to aldehydes and the reverse reaction , the reductive amination 
 of ketones , the reduction of alkenes using non - flavin - dependent enzymes , 
 and the regeneration of the oxidized form of the biomimetics .
activity 
 assays using the non - flavin - dependent double - bond reductase from nicotiana tabacum ( ntdbr ) showed that the biomimetics also support the 
 reduction of cinnamaldehyde to hydro - cinnamaldehyde at the expense 
 of biomimetics 14 , with conversion 
 ranging from 5 to 21% .
further kinetic data ( see the supporting information for more details ) showed that ntdbr has the highest preference for nadph as the hydride 
 donor for the reduction of cinnamaldehyde ( kcat 40 min , km 91 m ) .
conversely , the observed rate measured at 5 mm substrate 
 concentration revealed a reduced turnover number for nadh ( kobs 4 min ) as well as biomimetics 1 ( kobs 0.15 min ) and 3 ( kobs 0.08 min ) . additionally , a nadph oxidase isolated from b. subtilis(53,54 ) was also capable of 
 efficiently oxidizing biomimetics 14 using molecular oxygen as the only electron acceptor .
these are 
 examples that demonstrate the versatility of the cofactor analogues 
 in addition to the previously established system with a styrene monooxygenase , 
 greatly improving the efficiency of electron transfer for the selective 
 epoxidation of styrene derivatives .
biocatalyst 
 complexes , similar to that described herein , will guide further design 
 of new biomimetics , extending their reach to other useful transformations . 

this should extend the biomimetic series beyond compounds 15 and enable widespread application of nicotinamide 
 biomimetics in biocatalytic processes .
we have shown that synthetic coenzyme biomimetics can outperform 
 natural coenzymes in biotransformations reactions with widely used 
 redox biocatalysts . that laboratory - based designs can outperform
these synthetic coenzymes are inexpensive 
 to manufacture , are stable relative to their biological counterparts , 
 and are operational with a wide range of biocatalysts taken from the 
 er family and a few other oxidoreductases .
additionally , the biomimetics 
 can be recycled at the expense of formate and are therefore exploitable 
 using only catalytic amounts .
implementation of these synthetic biomimetics 
 as well as the design of more sophisticated analogues capable of operating 
 with a variety of other oxidoreductases will facilitate the use of 
 redox biocatalysts in chemicals production at low cost and thereby 
 transform the use of oxidoreductases more widely in industrial biocatalysis .
 the search for affordable , green 
 biocatalytic processes is a challenge 
 for chemicals manufacture . 
 redox biotransformations are potentially 
 attractive , but they rely on unstable and expensive nicotinamide coenzymes 
 that have prevented their widespread exploitation . 
 stoichiometric 
 use of natural coenzymes is not viable economically , and the instability 
 of these molecules hinders catalytic processes that employ coenzyme 
 recycling . here 
 , we investigate the efficiency of man - made synthetic 
 biomimetics of the natural coenzymes nad(p)h in redox biocatalysis . 
 extensive studies with a range of oxidoreductases belonging to the 
  ene  reductase family show that these biomimetics are 
 excellent analogues of the natural coenzymes , revealed also in crystal 
 structures of the ene reductase xena with selected biomimetics . 
 in 
 selected cases , these biomimetics outperform the natural coenzymes . 
  
 better - than - nature  
 biomimetics should find widespread 
 application in fine and specialty chemicals production by harnessing 
 the power of high stereo- , regio- , and chemoselective redox biocatalysts 
 and enabling reactions under mild conditions at low cost . 
Introduction
Results
and Discussion
Concluding Remarks