Patent Application: US-60935803-A

Abstract:
methods for preparative in vitro biocatalysis utilizing oxidoreductases . these methods are based on novel approaches to supply the oxidoreductases with reduction equivalents derived either from chemical or electrochemical sources . by utilization of organometallic mediators , the oxidoreductases are regenerated directly in the absence of nicotinamide coenzymes and biocatalysts generally used to transfer the reduction equivalents to the active site . these methods also include novel reactor design for continuous electro - chemical regeneration of these enzymes .

Description:
the present invention discloses a novel method to directly regenerate the oxygenase component of styrene monooxygenase for biocatalytic epoxidation of aryl - and alkyl - substiuted c — c double bonds in high enantiomeric purities ( fig1 ). [ 0065 ] fig1 shows the new regeneration concept for the monooxygenase component of the styrene monooxygenase enzyme system . in a traditional approach ( fig1 lower ), the reductase component ( styb ) is needed to transfer reduction equivalents from nadh to the monooxygenase part . for regeneration of nadh most commonly formate dehydrogenase ( fdh ) with formate as stoichiometric source of reduction equivalents is used . however , [ cp * rh ( bpy ) h ] + was shown to reduce fad which can be applied to stya thus eliminating the need for the reductase component , nad and the nadh regeneration system ( fig1 upper ). some non - limiting examples for reactions that can be performed are given in fig2 . the approach outlined is not limited to the application to styrene monooxygenase but generally to any fad - dependent monooxygenase and the reactions that it catalyzes . some non - limiting examples for fad - dependant oxygenases and dehydrogenases and reactions and products achievable are reactions catalyzed by : [ 29 ] monooxygenases comprising the e . c . number 1 . 14 . x . y that catalyze hydroxylation reaction at aromatic rings ( benzene derivatives and aromatics containing one or several heteroatoms such as o , s , n , p ), epoxidation reactions of olefins , beayer - villiger reactions and heteroatom oxygenations ( e . g . at b , al , ga , n , p , as , sb , s , se , te , cl , br , i ). fad containing oxidoreductases catalyzing the insertion of heteroatoms into organic compound such as tryptophan halogenase [ 30 ]. dehydrogenases dependant on fad catalyzing reduction reactions e . g . at organic acids , aldehydes , ketones , imines or c — c double bonds . the present invention also discloses a novel method to regenerate ( provide with reduction equivalents ) heme - and non - heme iron enzymes such as the catalytic reduction of cytochrome c ( fig3 ). this direct reduction of metal - containing enzymes can be applied to supply metal - containing monooxygenases with reduction equivalents necessary . thus , the electron - transport chain ( including nad ( p ) h and the regeneration thereof , the reductase and the mediator protein ) can be substituted by [ cp * rh ( bpy )( h 2 o )] 2 + . thus , this approach is , applicable to heme - and non - heme - iron monooxygenases and dioxygenases and oxidoreductases containing other metal cations . this invention discloses also a novel method for controllable in situ production of hydrogen peroxide and its coupling to p450 monooxygenases and peroxidases . instead of fad , any alloxazine - based structure , reacting with molecular oxygen to hydrogen peroxide can be used ( e . g . riboflavine , fmn , etc .). by adjusting reaction parameters ( especially c ([ cp * rh ( bpy )( h 2 o )] 2 + , c ( formate ), and temperature ) a hydrogen peroxide formation rate can be achieved that is suitable for a given hydrogen peroxide consuming enzymatic reaction . thus , this invention discloses a general approach for the regeneration of heme - containing mono - and dioxygenases as well as peroxidase ( based on heme - structures ). these enzymes can be regenerated by [ cp * rh ( bpy )( h 2 o )] 2 + either by direct reduction or by utilizing the hydrogen peroxide shunt ( fig4 ). this invention also discloses a novel method for in situ regeneration of enzymatically active nad ( p ) + utilizing cp * rh ( bpy )( h 2 o )] 2 + as oxidation catalyst and its application to dehydrogenase catalyzed oxidation reactions ( fig5 ). thus , it can be applied e . g . to kinetic resolution of racemic alcohols leading to enantiopure alcohols or regiospecific oxidation reactions . this invention also discloses methods to prevent the inactivation of enzymes and / or the redox - catalyst by mutual interaction . previously [ cp * rh ( bpy )( h 2 o )] 2 + has been reported to have no influence on the activity of several dehydrogenases ( hladh , tbadh , etc .) [ 13 ]. however , this is not generally the case for any given enzyme . for example incubation of [ cp * rh ( bpy )( h 2 o )] 2 + with 2 - hydroxybiphenyl - 3 - monooxygenase ( hbpa , e . c . 1 . 14 . 13 . 44 ) leads to complete loss of enzymatic activity . this inactivation can be prevented by utilizing nucleophilic buffers such as buffers containing ammonia or tris . this invention also discloses a novel reactor concept to circumvent the generation of freely diffusing reactive oxygen species generated by direct reduction of molecular oxygen at the cathode ( fig6 ). one of the major problems during electrochemical reactions in o 2 containing media is the direct cathodic reduction of o 2 leading to reactive reduced oxygen species ( superoxide , peroxide ) that are hazardous to enzyme activity . the biocatalyst is separated from the electrochemical cell by immobilization ( e . g . on eupergit ). o 2 supply can be adjusted to a concentration in the biocatalyst compartment so that it is completely consumed by the enzymatic reaction . thus , oxygen - free buffer is pumped through the electrolysis , and subsequently the reaction medium is lead through a hollow - fiber module to supply the medium with substrate for the enzymatic reaction and to withdraw the reaction product . in a typical experiment potassium phosphate buffer ( 50 mm , ph 7 . 0 , c ( nahco 2 )= 150 mm ) is supplemented with cytc - ( from horse heart , fluka , buchs switzerland ) and [ cp * rh ( bpy )( h 2 o )] 2 + - stocksolutions to 1 ml final volume in a quartz kuvette and placed into a thermostatted ( t = 30 ° c .) uv - spectrophotometer ( unicam ). after addition of the components , the absorption at λ = 550 nm is followed . concentrations of reduced cytc were determined after background correction according to the law of lamber - beer utilizing a molar extinction coefficient for reduced cytc of 21000 cm 2 * mol − 1 . [ 0092 ] fig7 displays the uv spectra recorded at intervals of 1 minute during one typical experiment . maximum absorptions at 550 nm ( originating from the absorption of reduced cytc ) fig8 correlate independently from c ([ cp * rh ( bpy )( h 2 o )] 2 + linearly to the concentration of cytc . duration of the lack phase and rate of cytc reduction however correlate to c ([ cp * rh ( bpy )( h 2 o )] 2 + ). result : heme - containing enzymes ( represented by cytc ) can be reduced by reduced [ cp * rh ( bpy ) h ] + ( in situ regenerated by formate ). quantitative reduction of the protein can be achieved . analogous experiments were carried out in the absence of the catalyst ([ cp * rh ( bpy )( h 2 o )] 2 + ), the source of reduction equivalents ( nahco 2 ) and cytc . none of these controls lead to significant increase in the absorption at 550 nm . result : these controls prove [ cp * rh ( bpy )( h 2 o )] 2 + catalyzed reduction of cytc . in another typical experiment , the reaction temperature was varied between 20 and 35 ° c . to exclude stoichiometric interaction of [ cp * rh ( bpy )( h 2 o )] 2 + with cytc , in another experiment low c ([ cp * rh ( bpy )( h 2 o )] 2 + ) were applied . in 50 ml degassed potassium phosphate buffer ( 50 mm , ph 7 . 0 , c ( nahco 2 )= 150 mm ) 10 μm ([ cp * rh ( bpy )( h 2 o )] 2 + ) were incubated with 80 μm cytc ( fig9 ). result : multiple turnovers of [ cp * rh ( bpy )( h 2 o )] 2 + as reduction catalyst can be obtained . turnover frequencies for [ cp * rh ( bpy )( h 2 o )] 2 + are in the range of 4 - 32 turnovers per hour and depend on the reaction temperature . the total turnover number of 6 for [ cp * rh ( bpy )( h 2 o )] 2 + the example given in fig9 is not optimized yet . [ cp * rh ( bpy ) h ] + catalyzed regeneration of the monooxygenase component of styrene monooxygenase ( stya ) in a typical experimental setup 1 ml total volume was placed in an eppendorf cup in a thermomixer . the temperature was set to 37 ° c . and the reaction mixture was shaken thoroughly to ensure o 2 intake . general compositions are given in table 2 : product formation was followed by reversed phase hplc . the hplc system ( merck ) consisted of a d - 7000 controller , l - 7200 autosampler , l - 7400 uv detector , l - 7100 liquid chromatography pump and was fitted with a cc250 / 4 nucleosil 100 - 5 c18 hd column . samples were eluted under isocratic conditions using 60 % acetonitrile and 40 % water at a flow rate of 1 ml × min − 1 . the elution pattern was monitored at 210 nm . hydrogen peroxide was determined enzymatically , based on the method by saito et al . [ 33 ]. samples were mixed with a solution of 0 . 6 mm 4 - amino antipyrine , 9 mm phenol , and 6 u × ml − 1 peroxidase from coprinus cinereus . after one minute incubation at room temperature the absorption at 550 nm was measured . experiments leaving out either [ cp * rh ( bpy )( h 2 o )] 2 + or stya yielded no detectable formation of styrene oxide . also the incubation of hydrogen peroxide in the presence of stya and fad did not lead to the formation of styrene oxide . result : reaction mechanisms other than outlined in fig1 ( upper ) can be excluded . 0 . 2 mm [ cp * rh ( bpy )( h 2 o )] 2 + , 0 . 05 mm fad , 0 . 005 mm fmn , 2 mm styrene , 280 u catalase , and 1 . 16 μm stya in 50 mm potassium phosphate buffer with 150 mm sodium formate were incubated at 37 ° c . and agitation at 1500 rpm . at intervals , reactions were stopped by addition of one equivalent volume of cold acetonitrile . after centrifugation ( 15 min at 4 ° c . and 23000 × g the upper phase was analyzed by reverse phase hplc . concentrations were corrected according to the phase ratio after centrifugation . results from this experiment are given in fig1 and table 3 . in a second experiment fmn was left out from the reaction mixture without detectable loss in styrene oxide productivity . result : the highly simplified regeneration approach for stya ( as member of fad - dependant monooxygenases ) is functional . multiple turnovers for both the enzyme and the regeneration catalyst can be achieved in the simplified in vitro regeneration approach ( fig1 ). a total volume of 500 μl 0 . 2 mm [ cp * rh ( bpy )([ h 2 o )] 2 + , 0 . 033 mm fad , fmn , 280 u catalase , and 1 . 16 μm stya in 50 mm potassium phosphate buffer with 150 mm sodium formate was incubated with 500 μl styrene ( 2 mm dodecane as internal standard ) at 37 ° c . and agitation at 1500 rpm . at intervals , samples from the styrene phase were withdrawn and analyzed by gc . results from this experiment are given in fig1 . result : biphasic reaction media containing the neat substrate as substrate - & amp ; product reservoir can be applied . initial productivities are in the range of one - phase reactions . experiment 4 : the role of non - stya supported reduction of fad ( leading to hydrogen peroxide ) 0 . 2 mm [ cp * rh ( bpy )( h 2 o )] 2 + , 0 . 05 mm fad , and 1 . 16 μm stya in . 50 mm potassium phosphate buffer with 150 mm sodium formate were incubated at 37 ° c . and agitation at 1500 rpm . at intervals samples were taken and analyzed for their hydrogen peroxide concentration . from the results a catalytic performance of 48 turnovers per hour for [ cp * rh ( bpy )( h 2 o )] 2 + was calculated . in a second experiments a reaction mixture of this composition was supplemented with 2 mm styrene . after 15 min the reaction was stopped and analyzed for hydrogen peroxide and styrene oxide . 0 . 28 mm styrene oxide and 2 . 07 were found at this time corresponding to an overall catalytic activity of [ cp * rh ( bpy )( h 2 o )] 2 + of 47 turnovers per hour . 0 . 4 mm [ cp * rh ( bpy )( h 2 o )] 2 + , 0 . 05 mm fad , 4 mm styrene , 280 u catalase , and 0 . 1 - 4 . 4 μm stya in 50 mm potassium phosphate buffer with 150 mm sodium formate were incubated 15 minutes at 37 ° c . and agitation at 1500 rpm ( fig1 ). in a second experiment , 0 . 1 - 0 . 6 mm [ cp * rh ( bpy )( h 2 o )] 2 + , 0 . 05 mm fad , 4 mm styrene , 280 u catalase , and 1 . 47 μm stya in 50 mm potassium phosphate buffer with 150 mm sodium formate were incubated 15 minutes at 37 ° c . and agitation at 1500 rpm ( fig1 ). experiment 6 : the role of fad concentration on styrene oxide formation 0 . 2 mm [ cp * rh ( bpy )( h 2 o )] 2 + , 280 u catalase , 1 . 25 μm stya , 2 mm styrene , and 1 - 200 μm fad in 50 mm potassium phosphate buffer with 150 mm sodium formate were incubated for 15 minutes at 37 ° c . and agitation at 1500 rpm . afterwards the reaction mixtures were analyzed by reverse phase hplc for their styrene oxide concentration ( fig1 ). result from experiments 4 - 6 : non - stya supported reduction of fad is the main competing reaction . however , this undesired reaction can be minimized by engineering of the reaction conditions . preliminary maximum space - time yield are as high as 4 . 8 mm /( l * h ). 0 . 2 mm [ cp * rh ( bpy )( h 2 o )] 2 + , 0 . 02 mm fad , 2 mm substrate , 200 u catalase , and 1 . 47 μm stya in 50 mm potassium phosphate buffer with 150 mm sodium formate were incubated at 37 ° c . and agitation at 1500 rpm . styrene oxide was analyzed using a cc200 / 4 nucleodex α - pm ( machery - nagel ). as eluent 60 % methanol and 40 % 6 . 2 mm teaa in water was used at a flow rate of 0 . 7 ml × min − 1 . 1 , 2 - dihydronaphthalene oxide , trans - β - methylstyrene oxide , indene oxide , and methyl phenyl sulfoxide were extracted from the reaction buffer with one aliquot hexane und analyzed unsing normal phase hplc with a chiracel ob — h column and hexane : isopropanol 95 : 5 ( 9 : 1 for methyl phenyl sulfoxide ) at a flow rate of 0 . 5 ml * min − 1 as mobile phase . as reference either authentical samples of racemic epoxides were used . ( table 4 ). result : the simplified regeneration approach does not limit the substrate range of stya catalyzed epoxidation reactions nor lowers it the enantiospecificity of the epoxidation reaction . furthermore , sulfoxidations catalyzed by stya have not been reported yet . 1 ml stya solution ( 2 . 2 mg * ml − 1 ) are incubated with 200 mg eupergit c ™ at 4 ° c . for 3 days . afterwards the immobilisate is washed to remove unbound proteins and stored in 50 mm phosphate buffer ( ph 7 ) at 4 ° c . in a typical experiment 100 mg of the immobilisate are incubated in potassium phosphate buffer ( 50 mm , ph 7 . 0 ) with 150 mm sodium formate , 50 μm fad , 2 mm styrene , and 0 . 2 mm [ cp * rh ( bpy )( h 2 o ) 2 + at 37 ° c . and 1500 rpm . at intervals , samples are taken from the supernatant and analyzed by reverse phase hplc ( fig1 ). result : stya covalently bound to a solid matrix is catalytically active . enzyme activities on the solid matrix are considerably lower than freely diffusing enzyme ( about 10 - 20 %) but can be optimized by advanced immobilization procedures and materials . [ cp * rh ( bpy ) h ] + catalyzed formation of hydrogen peroxide coupled to heme - containing enzymes hydrogen peroxide produced was determined enzymatically with a modified assay by method by saito et al . [ 33 ]. samples were mixed with a solution of 0 . 6 mm 4 - amino antipyrine , 9 mm phenol , and 6 u × ml − 1 peroxidase from coprinus cinereus . after one minute incubation at room temperature the absorption at 550 nm was measured . by using hydrogen peroxide samples of known concentration , a calibration curve was generated . fadh 2 concentrations were determined on the depleting absorption of fad at λ = 450 nm using a molar extinction coefficient of 12000 m − 1 cm − 2 . oxygen measurements were performed polarographically with an oxygen electrode , which was mounted to a gas - tight thermostatted 2 ml reaction vessel . prior to experiments , calibration of the electrode was performed in oxygen - saturated buffer and o 2 - free buffer . experiment 1 : depletion of molecular oxygen and accumulation of reduced fadh 2 0 . 2 mm [ cp * rh ( bpy )( h 2 o )] 2 + , 0 . 225 mm fad dissolved in o 2 saturated potassium phosphate buffer were thermostated at 37 ° c . in an air - tight reaction vessel ( o 2 - measurements : 2 ml , uv - measurements : 1 ml quartz kuvette ). reactions were started on addition of sodium formate stock solution ( final concentration : 150 mm ). [ 0139 ] fig1 shows the time course of o 2 - depletion and subsequent accumulation of reduced fadh 2 under reaction conditions where diffusion of o 2 into the reaction medium is prevented . result : fadh 2 can be in situ produced by [ cp * rh ( bpy ) h ] + . it reacts very fast ( diffusion limitation ) with molecular oxygen . 1 ml of 19 μm cp * rh ( bpy )( h 2 o )] 2 + and 0 - 200 μm fad in 50 mm potassium phosphate buffer ( ph 7 . 0 , c ( nahco 2 )= 150 mm ) are placed in a 1 . 5 ml eppendorf vial and incubated at 37 ° c . and agitation at 1500 rpm . at intervals 100 ml samples are withdrawn and analyzed for their h 2 o 2 content ( fig1 ). initial h 2 o 2 - formation rates correspond to catalytic activities for [ cp * rh ( bpy )( h 2 o )] 2 + of 69 . 9 ± 2 . 5 catalytic cycles per hour ( 37 ° c ., c ( nahco 2 )= 0 . 15 m ). these values were obtained from experiment 1 ( oxygen consumption and fadh 2 accumulation ) as well . the reaction rate depends linearly on c ([ cp * rh ( bpy )( h 2 o )] 2 + ). in analog experiments fad was substituted by fmn leading to identical results . result : the formation rate of hydrogen peroxide can be adjusted by varying the [ cp * rh ( bpy )( h 2 o )] 2 + concentration and are ( at first ) independent from the concentration of fad ( fmn ). however , over longer time scales ratios of fad /[ cp * rh ( bpy )( h 2 o )] 2 + higher than 1 are required to maintain [ cp * rh ( bpy )( h 2 o )] 2 + activity . experiment 3 : cytc as p450 - like protein ; its inactivation by hydrogen peroxide as described above , cytc exhibits monooxygenase activity in the presence of hydrogen peroxide ( hydrogen peroxide shunt ). on the other hand cytc is inactivated by hydrogen peroxide , which can be followed by the disappearing soret - peak ( heme absorbance at k = 408 nm ) [ 24 ]. in a typical experiment 50 mm cytc is incubated at 30 ° c . in the presence of 1 mm hydrogen peroxide . at intervals of 2 min the uv - spectrum ( between 300 and 650 nm ) is recorded ( fig1 ). in identical experiments , the absorbance at 408 nm was followed over time . table 5 gives the absolute rates of the decreasing absorption at 408 nm in the linear phase at varying cytc : h 2 o 2 ratios . after preincubation of cytc ( 15 μm ) in potassium phosphate buffer in the presence of 1 mm hydrogen peroxide for 10 minutes lead to almost complete loss of enzymatic activity ( measured on the sulfoxidation of thioanisol where only traces of methyl phenyl sulfoxide were detectable ). result : cytc ( representing the class of p450 - like monooxygenases ) is rapidly inactivated by hydrogen peroxide . this inactivation is a bimolecular process ; the inactivation rate linearly depends on c ( h 2 o 2 ) as well as on c ( cytc ). therefore , stochiometric addition of hydrogen peroxide is not applicable to preparative scale applications . experiment 4 : coupling of [ cd * rh ( bpy )( h 2 o )] 2 + catalyzed in situ production of hydrogen peroxide to cytc catalyzed oxidation of thioanisol . in a typical experiment 1 ml of 200 μm [ cp * rh ( bpy )( h 2 o )] 2 + , 50 μm fad , 2 mm thioanisol , and 5 - 20 μm cytc in 50 mm potassium phosphate buffer ( ph 7 . 0 , c ( nahco 2 )= 150 mm ) are placed in a 1 . 5 ml eppendorf vial and incubated at 37 ° c . and agitation at 1500 rpm . after 20 minutes the reactions are stopped by addition of 10 μl perchloric acid ( 10 %), centrifuged and the supernatant analyzed by reversed phase hplc ( fig1 ). in an otherwise identical experiment c ( cytc was set to 20 μm whereas the concentration of [ cp * rh ( bpy )( h 2 o )] 2 + was varied between 100 μm and 400 μm . a linear dependence of product concentration on the concentration of [ cp * rh ( bpy )( h 2 o )] 2 + was found . 1 ml of 200 μm [ cp * rh ( bpy )( h 2 o )] 2 + , 50 μm fad , 2 mm thioanisol , and 83 μm cytc in 50 mm potassium phosphate buffer ( ph 7 . 0 , c ( nahco 2 )= 150 mm ) are placed in a 1 . 5 ml eppendorf vial and incubated at 37 ° c . and agitation at 1500 rpm . after 20 minutes the reactions are stopped by addition of 10 μl perchloric acid ( 10 %), centrifuged and the supernatant analyzed by reversed phase hplc . experiments leaving out either cytc or [ cp * rh ( bpy )( h 2 o )] 2 + under otherwise identical conditions yielded no detectable formation of methyl - phenyl sulfoxide from thioanisol . in the presence of 560 u catalase yielded in the formation of traces of methyl - phenyl sulfoxide (& gt ; 0 . 01 mm after 20 minutes ). in another experiment 1 ml of 200 μm [ cp * rh ( bpy )( h 2 o )] 2 + , 50 μm fad , 2 mm thioanisol , and 20 μm cytc in 50 mm potassium phosphate buffer ( ph 7 . 0 , c ( nahco 2 )= 150 mm ) are placed in a 1 . 5 ml eppendorf vial and incubated at 37 ° c . and agitation at 1500 rpm . at intervals , samples are withdrawn and analyzed by reversed phase hplc ( fig2 ). result : the [ cp * rh ( bpy )( h 2 o )] 2 + catalyzed formation of hydrogen peroxide is applicable to supply a p450 - like monooxygenase with an appropriate amount of hydrogen peroxide for optimal stability and productivity . thus , long - term oxidation reactions become available . hbpa - activities were determined by uv - spectroscopy according to literature methods [ 34 ] by supplementing the experiment buffer with 0 . 1 mm nadh and observation of nadh - depletion at 340 nm for 1 minute , afterwards 2 mm of 2 - hydroxybiphenyl were added and nadh depletion was measured for 1 minute . hbpa activity was determined as difference of both rates . [ cp * rh ( bpy )( h 2 o )] 2 + activity was determined by uv - spectroscopy by supplementing the experiment buffer with 0 . 1 mm nad + and 150 mm final concentration of sodium formate and following the nadh formation at . 340 nm . experiment 1 : mutual inactivation of 2 - hydroxybiphenyl - 3 - monooxygenase ( hbpa e . c . 1 . 14 . 13 . 44 ) and [ cp * rh ( bpy )( h 2 o )] 2 + in a first experiment 0 . 11 u * ml − 1 hbpa of a partially enriched crude extract from recombinant e . coli overexpressing hbpa was incubated at 30 ° c . with 0 , 0 . 02 , 0 . 04 , and 0 . 1 mm [ cp * rh ( bpy )( h 2 o )] 2 + . in a second experiment c [ cp * rh ( bpy )( h 2 o )] 2 + ) was maintained at 0 . 02 mm whereas the hbpa content varied form 0 . 22 u * ml − 1 to 1 . 52 u * ml − 1 . at intervals , samples were analyzed with respect to residual hbpa activity table 6 displays half - live times and residual hbpa activities after 60 minutes of incubation . result : [ cp * rh ( bpy )( h 2 o )] 2 + can interact with proteins leading to loss of catalytic activity of both enzyme and [ cp * rh ( bpy )( h 2 o )] 2 + . the inactivation process is stochiometric . experiment 2 : prevention of hbpa - inactivation by [ cp * rh ( bpy )( h 2 o )] 2 + by blocking the rh - coordination sphere with nh 3 0 . 28 u * ml − 1 hbpa were incubated at varying ammonia concentrations ( 0 - 100 mm ) with 0 . 04 mm [ cp * rh ( bpy )( h 2 o )] 2 + . at intervals , samples were examined towards their residual enzyme activity ( fig2 ). result : since the nature of mutual inactivation of [ cp * rh ( bpy )( h 2 o )] 2 + and enzyme is coordination of nucleophilic groups at the enzymes surface , it can be prevented by blocking the free coordination space at the rhodium ion with nucleophilic ligands such as nh 3 . experiment 3 : [ cp * rh ( bpy )( h 2 o )] 2 + activity in the presence of 100 mm nh 4 ± under chemical and electrochemical reduction 0 . 04 mm [ cp * rh ( bpy )( h 2 o )] 2 + in 50 mm potassium phosphate buffer ( 50 mm , ph 7 . 5 ) are incubated at 30 ° c . with nh 4 + concentrations of 0 , 5 , 10 , 20 , 50 , 100 mm . at intervals , samples are analyzed by uv - spectroscopy towards their activity under form ate driven nadh regeneration . assay conditions were : 500 μl sample are given to 500 μl ( 2 mm nad + , 150 mm nahco 2 , t = 30 ° c . ): the absorption at 340 nm is followed for 1 minute ( fig2 ). in another experiment , 0 . 02 mm [ cp * rh ( bpy )( h 2 o )] 2 + at 30 ° c . in 50 mm potassium phosphate buffer ( c ( nad + )= 1 mm ) either in the presence or absence of 100 mm nh 4 + is reduced cathodical at a carbon - felt electrode ( e =− 750 mv vs . ag / agcl sat .) ( fig2 ). result : even though nh 3 containing buffers inhibit the formate - driven regeneration of [ cp * rh ( bpy ) h ] + ( and thus the nadh regeneration ), electro - chemical regeneration of nadh is possible . regeneration of oxidized nicotinamide coenzymes utilizing [ cp * rh ( bpy )( h 2 o )] 2 + as transhydrogenation catalyst between nad ( p ) h and fad in a typical experiment , a 0 . 1 mm nadh solution in 50 mm potassium phosphate buffer ( 50 mm , ph 7 . 0 ) is placed in a kuvette in a uv - spectrophotometer . the reaction mixture is thermostatted at 30 ° c . after supplementing with [ cp * rh ( bpy )( h 2 o )] 2 + ( 0 - 0 . 02 mm ) and fad ( 0 - 0 . 04 mm ) the absorption at 340 nm is followed . the initial rate is linearly dependent on c ([ cp * rh ( bpy )( h 2 o )] 2 + ). fad can be substituted by fmn . result : [ cp * rh ( bpy )( h 2 o )] 2 + serves as catalyst in the transhydrogenation reaction between reduced nicotinamide coenzymes and alloxazine - based structures ( such as fad or fmn ) in the presence of molecular oxygen the oxidized alloxazine is regenerated very fast ( see also fig1 ). this can serve as regeneration concept for oxidized nicotineamide coenzymes ( nad ( p ) + ) from their reduced forms . in a typical experiment , 20 mm of 3 - methyl cyclohenanol were incubated with 5 mm nadh , 50 μm ( cp * rh ( bpy )( h 2 o )] 2 + , 200 μm fad , 10000 u catalase and 38 nags of alcohol dehydrogenase from thermus sp . in 50 mm potassium phosphate buffer at 60 ° c . at intervals , samples were withdrawn and analyzed by gas chromatography ( fig2 ). in a control experiment under identical conditions and in the absence of tadh , only traces of 3 - methyl cyclohexanone were detectable . result : the proposed regeneration approach for oxidized nicotinamide coenzymes is applicable to enzymatic reaction ( here represented by the dehydrogenase from thermus sp .). in a typical experiment , 1 ml total volume of 200 μm [ cp * rh ( bpy )( h 2 o )] 2 + are incubated with 2 mm 3 - ethyl toluene and 100 μl of a cell crude extract of recombinant e . coli overexpressing alkb in a final volume of 1 ml 100 mm potassium phosphate buffer ( ph 7 . 4 ) supplemented with 150 mm sodium formate and 1 mg / ml bovine serum albumin at 30 ° c . and vigorous shaking . after 20 minutes the reaction is stopped by addition of 10 ml perchloric acid ( 10 %). after centrifugation the supernatant is extracted with one aliquot of hexane . the organic phase is analyzed by normal phase hplc ( nucleosil 250 / 3 100 - 5 c , mobile phase : hexane / isopropanol 95 : 5 , flow rate = 0 . 5 ml min − 1 ). from comparison with authentical samples , about 0 . 015 mm ( average of 4 experiments ) of 3 - methylphenethylalcohol were produced . [ 0186 ] table 4 substrates . ( reaction conditions : t = 37 ° c ., c ([ cp * rh ( bpy )( h 2 o )] 2 + ) = 0 . 2 mm , c ( stya ) = 1 . 47 μm , c ( nahco 2 ) = 150 mm , c ( fad ) = 20 μm , c ( catalase ) = 200 u * ml − 1 epoxides ee - value reaction time concentration of the substrate ( 2 mm ) [ min ] [ mm ] epoxides [%] styrene ( 4 mm ) 15 0 . 33 & lt ; 98 ( s ) [ a ] 1 , 2 - dihydronaphthalene 15 0 . 23 98 . 9 trans - β - methylstyrene 15 0 . 28 97 . 6 indene 15 0 . 42 97 . 9 3 - chloro - styrene 15 0 . 37 n . d . [ b ] 4 - methyl - styrene 15 0 . 29 n . d [ b ] 4 - fluoro styrene 15 positive [ c ] n . d . [ b ] 4 - chloro styrene 15 positive [ c ] n . d . [ b ] 4 - bromo styrene 15 positive [ c ] n . d . [ b ] 4 - methoxy styrene 15 positive [ c ] n . d . [ b ] thioanisole 15 0 . 33 ( methyl 23 . 3 phenyl sulfoxide ) phenyl ethyl sulfide 15 positive [ c ] n . d . [ b ] [ 0187 ] table 5 cytc inactivation as a function of [ cytc ] and [ h 2 o 2 ]. 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