h bonds in complex molecules 
 represents a most useful 
 but also one of the most challenging transformations in organic chemistry . over the past two decades , notable advances in the area of chemical 
 c
 h oxidation involving the use of oxidizing reagents , biomimetic supramolecular complexes , or transition metal catalysts have 
 disclosed opportunities to afford the oxidation of electronically 
 activated c 
h 
 sites could be targeted for oxidation by taking advantage of proximal 
  directing  groups pre - existing or preinstalled in the 
 target compound . despite this progress , 
 the vast majority of c 
h bonds in molecules of high complexity , 
 in particular isolated , ( stereo)electronically unbiased c 
the use of cytochrome p450 enzymes constitutes 
 an attractive alternative 
 strategy toward the oxyfunctionalization of unactivated c 
these biological 
 catalysts can provide a valuable complement to chemical oxidation 
 strategies as their site - selectivity could be , in principle , steered 
 toward a remote c  h site in a given compound by influencing 
 their molecular recognition properties via protein engineering . in practice , however , fine - tuning the regio- and stereoselectivity 
 of p450s toward a predefined position in a non - native substrate of 
 interest has represented a fundamental problem .
various factors contribute to 
 complicate these efforts including , among others , the difficulties 
 inherent to controlling the site - selectivity of the p450-catalyzed 
 oxidation reaction via rational design , the notoriously rare occurrence 
 of variants with the desired selectivity properties within engineered 
 p450 libraries , and the requirement for laborious and time - consuming 
 analyses ( i.e. , via hplc or gc ) for regio / stereoselectivity determination . as a result
, this goal has been historically 
 met with only limited or partial success also when ingenious ad hoc 
 high - throughput assays reporting on the p450 regio- or stereoselectivity 
 on a given substrate had been applied .
alternatively , 
 considerable screening efforts need to be invested for any single 
 substrate , as exemplified by a recent study focused on improving the 
 regioselectivity of a testosterone - hydroxylating p450 ( 50%  
 94% ) , an effort that required the analysis of several thousand engineered 
 variants ( > 9,000 ) by hplc . clearly , 
 more 
 general ( i.e. , substrate - independent ) and streamlined strategies to 
 gain access to p450 catalysts with refined regio- and stereoselectivity 
 are needed in order to make p450 catalysis accessible for chemical 
 synthesis .
artemisinin ( 1 , art ; figure 1a ) , a naturally occurring sesquiterpene 
 lactone of prominent value 
 in the pharmacological treatment of human malaria , exemplifies the scope and limitations of currently available 
 tools for late - stage functionalization of a complex molecule . over 
 the past two decades ,
extensive synthetic efforts directed at elaborating 
 this molecule in search for improved antimalarial agents have been 
 largely confined to the lactone ring , and primarily c10 and c9 , which 
 can be readily accessed through chemical methods . whereas substitutions at c10 have yielded derivatives of clinical 
 relevance such as artesunate , artemether , and artemisone ( figure 1a ) , methods to functionalize sp c 
h 
 sites in the upper hemisphere of the molecule , and in particular positions 
 c7 and c6a , would be highly desirable , as these positions constitute 
 major target sites during phase i metabolism of this molecule in humans , 
 a process that contributes to the rapid clearance and undesirably 
 short in vivo half - life of artemisinin - based antimalarials .
these sites have also remained 
 inaccessible to chemical oxidation via iron - based catalysts , which 
 have preference toward the more ( stereo)electronically activated tertiary 
 c  h bond in c6 . on the other hand , 

attempts to hydroxylate artemisinin by means of oxidizing microbial 
 strains have resulted in mixtures of multiple ( over)oxidation products 
 and/or a large extent of art deoxygenation to give deoxoartemisinin , 
 which lacks the endoperoxide bridge essential for biological activity .
( a ) chemical structure of artemisinin ( 1 , 
 art ) and 
 artemisinin - based antimalarial drugs .
using artemisinin as a model substrate , we developed 
 and report 
 here an efficient , new strategy for developing highly regio- and stereoselective 
 p450 catalysts for the oxyfunctionalization of a set of predefined , 
 isolated c 
the described approach 
 hinges upon three key elements , namely ( 1 ) the generation of p450 
 catalyst diversity via mutagenesis of first - sphere active site residues 
 in a parent p450 ; ( 2 ) mapping of the active site configurations of 
 the resulting p450 variants via high - throughput  fingerprinting  to rapidly identify the library members with 
 potentially unique regio / stereoselectivity features ; and ( 3 ) prioritization 
 of these variants via a general method of fingerprint - based prediction 
 of p450 reactivity toward the target substrate ( i.e. , artemisinin ) . 

we demonstrate how this rationally driven , systematic strategy enabled 
 the rapid optimization of an unselective artemisinin - hydroxylating 
 p450 variant ( figure 1c ) into a set of p450 
 oxidation catalysts with highly refined regio- and stereoselectivity 
 for the hydroxylation of each of the three unactivated sp c  h bonds in positions c7 and c6a of artemisinin .
importantly , 
 the entire procedure required the empirical testing of only a handful 
 of p450 variants via conventional hplc - based analyses and yielded 
 a set of synthetically useful p450 catalysts for the selective , late - stage 
 oxyfunctionalization of a relevant yet previously inaccessible region 
 of artemisinin scaffold at both preparative scales and in high yields .
the starting point of this work 
 was fl#62 , an engineered variant of the 
 catalytically self - sufficient fatty acid monooxygenase p450bm3 from bacillus megaterium . in previous studies
, fl#62 was found 
 to exhibit a broad substrate profile that comprises large decaline- 
 and steroid - based compounds .
accordingly , we envisioned this variant 
 could also accept the bulky tricyclic terpene artemisinin ( figure 1b ) as a substrate for oxidation .
upon testing , fl#62 
 was indeed found to be capable of hydroxylating art with high activity , 
 i.e. , supporting about 340 total turnovers ( ttn ) in the presence of 
 a nadph cofactor regeneration system . in comparison , wild - type p450bm3 showed no art - oxidation activity . the site - selectivity 
 of fl#62 was poor , however , producing a mixture of 7(s)-hydroxy - art ( 2 ) , 7(r)-hydroxy - art 
 ( 3 ) , and 6a - hydroxy - art ( 4 ) in a 83:10:7 
 ratio ( figure 1c ) . this notwithstanding , the 
 aliphatic c 
h bonds targeted by this enzyme resided in the 
 relevant , upper hemisphere of the art scaffold , overlapping in two 
 cases ( 3 , 4 ) with those targeted by human 
 liver p450s , making fl#62 a promising starting point toward evolving 
 selective p450 catalysts for the oxidative activation of each of these 
 sites . to this end
, our first step involved altering the 
 fl#62 active site via mutagenesis . in p450bm3 , more than 
 a dozen amino acids extend their side chains into the heme cavity 
 and the long hydrophobic substrate channel connecting the latter to 
 the protein surface . among these ,
mutation of those 
 that are most proximal to the heme ( referred to here as  first - sphere  

residues ) was expected to have the largest impact on the orientation 
 of the enzyme - bound artemisinin above the reactive iron center and , 
 thus , on the regio- and stereoselectivity of fl#62 .
accordingly , a 
 series of active site libraries were constructed via site - saturation 
 mutagenesis ( nnk ) of first - sphere residues 74 , 78 , 81 , 82 , 87 , 181 , 
 and 184 , which lie within 12  from the heme iron and within 
 5  from the terminal carbons of n - palmitoylglycine 
 in the substrate - bound structure of p450bm3 heme domain ( figure 2 ) .
first - sphere 
 residues along the i helix ( e.g. , a264 , t268 ) were not taken into 
 consideration because of their structural role or involvement in catalysis , while a328 was excluded because most substitutions at this site 
 would likely disfavor access of the bulky terpene to the heme .
overall , 
 the size of the resulting libraries included more than 10 members , many of which , in all likelihood , would be misfolded or 
 functionally redundant as a result of deleterious or neutral mutations , 
 respectively .
crystal structure of p450bm3 ( heme domain ) 
 in complex 
 with n - palmitoylglycine ( pdb code 1jpz(24 ) ) .
the bound substrate is displayed as sphere models ( orange ) , 
 and the heme is displayed as stick model ( red ) .
the dotted circle 
 highlights the first - sphere active site residues targeted for mutagenesis 
 in this study . to rapidly identify the most relevant fraction of 
 these libraries , namely , the variants that not only are functional 
 but also have the highest probability to exhibit altered regio- and 
 stereoselectivity in art oxidation
this method provides a means to quickly map the active 
 site geometry of these enzymes via a set of structurally diverse and 
 conformationally rigid chromogenic probes ( compounds p1p5 , figure s1 in the supporting 
 information ) .
accordingly , a total of 12,500 recombinants from 
 the mutagenesis libraries were fingerprinted in 96-well plates using 
 probes p1p5 and a purpald - based 
 colorimetric assay for quantification of probe activity ( figure 3a ) .
the acquired fingerprints were then compared 
 against each other in order to identify the p450 variants with a unique 
 profile , this feature indicating that the corresponding enzyme possesses 
 a unique active site configuration . using 
 this approach , about 1,950 out of the 12,500 screened variants were 
 determined to be catalytically competent ( criterion : > 10% of parent 
 enzyme activity on at least one of the fingerprint probes ) .
among 
 these , 522 variants were established to be functionally unique ( criterion : 
 larger than 20% variation on at least one of the five fingerprint 
 components compared to the parent enzyme and any other member of the 
 library ) and thus worthy of further consideration .
notably , this fraction 
 represented only the 4.1% of the original library , highlighting the 
 efficiency of this tier-2 procedure in drastically reducing the number 
 of candidate enzymes to consider in the subsequent steps .
fingerprint - based 
 strategy to guide the discovery of regio- and 
 stereoselective artemisinin - hydroxylating p450 catalysts .
( a ) schematic 
 summary of the process of identification of the functionally diverse 
 p450 variants from the active site libraries via high - throughput fingerprinting 
 followed by fingerprint comparative analysis ( tier 2 ) .
( c ) outline 
 of the method applied to predict art reactivity via multivariate fingerprint 
 analysis ( tier 3 ) .
, we sought to develop a tool 
 to further guide our search for art - hydroxylating variants within 
 this collection of 522 functionally diverse p450 catalysts .
since 
 art shares little or no structural similarity with any of the fingerprint 
 probes , a p450 reactivity prediction approach alternative to the one 
 previously implemented in the context of probe - related substrates was required . to this end
, we envisioned that 
 a more general strategy based on fingerprint multivariate analysis 
 could prove useful in the context of a probe - unrelated target substrate 
 like art .
as outlined in figure 3b , this method 
 relies on generating a fingerprint - based model predictive of art reactivity 
 by correlating p450 fingerprints with experimental art oxidation activities 
 across a small subset (  training set  ) of the enzyme 
 collection .
since the fingerprints relay information on the p450 active 
 site shape and geometry , this process translates into finding a fingerprint 
 signature that defines an active site configuration compatible with 
 art binding and , thus , its oxidation .
the model is then applied to 
 rank the entire collection of fingerprinted p450s according to their 
 predicted art hydroxylation activity . according to this design , 

a training set of 20 p450s was assembled using fl#62 and 19 randomly 
 chosen variants from the 522-member p450 collection ( training set 
 a , table s2 in the supporting information ) .
the art - hydroxylating activity for each of these enzymes was measured 
 in ttn from small - scale reactions followed by derivatization of the 
 art hydroxylation products with benzoyl chloride and hplc analysis . 

the derivatization procedure was implemented to enable sensitive and 
 precise quantification of the reaction products as art contains no 
 strong chromophores . out of the 19 variants , four exhibited art hydroxylation 
 activity , supporting up to about 110 total turnovers and showing varying 
 regio- and stereoselectivity ( table s2 in the supporting information ) .
these experiments also indicated 
 that only a small fraction ( 20% ) of this p450 catalyst collection 
 would be expected to be active on art . to generate the fingerprint - based 
 model predictive of art reactivity , 
 the experimental art activities
were then correlated with the fingerprints 
 of the corresponding p450s across the whole training set via multiple 
 linear regression analysis ( mlr ) ( figure 4a ) , 
 which provides a statistical tool to analyze the correlation between 
 a dependent variable y and multiple independent variables 
 potentially correlated with y ( equation : y = b0 + b1x1 + b2x2 + ... + bnxn ) . 
 in the applied mathematical function ,
y corresponded 
 to relative art activity and the five fingerprint components were 
 the five independent variables x1 ... x5 . from the resulting best - fit regression coefficients , 

a fingerprint - based model was obtained , which was then applied to 
 rank the remaining p450s in the collection according to their predicted 
 art reactivity ( high - scoring = predicted art active , low - scoring = 
 predicted art inactive ) ( figure 4b ) .
( a ) plot 
 of experimental versus calculated art activity from multiple linear 
 regression analysis of art reactivity / fingerprint correlation across 
 p450 training set a ( table s1 in the supporting 
 information ) .
( b ) 
 ranking of the 522 p450 variants according to their predicted artemisinin 
 activity calculated based on the fingerprint - based model ( training 
 set a ) .
( c ) total turnovers in art hydroxylation for the 50 top - scoring 
 p450 variants ( solid box in panel b ) arranged from the most to the 
 least active variant .
guided by these predictions , we focused our attention on the 50 
 best - scoring p450 variants from the ranking list ( figure 4b , solid box ) , which were characterized for art hydroxylation 
 activity .
gratifyingly , we found that 78% ( 39/50 ) of these variants 
 showed art - hydroxylation activity at synthetically useful levels ( > 100 
 ttn ; average = 323 ttn , figure 4c ) .
furthermore , 
 nearly half of the correctly identified variants ( 22/50 ) were found 
 to possess higher art - hydroxylating activity than the parent enzyme , 
 with one ( ii - c3 ) supporting almost twice as many total turnovers ( 602 
 vs 339 ) ( figure 4c ) .
notably , the  hit  
 rate achieved using this method ( 78% ) greatly exceeded that expected 
 from random sampling of the collection ( 20% ) . in order to 
 further assess the quality of these predictions
, we then tested the 
 twenty lowest - scoring variants from the ranking list ( figure 4b , dotted box ) , which were expected to have minimal 
 or no activity on art .
90% of these p450s ( 18/20 ) show no detectable 
 oxidation activity on the compound , with the two active variants supporting 
 only up to 130 ttn .
the fraction of false negatives ( 10% ) within this 
 group was thus lower than the expected occurrence of art - active p450s 
 from random sampling ( 20% ) , further supporting the reliability of 
 the predictions and functionality of the method to generate them .
since the p450 
 catalysts in the collection were preselected to possess unique active 
 site geometries ( figure 3a ) , the 42 art - reactive 
 variants identified using the strategy described above were expected 
 to exhibit markedly different regio- and stereoselectivity in art 
 oxidation , ideally displaying the sought - after high selectivity toward 
 c7- , with both s and r stereoselectivity , 
 and c6a - hydroxylation .
inspection of the product distribution of these 
 variants confirmed our expectations ( table s3 in the supporting information ) .
importantly , as many as five variants 
 ( 12% of the pool ) were found to exhibit 95% selectivity for 
 c7 hydroxylation with s - stereoselectivity . among 
 these
, the triple mutant iv - h4 displayed the desired absolute regio- 
 and stereoselectivity for this transformation , in addition to supporting 
 higher ttn than the parent enzyme ( table 1 ) . 

on the other hand , three variants showed much improved ( > 70% ) selectivity 
 for 7(r)-hydroxylation ( table s3 in the supporting information ) , which is produced in 
 only 10% relative ratio with the parent enzyme . among these
, variant 
 ii - h10 displayed absolute regio- and r - stereoselectivity 
 for hydroxylation of this site , thereby providing a second , exquisitely 
 selective p450 catalyst for c7-oxyfunctionalization with complementary 
 stereoselectivity compared to iv - h4 ( figure 5 ) .
finally , p450 catalysts with improved 6a - hydroxylation activity 
 were also captured through the strategy , with the best variant ( ii - e2 ) 
 hydroxylating this position with considerably higher ( 48% ) , albeit 
 still suboptimal , regioselectivity compared to fl#62 ( 7% ) ( table s3 
 in the supporting information ) .
the comparatively 
 lower occurrence of 6a - hydroxylating p450s within these first - generation 
 variants could be explained on the basis of the low level of 6a - hydroxylation 
 activity in the parent enzyme combined with the higher strength of 
 this primary c  h bond compared to the secondary ones in c7 . 

importantly , the improvement in 6a - selectivity in ii - e2 was accompanied 
 by an increase in catalytic activity ( 338  393 ttn , table 1 ) , making this variant a promising intermediate 
 toward further refining 6a - selectivity via another round of directed 
 evolution .
mutations in fl#62 vs p450bm3 are v78a , 
 f81s , a82 v , f87a , p142s , t175i , a180 t , a184v , a197v , 
 f205c , s226r , h236q , e252 g , r255s , a290v , l353v .
rates are measured over initial 30 s. ratio between product formation 
 rate and nadph oxidation rate in the presence of artemisinin .
overview of the divergent evolution process leading to the selective 
 art - hydroxylating p450 variants . by sequencing , 
 ii - e2 was determined to carry three active site mutations ( a78n / s81f / v82a ) , 
 which left additional first - sphere positions available for mutagenesis 
 ( figure 2 ) .
accordingly , a triple site - saturation 
 library ( 74/181/184 ) was constructed based on this enzyme , from which 
 3,000 recombinants were subjected to high - throughput fingerprinting 
 followed by fingerprint comparative analysis according to the tier-2 
 procedure of figure 3a .
this step revealed 
 the occurrence of just 50 unique - fingerprint p450s out of a total 
 of 120 catalytically active variants ( 1.6% of the library ) .
after 
 expanding the training set to include ii - e2 (= training set b ) , these 
 50 variants were scored based on their predicted art reactivity according 
 to the tier-3 method of figure 3b as described 
 above for the fl#62-derived variants ( figures s2 and s3 in the supporting information ) . upon evaluation of the 
 25 top - scoring variants , we found that about half ( 52% ) were capable 
 of hydroxylating art , supporting up to 474 ttn ( average : 146 ttn ) 
 ( table s4 in the supporting information ) . on the other hand , 90% ( 9/10 ) of the predicted inactive ones ( bottom - ranking ) 

furthermore , nearly two - thirds 
 ( 8/13 ) of the correctly identified art - active variants exhibited significantly 
 improved 6a - selectivity ( > 80% ) compared to ii - e2 ( table s4 in the supporting information ) .
most importantly , this 
 approach enabled the identification of two highly regioselective p450 
 catalysts for c6a hydroxylation , namely , x - e12 ( 94% selectivity ) and 
 x - f11 ( 92% selectivity ) , thereby completing our desired divergent 
 evolution process ( figure 5 ) .
for x - e12 , the 
 improvement in 6a - selectivity was accompanied by a reduction in catalytic 
 activity ( 113 vs 393 ttn in ii - e2 ) .
in contrast , x - f11 was found to 
 support nearly as many turnovers as ii - e2 and higher ttn than the 
 initial enzyme fl#62 ( table 1 ) . to determine 
 whether potentially interesting catalysts could have been missed in 
 this process ,
these studies revealed the occurrence of only 
 two additional art - oxidizing variants , none of which showed superior 
 performance in terms of either regioselectivity or ttn compared to 
 those already identified ( table s4 in the supporting 
 information ) .
based on these results , we concluded that the 
 method allowed efficient capturing , within the top one - fifth ( 10/50 ) 
 and half ( 25/50 ) of the ranked variants , of 37% ( 6/16 ) and 81% ( 13/16 ) , 
 respectively , of the art - active p450 catalysts in the pool , with the 
 ten top - ranking p450s comprising those with the highest 6a - regioselectivity .
consistent with their divergent reactivity , 
 the best variants for 7(s)-hydroxylation ( iv - h4 , 
 v - h2 ) , 7(r)-hydroxylation ( ii - h10 , iii - b1 ) , and 6a - hydroxylation 
 ( x - e12 , x - f11 ) show distinct fingerprints compared to each other , 
 the parent fl#62 , and the evolutionary intermediate ii - e2 ( figure 6 ) .
these variants were further characterized to 
 gain insights into the effect of the mutations on their catalytic 
 and substrate binding properties .
interestingly , the selective 7(s)-hydroxylating variants were found to share a similar 
 mutational pattern ( table 1 ) , both carrying 
 three active site mutations with two identical substitutions at position 
 78 and 82 and a similar  solution 
( val vs ile ) at the 
 neighboring site 81 ( figure 2 ) . in comparison , 

refinement of 7(r)- and c6a - selectivity required 
 a more extensive remodeling of the enzyme active site as judged by 
 the five to six mutations occurring in ii - h10 , x - e12 , and x - f11 ( table 1 ) .
overall , this trend well reflected the functional 
 distance of these variants , in terms of site - selectivity , from the 
 parent enzyme .
ii - h10 mutations were found to comprise a triad of 
 phenylalanine substitutions within the heme pocket ( s81f , a87f , l181f ) . 

interestingly , iii - b1 , the second best ( r)7-hydroxylating 
 variant found in this study , also carries a pair of phenylalanine 
 mutations in proximity of the heme .
these common features suggest 
 a distinctive requirement for bulky substitutions at the level of 
 the heme pocket to favor art hydroxylation at c7 with r stereoselectivity , when compared to iv - h4 and v - h2 .
another interesting 
 observation emerged from comparing the mutations in the 6a - hydroxylating 
 variants with those of ii - h10 , which evidenced the importance of substitutions 
 at positions 74 and 181 in steering the enzyme selectivity toward 
 c6a .
probe activities correspond to mean values from 
 triplicate experiments ( se within 10% ) after normalization to those 
 of the reference enzyme p450bm3(f87a ) . upon incubation with art
, all the p450 variants 
 were found to exhibit 
 a 5 to 20% shift of the heme iron spin - state equilibrium ( figure 7 and figure s4 in the supporting 
 information ) , which is indicative of the ability of this substrate 
 to displace the heme - bound water ligand upon complex formation .
this 
 property also enabled estimation of the binding affinity ( kd ) of these variants for art via titration experiments . 

for all the p450 variants ( figure 7 and figure 
 s5 in the supporting information ) , hyperbolic 
 curves were observed , suggesting a noncooperative binding interaction . 

interestingly , the 7(s)- and the 7(r)-selective variants showed kd values 
 similar to that of fl#62 ( 3060 m ; table 1 ) , indicating that the acquired mutations had little impact 
 on art binding affinity compared to the parent enzyme . 5- to 10-fold 
 higher kd values were observed instead 
 for variants x - e12 and x - f11 as well as for ii - e2 , indicating that 
 the mutations responsible for improving 6a - hydroxylation selectivity 
 resulted in a somewhat weaker interaction with art .
these equilibrium 
 dissociation constants fall however in the range of that observed 
 for wild - type p450bm3 and the fatty acid laurate ( 270 m ) .
artemisinin binding experiments with ( a ) variant iv - h4 , 
 ( b ) variant 
 ii - h10 , and ( c ) variant x - e12 .
left panel : overlay of the enzyme absorbance 
 spectrum before ( gray line ) and after ( red line ) addition of artemisinin 
 ( 1 mm ) , illustrating the substrate - induced shift of the heme spin 
 state equilibrium .
right panel : representative plot of the artemisinin - induced 
 heme spin shift versus artemisinin concentration . the dissociation 
 constant ( kd ) for the enzyme 
artemisinin 
 complex was calculated via nonlinear fitting of the experimental data 
 ( dots ) to a noncooperative 1:1 binding model equation ( solid line ) . 

data relative to the other variants of table 1 are provided in the supporting information .
further experiments were carried out to analyze 
 the impact of the 
 mutations on the catalytic rates of the enzymes .
these analyses revealed 
 that all the selective 7(s)- , 7(r)- , and 6a - hydroxylating variants exhibited a 3- to 10-fold lower 
 initial product formation rate compared to fl#62 and the less selective 
 ii - e2 ( table 1 ) .
this trade - off between oxidation 
 rate and site - selectivity has been observed in other studies as well , and it can be rationalized here considering 
 that the improved site - selectivity in the former enzymes must inherently 
 involve a more specific enzyme  substrate interaction and thus 
 a restriction of the ( productive ) binding orientations available to 
 art during oxidation . in the case of
ii - h10 and v - h2 , the observed 
 reduction in product formation rate is likely to result also from 
 a reduction in their coupling efficiency (= ratio of product formation 
 rate / nadph oxidation rate ) as compared to the parent enzyme .
for all 
 the other selective p450 variants ( iv - h4 , x - e12 , x - f11 ) , however , 
 the improvement in regio / stereoselectivity came at no cost of the 
 coupling efficiency , as evidenced by the values reported in table 1 .
notably , for both the 6a - hydroxylating variant 
 x - f11 and the 7(s)-hydroxylating variant iv - h4 the 
 coupling efficiency significantly improved compared to fl#62 , exceeding 
 values of 55% and 70% , respectively .
the catalytic turnovers achieved 
 in analytical - scale settings indicated that the evolved p450 variants 
 could be valuable for synthetic purposes .
prior attempts toward enzymatic 
 hydroxylation of art have involved the use of oxidizing strains . besides requiring long reaction times ( 314 
 days ) ,
these transformations have however resulted in the unselective 
 oxidation of the natural product and/or its degradation to the biologically 
 inactive deoxoartemisinin via deoxygenation of the endoperoxide group . to assess the synthetic utility of the engineered 
 p450 catalysts developed here , large - scale reactions involving up 
 to 0.4 g of art
were carried out for 12 h using iv - h4 ( 0.19 mol % ) , 
 ii - h10 ( 0.25 mol % ) , and x - f11 ( 0.28 mol % ) in buffer ( 50 mm kpi , 
 ph 8.0 ) in the presence of a cost - effective nadph regeneration system 
 consisting of a thermostable phosphite dehydrogenase and sodium phosphite as sacrificial reductant . from these 
 reactions , 0.26 to 0.41
g of the three desired products ( 7(s)- , 7(r)- , and 6a - hydroxy - art , respectively ) 
 could be isolated in over 90% yields .
interestingly , both iv - h4 and 
 ii - h10 were found to support higher total turnovers under these settings 
 ( 485 and 350 ttn , respectively ) , likely due to more favorable oxygen 
 transfer conditions .
importantly , access to preparative amounts 
 of 3 and 4 could now allow for the direct 
 modification of two metabolically labile sites in artemisinin and 
 derivatives thereof via stereoconservative hydroxyl group functionalizations ( e.g. , 
 alkylation or acylation ) .
at the same time , the complementary stereoselectivity 
 of iv - h4 for c7 hydroxylation can be leveraged upon to chemically 
 protect , in a two - step sequence , the pro - r c  h 
 bond in c7 via deoxofluorination , a transformation that typically 
 proceeds through inversion of configuration at secondary carbon centers . to illustrate this point , the enzymatically 
 produced 7(s)-hydroxy - artemisinin 2
was 
 reacted with dast to afford enantiopure 7(r)-fluoro - artemisinin 5 in 82% yield ( scheme 1 ) .
5 was then converted in two steps into 6 and 7 , which correspond , respectively , to the clinical antimalarial drugs 
 artemether and artesunate bearing a c  h to c  f substitution , 
 with the ideal configuration , at the major site of metabolic attack 
 of these drugs by human hepatic p450s .
altogether , these results demonstrated the viability 
 of these p450 catalysts for oxyfunctionalization of art at preparative 
 scales as well as their utility toward enabling relevant , late - stage 
 elaborations of art scaffold via p450-mediated c  h functionalization .
in summary , we have demonstrated how a 
 systematic approach based 
 on first - sphere active site mutagenesis , high - throughput p450 fingerprinting , 
 and fingerprint - based p450 reactivity predictions can provide an efficient 
 solution to the problem of developing p450 catalysts with fine - tuned 
 site - selectivity for the oxidative activation of multiple , isolated 
 sp c  h bonds in a complex molecule . by guiding 
 and
focusing our search for the desired catalysts across a large engineered 
 p450 library , this method allowed us to accomplish our goal of refining 
 the site - selectivity of a p450 enzyme in a time- and cost - effective 
 manner . to our knowledge , the complete inversion in stereoselectivity 
 achieved with ii - h10 ( 100% ee ( r ) from 78% ee ( s ) in fl#62 ) , the absolute regioselectivity and stereocomplementarity 
 obtained with the iv - h4/ii - h10 pair , and the regioselectivity shift 
 achieved with x - e12 to afford the selective hydroxylation of a primary , 
 nonallylic / nonbenzylic c  h site ( 7%  94% ) remain unprecedented 
 in previous p450 engineering efforts .
it is also 
 worth noting that the overall process required the empirical testing 
 of only 75 p450 variants via laborious hplc - based methods , thereby 
 reducing by orders of magnitude the screening efforts currently associated 
 with the search for selective p450 catalysts . from a methodological standpoint , another important aspect is that 
 the tier-2 component of this approach is independent of the nature 
 of the target substrate whereas the tier-3 method could be readily 
 extended to any probe - unrelated compound of interest , making this 
 strategy potentially very general in terms of substrate scope . from 
 the characterization of the evolved artemisinin - hydroxylating variants , 
 intriguing trends
have emerged with respect to the active site positions 
 and mutations responsible for steering the selectivity of these enzymes 
 toward each one of the three , spatially close sp c  h 
 bonds in c7 and c6a of artemisinin ( figure 1b ) .
these findings call for future studies to explore in more detail 
 the molecular mechanisms that permit such a subtle control on the 
 c  h oxidation reaction catalyzed by this group of related variants . 

finally , the p450-based oxidation catalysts developed in this work 
 disclose now the opportunity to selectively functionalize relevant 
 aliphatic positions within the artemisinin scaffold that have so far 
 remained inaccessible to chemical methods / catalysts . as demonstrated with the preparation 
 of 7(r)-fluoroartemether and 7(r)-fluoroartesunate , the suitability of this set of engineered p450s 
 for late - stage c  h functionalization of artemisinin at practically 
 useful scales is expected to render them of particularly high value 
 toward the synthesis and investigation of new classes of c7- and c6a - modified 
 artemisinin - based antimalarial agents .
multiple 
 site - saturation ( nnk ) mutagenesis libraries were prepared using pcwori_fl#62 
 as template , primers bamhi_2_fwd ( 5-ggaaacaggatccatcgatgc-3 ) 
 and saci_2_rev ( 5-aatatcgagctcgtagtttgtatgatc-3 ) 
 as megaprimers , and the oligonucleotides of table s1 in the supporting information as mutagenizing primers . 

construction of libraries 78/87 , 78/81/87 , 78/87/181 , 78/87/184 , 78/81/82/87 , 
 and 81/82/87/184 was described earlier .
additional libraries ( 78/81/82/87/181/184 and 74/81/82/87/181/184 ) 
 were constructed in a similar manner by pcr overlap extension mutagenesis . 

the soe products ( 1.5 kbp ) were digested with bamhi and saci restriction enzymes and ligated to bamhi / saci double - digested pcwori_fl#62 
 vector .
the ligation mixtures were transformed in chemically competent 
 dh5 cells and plated on lb agar plates containing ampicillin 
 ( 100 mg l ) followed by overnight incubation at 
 37 c .
96-deep - well plates containing 400 l of lb medium ( 100 mg 
 of ampicillin l ) per well were inoculated with 
 single colonies from the p450 libraries and shaken overnight at 37 
 c and 200 rpm . a row ( 8 wells ) in each plate was inoculated 
 with dh cells expressing fl#62 ( or ii - e2 in the case of ii - e2-based 
 libraries ) as a reference .
after overnight growth , the lb plates were 
 used to inoculate a second set of 96-deep - well plates containing 900 
 l of terrific broth ( tb ) medium ( 100 mg of ampicillin l ) . at od600 = 1.0
, the tb cultures were 
 induced with 0.25 mm -d-1-thiogalactopyranoside ( iptg ) 
 and 0.3 mm -aminolevulinic acid ( ala ) .
after incubation at 
 30 c and 200 rpm for 18 h , the plates were centrifuged at 3500 
 rpm and the pellets were separated from the supernatant and frozen 
 at 80 c .
cell lysates were prepared by adding 400 l 
 of lysis solution ( 4 u of deoxyribonuclease i , 0.8 mg / ml lysozyme , 
 10 mm mgcl2 , 50 mm phosphate buffer , ph 7.5 ) to each well . 

after incubation at 37 c for 70 min , clarified lysates for the 
 screening were obtained by centrifuging the plates at 4,000 rpm .
p450 
 demethylation activity on probe p1p5 ( figure s2 in the supporting information ) was measured in parallel reactions with the aid of a beckman coulter 
 multimek 96 automated pipettor and a tecan infinity plate reader . 

reactions were carried out by mixing 50 l of clarified cell 
 lysate with 150 l of 50 mm phosphate buffer ( ph 7.5 ) containing 
 the probe ( final concentration : 1 mm ) and a phosphite dehydrogenase 
 ( ptdh)-based cofactor regeneration system ( final concentrations : 1.8 
 m ptdh , 50 mm sodium phosphite , 150 m nadp ) .
after incubation for 1 h at room temperature , to each well was 
 added 50 l of 2 m naoh containing 150 mm purpald following 
 by reading of the absorbance at 550 nm with the plate reader .
the 
 measured demethylation activity of each member of the library on the 
 probes p1p5 was then normalized 
 to the activity of the parent enzyme from the same plate .
the p450 enzymes 
 were expressed from pcwori - based vectors and purified by ion - exchange 
 chromatography as described previously .
p450 concentration was determined from co binding difference spectra 
 ( 450500 = 91,000 m cm ) .
the vector encoding for the thermostable phosphite 
 dehydrogenase ( ptdh ) was kindly provided by the zhao group .
ptdh was expressed and purified via ni - affinity 
 chromatography according to the published procedure .
a 200 ml scale reaction was set up adding fl#62 
 ( 1 m ) to a buffered solution ( 50 mm potassium phosphate , ph 
 8.0 ) containing 32 mg of artemisinin ( 0.53 mm ) , ptdh ( 2 m ) , 
 nadp ( 150 m ) , and sodium phosphite ( 50 mm ) .
the 
 reaction mixture was stirred overnight at room temperature and then 
 extracted with dichloromethane ( 3  30 ml ) .
the collected organic 
 layers were dried with na2so4 , concentrated 
 under reduced pressure , and purified by flash chromatography ( dichloromethane / hexanes / ethyl 
 acetate : 1/1/1 ) to afford 2 ( 18 mg ) , 3 ( 2 
 mg ) , 4 ( 1 mg ) , and recovered artemisinin ( 10 mg ) .
h nmr ( 500 mhz , cdcl3 ) :  = 1.18 ( 3 
 h , d , j = 7.2 hz ) , 1.23 ( 1 h , m ) , 1.27 ( 3 h , d , j = 7.2 hz ) , 1.44 ( 1 h , m ) , 1.50 ( 3 h , s ) , 1.57 ( 2 h , m ) , 
 1.79 ( oh , br ) , 1.95 ( 1 h , m ) , 2.022.20 ( 3 h , m ) , 2.48 ( 1 h , 
 m ) , 3.32 ( 1 h , ddd , j = 10.6 hz , j = 10.6 hz , j = 4.5 hz ) , 3.42 ( 1 h , m ) , 5.98 ( 1 
 h , s ) . c nmr ( 100 mhz , cdcl3 ) :  = 12.6 , 
 15.5 , 24.8 , 25.1 , 32.1 , 32.6 , 35.8 , 42.3 , 44.5 , 47.9 , 73.5 , 78.8 , 
 93.5 , 105.5 , 171.6 . ms ( esi ) calcd for c15h23o6 [ m + h]m / z : 299.15 , found 299.47 .
the 7(s ) configuration of 2 was confirmed based on the j coupling constant between the 7(h ) proton and 6(h ) and 8(h ) protons 
 and the known chair conformation of the cyclohexyl ring in artemisinin .
the observed coupling constants ( j = 10.6 hz ; j = 10.6 hz ; j = 4.5 hz ) are consistent with 7(h ) being in trans to 6(h ) and thus with c7 atom having s configuration .
h nmr ( 500 mhz , cdcl3 ) :  = 1.12 ( 3 
 h , d , j = 6.7 hz ) , 1.23 ( 3 h , d , j = 7.3 hz ) , 1.34 ( 1 h , dd , j = 13.7 hz , j = 2.1 hz ) , 1.461.51 ( 4 h , m ) , 1.56 ( 1 h , m ) , 1.81 
 ( oh , br ) , 1.95 ( 2 h , m ) , 2.10 ( 2 h , m ) , 2.402.53 ( 2 h , m ) , 
 3.45 ( 1 h , m ) , 3 .
89 ( 1 h , ddd , j = 2.8 hz , j = 2.8 hz , j = 2.5 hz ) , 5.90 ( 1 h , s ) .
c nmr ( 100 mhz , cdcl3 ) :  = 12.5 , 15.8 , 
 24.6 , 25.2 , 30.7 , 32.4 , 36.0 , 37.6 , 41.3 , 43.4 , 69.1 , 79.3 , 93.4 , 
 105.5 , 172.4 . ms ( esi ) calcd for c15h23o6 [ m + h]m / z : 299.15 , found 299.41 .
the 7(r ) configuration of 3 was confirmed based on the j coupling constant between the 7(h ) proton and 6(h ) and 8(h ) protons 
 and the known chair conformation of the cyclohexyl ring in artemisinin . the observed coupling constants ( j = 2.8 hz ; j = 2.8 hz ; j = 2.5 hz ) are consistent with 7(h ) being in cis to 6(h ) and thus with c7 atom having r configuration .
h nmr ( 500 mhz , cdcl3 ) :  = 1.15 ( 1 h , m ) , 1.26 
 ( 3 h , d , j = 6.6 hz ) , 1.361.49 ( 4 h , m ) , 
 1 .
1.541.61 ( 3 h , m ) , 1.65 ( oh , br ) , 1.761.85 ( 2 h , 
 m ) , 1.952.14 ( 3 h , m ) , 2.50 ( 1 h , ddd , j = 
 17.3 hz , j = 13.3 hz , j = 3.9 hz ) , 
 3.45 ( 1 h , m ) , 3 .
68 ( 1 h , dd , j = 10.6 hz , j = 5.6 hz ) , 3.79 ( 1 h , dd , j = 10.6 hz , j = 3.0 hz ) , 5.93 ( 1 h , s ) .
c nmr ( 100 mhz , 
 cdcl3 ) :  = 12.6 , 23.0 , 24.5 , 25.2 , 27.9 , 32.9 , 35.9 , 
 44.2 , 44.5 , 44.8 , 64.1 , 79.5 , 93.6 , 105.4 , 172.0 . ms ( esi ) calcd for 
 c15h23o6 [ m + h]m / z : 299.15 , found 299.39 .
analytical - scale reactions ( 1 ml ) were 
 carried using 0.51 m p450 , 0.5 mm artemisinin , 2 m 
 ptdh , 100 m nadp , and 50 mm sodium phosphite in 
 potassium phosphate buffer ( 50 mm , ph 8.0 ) .
the p450 variants described 
 in table 1 were characterized in purified form , 
 while those described in tables s2s4 in the supporting information were characterized directly from cell 
 lysates .
the hydroxylation products were functionalized with benzoyl 
 chloride followed by hplc analysis as described in more detail in 
 the supporting information .
ttn values 
 were calculated based on the total amount of hydroxylated products 
 in the reactions as quantified based on the calibration curves generated 
 using purified 24 .
the enzyme regio- 
 and stereoselectivity was determined based on the peak areas corresponding 
 to the hydroxylation products 2 , 3 , and 4 .
mean and standard deviation values reported for p450 variants 
 in table 1 were calculated from experiments 
 performed at least in triplicate .
initial product formation rates were measured from 1 ml scale reactions 
 containing 250 m artemisinin , 0.10.5 m purified 
 p450 , and 200 m nadph in potassium phosphate buffer ( 50 mm , 
 ph 8.0 ) at room temperature .
after 30 s , the samples were extracted 
 with dichloromethane and subjected to benzoyl chloride derivatization 
 followed by hplc analysis as described above
. cofactor oxidation rate 
 in the presence of artemisinin was measured by monitoring nadph depletion 
 at 340 nm (  = 6.22 mm cm ) using 0.1 m purified p450 , 250 m artemisinin , and 
 200 m nadph .
coupling efficiency was calculated from the ratio 
 between the initial product formation rate and the initial nadph oxidation 
 rate . reported mean and standard deviation values were calculated 
 from experiments performed at least in triplicate .
binding experiments were performed using 3 m 
 purified p450 in potassium phosphate buffer ( 50 mm , ph 8.0 ) by titrating 
 increasing amounts of artemisinin ( 5 m to 1 mm ) from an ethanol 
 stock solution ( 50 mm ) . at each concentration ,
a difference spectrum 
 from 350 to 500 nm was recorded and binding curves were generated 
 by plotting the change in absorbance at 390 and 420 nm corresponding 
 to the high - spin and low - spin state of the enzyme , respectively , against 
 the artemisinin concentration .
kd values 
 were calculated using sigma plot via nonlinear fitting of the experimental 
 binding curves to an equation describing a standard 1:1 binding interaction . 
 reported mean and standard deviation values were calculated from experiments 
 performed at least in triplicate .
after high - throughput fingerprinting , p450 variants displaying a 
 unique fingerprint were identified by selecting the parent - normalized 
 fingerprints according to the following criteria : ( a ) > 10% of parent 
 activity on at least one probe ; and ( b ) larger than 20% variation 
 in activity on at least one probe compared to the parent or any other 
 member of the library .
the p450 variants with a unique fingerprint 
 were transferred from the library plates to new 96-well plates containing 
 p450bm3(f87a ) as reference enzyme .
the p450 variants of 
 this collection were then fingerprinted in triplicate using the probe 
 activity of p450bm3(f87a ) for fingerprint normalization . 

the standard error in the values of the fingerprints provided in figure 3b is within 10% . to generate the fingerprint - based 
 model predictive of artemisinin reactivity , 19 randomly chosen p450 
 variants from the collection were characterized for artemisinin hydroxylation 
 activity measuring their ttn values ( table s2 in the supporting information ) .
after normalization against the activity 
 of the parent enzyme ( ttn(p450 variant)/ttn(fl#62 ) ) , the normalized 
 activities were then correlated with the corresponding fingerprints 
 using multiple linear regression ( mlr ) analysis using the equation y = b0 + b1x1 + b2x2 + b4x4 + b4x4 + b5x5 , where y corresponds to relative artemisinin 
 reactivity , x1 to x5 correspond to the five fingerprint components ( activity on 
 probes 1 to 5 , respectively ) , and b1 to b5 correspond 
 to the regression coefficients for the five independent variables x1 to x5 .
this process 
 yielded the plot of calculated versus experimental artemisinin activity 
 presented in figure 2a , with the calculated 
 regression coefficients being b1 = 0.0109 , b2 = 0.0016 , b3 = 
 0.0551 , b4 = 0.0338 , and b5 = 0.01271 .
the low rmsd value ( 0.0859 ) indicated 
 an excellent fit between the calculated and experimental values .
this 
 model was then used to rank the p450 variants of the collection according 
 to their predicted artemisinin reactivity .
the same method was applied 
 for generating a fingerprint - based predictive model for ranking the 
 second - generation variants derived from ii - e2 . in this case , the training 
 set used for calculations was expanded to include ii - e2 ( training 
 set b , table s2 in the supporting information ) as this variant exhibited the highest selectivity toward 6a - hydroxylation . 

mlr analysis of the correlation between fingerprints and artemisinin 
 hydroxylation activities across this training data set yielded a best - fitting 
 model with regression coefficients b1 = 
 0.0037 , b2 = 0.0278 , b3 = 0.0839 , b4 = 0.1158 , and b5 = 0.1202 and a rmsd 
 of 0.1789 ( figure s2 in the supporting information ) .
based on this model , the 50 ii - e2 derived variants were scored 
 according to their predicted artemisinin reactivity ( figure s3 in 
 the supporting information ) and subsequently 
 prioritized according to their score . to prepare 2 , purified p450 variant iv - h4 ( final concentration : 1 m ; 
 0.19 mol % )
was dissolved in 1 l of 50 mm phosphate buffer ( ph 8.0 ) 
 in the presence of artemisinin ( 150 mg , final concentration : 0.53 
 mm ) , ptdh ( 2 m ) , nadp ( 150 m ) , and sodium 
 phosphite ( 50 mm ) .
the collected organic layers were dried with na2so4 , concentrated , and purified by flash chromatography 
 ( dichloromethane / hexanes / ethyl acetate : 1/1/1.5 ) to afford 2 ( 138 mg , 92% ) .
the same procedure was repeated two more times to 
 yield a total of 410 mg of 2 . to prepare 3 , purified p450 variant ii - h10 ( final concentration 1 m ; 0.25 
 mol % )
was dissolved in 1 l of 50 mm phosphate buffer ( ph 8.0 ) in 
 the presence of artemisinin ( 110 mg , final concentration : 0.39 mm ) , 
 ptdh ( 2 m ) , nadp ( 150 m ) , and sodium phosphite 
 ( 50 mm ) .
the collected organic layers were dried with na2so4 , concentrated , and purified by flash chromatography ( dichloromethane / hexanes / ethyl 
 acetate : 1/1/1.5 ) to afford 2 ( 100 mg , 91% , ttn : 350 ) . 

the same procedure was repeated two more times to yield a total of 
 305 mg of 2 . to prepare 4 ,
purified p450 
 variant x - f11 ( final concentration : 1 m ; 0.28 mol % ) was dissolved 
 in 1 l of 50 mm phosphate buffer ( ph 8.0 ) in the presence of artemisinin 
 ( 100 mg , final concentration : 0.35 mm ) , ptdh ( 2 m ) , nadp ( 150 m ) , and sodium phosphite ( 50 mm ) .
the collected 
 organic layers were dried with na2so4 , concentrated , 
 and purified by flash chromatography ( dichloromethane / hexanes / ethyl 
 acetate : 1/1/2 ) to afford 4 ( 90 mg , 90% ) .
the same procedure 
 was repeated two more times to yield a total of 260 mg of 4 . to a solution of 3 ( 50 mg , 0.17 mmol ) 
 in anhydrous ch2cl2 ( 5 ml ) was added ( diethylamino)sulfur 
 trifluoride ( 55 mg , 0.34 mmol ) dropwise at 78 c . the 
 reaction mixture was warmed up to 0 c and stirred until the 
 starting material disappeared as determined by tlc .
the reaction mixture 
 was quenched with 60 l of triethylamine and extracted with 
 ch2cl2 ( 2  5 ml ) .
the collected organic 
 layers were dried with na2so4 and concentrated , 
 and the residue was purified by flash chromatography ( hexanes / ethyl 
 acetate : 3/1 ) to afford 5 ( 42 mg , 82% ) .
h 
 nmr ( 500 mhz , cdcl3 ) :  = 1.20 ( 3 h , d , j = 7.0 hz ) , 1.27 ( 3 h , d , j = 7.4 hz ) , 1.281.50 
 ( 6 h , m ) , 1.85 ( 1 h , m ) , 1.94 ( 1 h , m ) , 2.03 ( 1 h , m ) , 2.292.36 
 ( 2 h , m ) , 2.53 ( 1 h , m ) , 3.49 ( 1 h , m ) , 4.67 ( 1 h , m ) , 5.87 ( 1 h , 
 s ) .
c nmr ( 100 mhz , cdcl3 ) :  = 10.12 , 
 12.4 , 24.7 , 25.2 , 28.7 ( d , j = 21.8 hz ) , 32.3 , 35.9 , 
 37.9 , 40.7 ( d , j = 20.6 hz ) , 43.9 , 78.7 , 90.6 ( d , j = 166.0 hz ) , 93.0 , 105.5 , 171.9 .
f nmr ( 376.5 
 mhz , cdcl3 ) :  = 133.9 . ms ( esi ) calcd for 
 c15h22fo5 [ m + h]m / z : 301.15 , found 301.42 . to a solution of 5 ( 20 mg , 0.07 mmol ) in 
 2 ml of anhydrous methanol was added sodium borohydride ( 8 mg , 0.21 
 mmol ) at 0 c .
the reaction mixture was stirred at 0 c 
 until the starting material disappeared as determined by tlc .
the 
 reaction mixture was quenched with 5 ml of ice - cold water , and methanol 
 was removed by evaporation in vacuo .
the aqueous layer was then extracted 
 with ch2cl2 ( 3  5 ml ) , and the collected 
 organic layers were dried with na2so4 and concentrated . 
 the crude product was dissolved in 2 ml of anhydrous meoh followed 
 by the addition of sulfuric acid ( 11 l , 0.21 mmol ) .
the mixture 
 was stirred overnight at room temperature and then quenched with 50 
 l of triethylamine .
the mixture was concentrated under reduced 
 pressure and purified by flash chromatography ( hexanes / ethyl acetate : 
 3/1 ) to afford 6 ( 11 mg , 55% ) .
h nmr ( 500 
 mhz , cdcl3 ) :  = 0.97 ( 3 h , d , j = 7.5 hz ) , 1.15 ( 3 h , d , j = 6.8 hz ) , 1.50 ( 5 h , 
 m ) , 1.821.92 ( 2 h , m ) , 1.98 ( 1 h , m ) , 2.052.20 ( 3 
 h , m ) , 2.47 ( 1 h , m ) , 2.72 ( 1 h , m ) , 3.48 ( 3 h , s ) , 4.57 ( 1 h , d , j = 49.5 hz ) 4.76 ( 1 h , d , j = 3.3 hz ) , 
 5.43 ( 1 h , s ) .
c nmr ( 100 mhz , cdcl3 ) :  
 = 12.7 , 16.1 , 24.6 , 26.1 , 29.7 ( d , j = 20.7 hz ) , 
 36.4 , 37.3 , 40.6 ( d , j = 19.9 hz ) , 45.1 , 56.1 , 80.2 , 
 86.9 , 91.9 ( d , j = 172.5 hz ) , 92.3 , 103.6 , 104.2 .
ms ( esi ) calcd for c16h25fnao5 [ m 
 + na]m / z : 339.16 , found 
 339.25 . to a solution of 5
( 20 mg , 0.07 mmol ) in 
 2 ml of anhydrous methanol was added sodium borohydride ( 8 mg , 0.21 
 mmol ) at 0 c .
the reaction mixture was stirred until the starting 
 material disappeared as determined by tlc , followed by quenching with 
 5 ml of ice - cold water .
the mixture was evaporated in vacuo to remove 
 methanol , and the remaining aqueous layer was extracted with ch2cl2 ( 3  5 ml ) .
the 
 crude product was dissolved in 2 ml of 1,4-dioxane followed by the 
 addition of succinic anhydride ( 8 mg , 0.077 mmol ) and triethylamine 
 ( 50 l , 0.35 mmol ) .
the mixture was then concentrated 
 under reduced pressure and purified by flash chromatography 
 ( dichloromethane / methanol : 20/1 ) to afford 7 ( 24 mg , 
 90% ) .
h nmr ( 500 mhz , cdcl3 ) :  = 0.90 
 ( 3 h , d , j = 7.1 hz ) , 1.051.11 ( 4 h , m),1.321.60 
 ( 6 h , m ) , 1.90 ( 2 h , m ) , 2.102.22 ( 3 h , m ) , 2.47 ( 1 h , m ) , 
 2.662.79 ( 4 h , m ) , 4.65 ( 1 h , d , j = 50.1 
 hz ) , 5.46 ( 1 h , s ) , 5.82 ( 1 h , d , j = 10.1 hz ) .
c nmr ( 100 mhz , cdcl3 ) :  = 11.7 , 15.9 , 
 24.4 , 25.9 , 27.2 ( d , j = 20.6 hz ) , 28.5 , 28.7 , 31.0 , 
 36.1 , 38.3 , 40.1 ( d , j = 19.2 hz ) , 44.3 , 52.1 , 79.2 , 
 91.5 ( d , j = 146.5 hz ) , 93.2 , 104.6 , 171.1 , 172.6 . f nmr ( 376.5 mhz , cdcl3 ) :  = 135.2 . 
 ms ( esi ) calcd for c19h27fnao8 [ m 
 + na]m / z : 402.17 , found 
 402.33 .
 the selective oxyfunctionalization of isolated sp3 c  h 
 bonds in complex molecules represents a formidable challenge in organic 
 chemistry . here , we describe a rational , systematic strategy to expedite 
 the development of p450 oxidation catalysts with refined regio- and 
 stereoselectivity for the hydroxylation of remote , unactivated c  
 h 
 sites in a complex scaffold . using artemisinin as model substrate 
 , 
 we demonstrate how a three - tier strategy involving first - sphere active 
 site mutagenesis , high - throughput p450 fingerprinting , and fingerprint - driven 
 p450 reactivity predictions enabled the rapid evolution of three efficient 
 biocatalysts for the selective hydroxylation of a primary and a secondary 
 c  h site ( with both s and r stereoselectivity ) in a relevant yet previously inaccessible region 
 of this complex natural product . 
 the evolved p450 variants could be 
 applied to provide direct access to the desired hydroxylated derivatives 
 at preparative scales ( 0.4 g ) and in high isolated yields ( > 90% ) , 
 thereby enabling further elaboration of this molecule . as an example 
 , 
 enantiopure c7-fluorinated derivatives of the clinical antimalarial 
 drugs artesunate and artemether , in which a major metabolically sensitive 
 site is protected by means of a c  h to c  f substitution , 
 were afforded via p450-mediated chemoenzymatic synthesis . 