document: the oh functionality is commonly activated by attachment of a good leaving group , e.g. , by conversion into an ester derivative of a strong acid ( tosylate , mesylate , triflate ) , usually employing mitsunobu or appel conditions.1 this classic methodology suffers from poor atom economy and creates large amounts of waste , thus rendering these processes inefficient and unattractive , although remarkable progress towards catalytic processes was made in the past decade.2 in nature , activation of hydroxy groups often proceeds through enzymatic phosphorylation by kinases using activated phosphate ( adenosine triphosphate , atp ) or sulfation catalyzed by mammalian sulfotransferases in conjunction with the respective cofactor , i.e. , 3phosphoadenosine5phosphosulfate ( paps).3 nonspecific bacterial arylsulfotransferases are also able to sulfate hydroxy groups by using organic sulfate donors.4 the requirement for molar equivalents of these cofactors in synthetic applications is problematic from a scaleup perspective . 
   although enzymatic atp recycling is feasible with reasonable efficiency ( up to several hundred ttns),4a,5 paps can not be recycled efficiently so far.4 most importantly , atpdependent kinases and papsdependent sulfotransferases are typically involved in biosynthetic pathways and signal messaging and thus are often highly substratespecific and hence do not allow broad synthetic applications . 
   an efficient alternative to kinases is the use of atpindependent phosphatases using cheap inorganic polyphosphates ( pyrophosphate , ppi ; triphosphate , pppi ; polyphosphoric acid , polyp ) as activated phosphate donor ( pdonor ) , which circumvents the need for cofactor recycling.6 additionally , due to their occurrence in biodegradation pathways , phosphatases usually display a broad substrate spectrum , and inorganic phosphate formed as byproduct is environmentally benign . 
   a major drawback resides in the reversibility of phosphate transfer , causing the undesired hydrolysis of the newly formed phosphate ester , which requires a kinetically tightly controlled system ( figure  1 ) . 
   mechanism of ( a ) competing hydrolysis and ( b ) transphosphorylation involving a covalent phosphoenzyme intermediate catalyzed by phonse.26 
    the use of phosphatases for synthetic applications dates back to the late 1940s , when axelrod and appleyard achieved transphosphorylation using phosphataseenriched extracts and pnitrophenyl phosphate ( pnpp ) or phenolphthalein phosphate as donor.7 later , nordlie identified glucosepyrophosphotransferase activity of e. coli alkaline phosphatase;8 however , the method was applied on a preparativescale only decades later.9 asano et al . 
   applied acid phosphatases from enteric bacteria to the phosphorylation of nucleosides and designed enzyme mutants with reduced hydrolytic activity.10 acid phosphatases from shigella flexneri ( phonsf ) and salmonella enterica ser . 
   typhimurium lt2 ( phonse ) were found to have a broad substrate spectrum11 and were applied in combination with aldolases to synthesize natural and unnatural sugar molecules on a gramscale.12 
    in drug discovery , phosphorylation is a common prodrug strategy to enhance the accessibility of drug candidates within the body by enhancing their polarity and solubility.13 in some cases , the phosphorylated form may be the active species.14 in synthetic chemistry , phosphate di and triesters are widely applied as brnsted acid catalysts for various asymmetric transformations15 and as leaving groups in transitionmetalcatalyzed coupling reactions16 as well as in organolithium17 and alkene synthesis.18 in contrast , phosphate monoesters have attracted so far little attention as tools for functionalgroup modification in classic chemical processes , most likely as a consequence of their insolubility in organic solvents resulting from their negative charge . 
   current chemical routes to phosphate monoesters often proceed via the corresponding phosphate di or triesters and require harsh reagents and conditions leading to hardly separable product mixtures . in this study , we aimed to evaluate a set of phosphatases as potential candidates for synthetic applications . 
   various techniques were investigated to enhance product titers of phosphorylated alcohols and to minimize the thermodynamically favored hydrolytic reaction . 
   enzyme candidates were selected from the family of acid phosphatases , which have been shown to transfer efficiently a phosphate moiety from a highenergy inorganic di / tri / polyphosphate onto various hydroxylated compounds . 
   nonspecific acid phosphatase phonse from salmonella typhimurium and acid phosphatase phonsf from shigella flexneri have been reported to convert various primary and secondary alcohols and sugars11 and were implemented in cascade reactions involving glucose6phosphate19 and dihydroxyacetone phosphate as intermediates.12 acid phosphatase phytase catalyzes the hydrolysis of phytate , a phosphorylated natural cyclitol by liberating inorganic phosphate and is therefore commonly used as animal feedstock additive.20 acid phosphatase aphast from salmonella typhimurium lt221 was selected due to its phosphotransferase activity on alcohols using pnpp as donor.22 piacp from prevotella intermedia 
   23 was selected based on high sequence similarity with robust phocmm developed for nucleoside phosphorylation.24 all enzymes except phytase were overexpressed in e. coli and subsequently purified to homogeneity by using histag or streptag techniques ( figure s1 ) . 
   a common mechanistic feature of phosphatetransferring phosphatases is the formation of a covalent phosphoenzyme intermediate , which undergoes either nucleophilic attack by water ( hydrolysis ) or accepts a suitable nucleophile ( transphosphorylation ) ( figure  1 ) . to this end , most enzymes use histidine as catalytic residue ( phonsf , phonse , piacp and phytase ) except aphast which uses aspartic acid.25 due to the competition between hydrolysis and transphosphorylation in aqueous medium , high substrate loads are required to achieve high product titers , while kinetic control is crucial to keep the phosphorylation / hydrolysis ratio as high as possible . in order to verify the activity of all recombinant proteins , a spectrophotometric assay based on the hydrolysis of pnpp was used . 
   a range of alcohols was investigated , including primary and secondary mono and dihydroxy compounds , as well as cyclic substrates ( table  1 ) . 
   although phytase has been successfully employed in a cascade reaction to convert glycerol into glycerol1phosphate in a nonstereoselective fashion,27 under our assay conditions , the enzyme showed no transphosphorylation activity at ph = 2.5 and 4.2 on any substrate . 
   strong hydrolytic activity on inorganic phosphate donors was still observed , confirming active protein ( data not shown ) . 
   aphast also contains mg ( in addition to the catalytically active nucleophile aspartate21 ) , whose activity is affected by inorganic polyphosphates by metal chelation . 
   although the addition of 10 mm mgcl2 could partly restore the hydrolytic activity on pnpp in the presence of 250 mm ppi ( figure s2 ) , only traces of product and marginal hydrolytic activity were obtained in the transphosphorylation of 1,4butanediol ( 500 mm ) by using 100 mm ppi as donor at ph = 4.2 and 5.5 , independent of the mgcl2 concentration ( 10 , 50 , 100 mm ) ( data not shown ) . 
   this suggests that inorganic oligophosphates ( as opposed to pnpp ) are not ideal phosphate donors for metalcontaining phosphatases , such as aphast . on the contrary , phonsf , phonse and piacp 
   all proved to be efficient catalysts in the phosphorylation of various alcohols ( table  1 ) . 
   substrate scope of phonsf , phonse and piacp in transphosphorylation.[a ] , [ b ] , [ c ] , [ d ] , [ e ] , [ f ] , [ g ] 
    reaction conditions : ph = 4.2 , 50 g  ml enzyme , 1 ml of volume , 1  % dmso as internal standard , 30 c , 600 rpm shaking . 
   product levels were determined by consumption of substrate using hplc with ri detector ( control experiments used as reference ) . 
   phosphorylation of primdiols proceeded to various degrees depending on the chain length : while shortchain ethylene glycol ( 1 ) was selectively monophosphorylated , 1,4butanediol ( 2 ) and the unsaturated ( z ) analog 4 yielded the corresponding mono / bisphosphorylated products in a ratio of 90:10 . extending the chain length even further ( 1,6hexanediol , 3 ) gave 85:15 of mono / diphosphate as determined by lcri chromatography ( see supporting information ) . 
   it is obvious , that the presence of a ( negatively charged ) phosphate group in the proximity of the reacting hydroxy moiety determines whether an ,diol undergoes bisphosphorylation : while shortchain ethylene glycol is exclusively monophosphorylated , longchain 1,6hexanediol yields the bisphosphate in up to 15  % . 
   haloalcohol 5 was also converted : phonse tolerated higher substrate concentrations than phonsf and piacp ( 500 mm vs. 300 mm ) resulting in the formation of 113 mm product in 60 min . to probe the chemoselectivity of the enzymes for o vs. nnucleophiles , 6amino1hexanol ( 6 ) 
   the three enzymes exclusively furnished the ophosphate monoester at very similar product levels ( ca . 
   this was proven by p nmr spectroscopy resulting in a chemical shift typical for signals of phosphorylated primary hydroxy groups ( figure s5).28 in line with this observation , 1,6diaminohexane ( 12 ) was not converted , only pdonor hydrolysis was observed ( data not shown ) , proving that these enzymes do not accept amines in place of alcohols . 
   sterically demanding secondary alcohol 7 was transformed only with phonse , which is in line with the previously observed preference of this enzyme for secalcohols.11 similar observations were made with 1,2propanediol ( 8) , which was exclusively phosphorylated at the primalcohol by phonsf and piacp , while phonse showed reduced selectivity ( ca . 
   90:10 prim / secphosphate , determined by p nmr spectroscopy ; figure s6 ) . in order to elucidate the enantioselectivity of phosphatases , 
   the two enantiomers of 8 were tested separately . in case of phonsf and piacp , similar activities on both enantiomers 
   were observed indicating nonenantioselective phosphorylation . with phonse , a marginal enantiopreference for ( r)8 was detected ( figure s7 ) . 
   to further investigate the enantioselectivity of phonse , both enantiomers of 2butanol ( 9 ) were subjected to screening conditions ; however , no significant difference in the reaction course was observed . 
   furthermore , racemic cyclic transdiol 10 was tested , and beside a moderate amount of monophosphorylated product , no stereoselectivity could be observed ( as determined by measurement of the ee value of remaining 10 by gc after derivatization of both oh functionalities ; see supporting information ) . 
   substrates containing n or snucleophiles or charged groups were not converted ( figure  2 ) , while ppi was still hydrolyzed indicating that the enzymes were still active ( data not shown ) . 
   the absolute chemoselectivity of ophosphorylation vs. n and snucleophiles was corroborated by the nonacceptance of substrates 11 and 12 . 
   mevalonolactone ( rac13 ) and charged substrates , like lactic ( rac14 ) and tartaric acid ( 15 ) ( both enantiomers tested separately ) were not converted . 
   although ophosphod , lserine could be stereoselectively hydrolyzed by a mutant of phonse,29 the wildtype enzyme was not active in the phosphorylation of racserine ( 16 ) . 
   substrates not converted by phonsf , phonse , piacp and phytase [ 300 mm substrate concentration except for rac14 ( 200 mm ) and 17 ( 15 mm ) ] . due to the reversibility of the reaction ( figure  1 ) , kinetic control of the reaction is necessary to ensure optimal product titers by avoiding product depletion by ( reverse ) hydrolysis and to ensure optimal product titers . this may be achieved by adjustment of reaction parameters such as ph or selection of oligophosphate donor.30 therefore , 1,4butanediol ( 2 ) was selected as a model substrate , and enzymes were tested with tri and polyphosphate as alternative to ppi ( table  1 and figures s810 ) . 
   the amount of 4hydroxybutyl phosphate formed could be significantly increased by replacing ppi by high amounts of pppi and polyp in the reaction catalyzed by phonsf and piacp , allowing up to 61  % conversion ( 305 mm product ) with respect to the theoretical maximum . 
   the difference in the slope in product disappearance clearly shows that product hydrolysis is decreased in the presence of tri and polyphosphate compared to ppi . 
   in contrast , these donors had a negative influence on the performance of phonse , where a product maximum was obtained with ppi . moreover , hplc monitoring of the reaction indicated incomplete consumption of polyphosphoric acid . 
   this suggests that this enzyme accepts only short chain oligophosphates as reported for calf intestine alkaline phosphatase.9 
    since phonsf generally delivered the highest product titers with primary alcohols as acceptors , the enzyme was selected for further investigations with 1,4butanediol concerning the effect of ph ( range 3.84.8 ) by using different pdonors ( figure  3 ) . 
   on the other hand , pppi and polyp usually yielded higher product concentrations at lower ph , due to slower product hydrolysis . 
   however , the poorly defined oligomeric composition of the polyphosphate donor renders precise analysis as well as product isolation challenging , and polyp was not investigated further . 
   effect of pdonor type and ph on the phosphorylation of 2 ( product formation over time ) . 
   conditions : 500 mm 
   2 , 250 mm ppi or pppi or 100 g  l polyp , 50 g ml phonsf , 1 ml volume , 1  % dmso as internal standard , 30 c , 600 rpm shaking . 
   the results depicted in figure  3 using phonsf suggest that a high number of transferable highenergy phosphate moieties as well as low ph values are crucial to achieve and maintain high product titers . 
   inspired by this observation , reactions were conducted at variuos ph values using high amounts of ppi and pppi overnight ( table  2 ) . 
   it should be noted that within the incubation time of 16.5 h both transphosphorylation and hydrolysis of product and phosphate donor will occur , and the balance between the two processes is determined by the activity of the enzymes and the ph . 
   although identical amounts of transferable phosphate moieties are available from 500 mm ppi and 250 mm pppi , significant differences in product formation were obtained after 16.5 h reaction time . using 500 mm ppi as a donor , 
   product yields of 2 for phonsf and piacp were similar at the three ph values . using ppi , up to 292 mm product ( up to 58  % conversion ) 
   could be obtained accompanied by nearly total consumption of ppi ( > 90  % ) , and the product / pi ratio was ca . 
   1:2 ( the ideal ratio is 1:1 in the absence of product hydrolysis and complete phosphate transfer ) . 
   however , under these conditions phonse furnished only inorganic monophosphate , indicating total hydrolysis of ppi and product . in the presence of 250 mm pppi the picture changed : the transphosporylation activity at ph = 3.5 of phonsf dropped considerably , that of piacp also dropped , but surprisingly considerable phosphorylating activity of phonse was maintained ( 164 mm , 33  % conversion and 54  % pdonor consumption ) . at higher ph values 
   this enzyme was still active , but only hydrolysis was observed , while phonsf and piacp delivered moderate to good amounts of product at full consumption of the pdonor . at 400 mm pppi and ph = 4.2 
   both phonsf and piacp showed the highest phosphorylating activity resulting in up to 393 mm product ( 79  % conversion , product / pi ratio ca . 
   4:5 ) at 75  % pdonor consumption . amount of product and pi formed in acid phosphatase catalyzed phosphorylation of 2 with ppi or pppi as donor , at three ph values with phonsf , piacp and phons . 
   reaction conditions : 500 mm 
   2 , phosphate donor as indicated , 50 g  ml phonsf , ph as indicated , 1 ml volume , 1  % dmso as internal standard , 30 c , 600 rpm shaking , 16.5 h. ideal ( maximum obtainable ) product / pi ratio is 1:1 with ppi and 2:1 with pppi . 
   cons.donor : consumption of pdonor calculated as follows : ( c 
   prod + c 
   pi)/c 
   theoretical max . 
   , higher pppi concentrations translate into higher product concentration and lower pi concentration , indicating more efficient phosphate transfer due to reduced product hydrolysis . from the data it is clear that using these phosphorylating enzymes in synthesis effectively requires a careful study of the donor concentrations , the type of donor , and the ph ; in batch processes also the time of incubation should be carefully chosen . 
   a long incubation time will result in low yields or near complete hydrolysis of the phosphorylated product . 
   consequently , kinetic control of the phosphorylation is very important , and physical separation of the phosphatase by employing a continuous flowreactor has proven to be an efficient tool to maintain high product levels ( by minimizing hydrolysis ) and scaleup the reaction.12c,31 under optimized conditions , the contact time between enzyme and substrate(s ) is controlled by the flow rate to ensure that ( rapid ) phosphorylation is complete , but ( slow ) product hydrolysis can not take place after the product has left the enzymecontaining column reactor . 
   we evaluated several epoxy and aminofunctionalized polymeric beads for the covalent immobilization of phonsf and piacp ( table s3 ) . 
   relizyme ha403/m , an aminofunctionalized resin , resulted in the highest specific activity for both enzymes ( 159 and 215 u  g dry beads for phonsf and piacp , respectively ) . 
   phonsf ( 300 u ) immobilized on relizyme ha ( 300 mg ) was applied to the preparativescale phosphorylation of 2 , yielding 6.86 g ( 22.5 mmol ) of the corresponding barium salt of 4hydroxybutyl phosphate ( 41  % isolated yield , for details see supporting information ) . 
   the enzymatic phosphorylation of primary and secondary alcohols in aqueous medium using cheap inorganic di , tri and polyphosphate as pdonors catalyzed by three acid phosphatases proceeded with absolute chemoselectivity for o vs. n and snucleophiles and with high regioselectivity for prim over sechydroxy groups . 
   the use of mild reaction conditions allowed the formation of phosphate monoesters as sole products , yielding environmentally innocuous inorganic monophosphate and unreacted phosphate donor as byproducts . 
   the thermodynamically favored hydrolysis of phosphate esters could be minimized by controlling various reaction parameters , thus allowing the use of phosphatases in the synthetic direction . 
   scaleup was demonstrated by the synthesis of 4hydroxybutyl phosphate ( 6.86 g , 41  % yield ) using an immobilized enzyme in flow mode . 
   
summary: abstracta set of phosphatases was evaluated for their potential to catalyze the regio and stereoselective phosphorylation of alcohols using a highenergy inorganic phosphate donor , such as di , tri and polyphosphate . 
   parameters such as type and amount of phosphate donor and ph of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester . diols were monophosphorylated with high selectivities . this biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale ( 6.86 g ) in a flow setup with immobilized phosphatase .