the development of 
 site - selective catalysts that are able to deliver 
 different products , derived from a common scaffold , is emerging as 
 an important strategy to the diversification of biologically active 
 scaffolds . significant chemical challenges 
 arise when the scaffold to be modified is a complex natural product , 
 especially in situations where the structure of the natural product 
 contains multiple copies of the same functional group . polyhydroxylated compounds are but one example of molecules 
 of this type ; attempted derivatization of the hydroxyl group array 
 ( for example , through acylations , phosphorylations , sulfonylations , thiocarbonylations , phosphoramiditation , glycosylations , silylations , inter alia ) can lead to mixtures 
 of different monofunctionalized products as well as mixtures of bis- , 
 tris- , or even higher functionalized products . the chromatographic 
 separation of complex mixtures , as well as the precise structural 
 assignment of each component , can be a daunting task , independent 
 of any attempt to describe a biological function to each new analogue 
 that might be prepared in this way . catalysis provides an opportunity 
 to simplify the challenge , provided 
 that catalysts can be found that are selective for unique molecular 
 products . many types of catalysts have been applied to this problem , 
 including enzymes and , increasingly , 
 small molecules . numerous catalyst discovery 
 efforts of this type have involved a healthy dose of screening catalyst 
 libraries . aside from often providing successful 
 outcomes , nominal combinatorial techniques have been reasonable to 
 apply to the problem , given the complexity of the reactions in terms 
 of scaffold complexity , as well as the numerics of possible products 
 that can be formed . on the other hand , so - called  rational 
 design  of catalysts for the objective of site - selective modification 
 of a complex scaffold may also be possible in situations where ample 
 information may be available about how a particular natural product 
 scaffold might associate with a sector of a catalyst candidate . we 
 have been exploring the possibility of rational design of site - selective 
 catalysts for the modification of the glycopeptide antibiotics vancomycin and teicoplanin . our choice of these 
 scaffolds was based on two considerations : ( 1 ) antibiotic resistance 
 and the need for new antibiotics and ( 2 ) the 
 opportunity to exploit the well - known mechanism of action for inspiration 
 in terms of catalyst design . structure of 
 teicoplanin a2 - 2 ( 1 ) and protected 
 teicoplanin a2 - 2 derivative 2 . in this context , we recently reported the peptide - based 
 site - selective 
 phosphorylation of teicoplanin derivative 2 ( figure 1 ) . from these studies , 
 we identified three catalysts ( 35 ) that selectively phosphorylated three distinct sugar units present 
 in teicoplanin derivative 2 ( table 1 ) . specifically , the design of catalysts 3 and 5 was inspired by the well - precedented interactions between 
 the dala - dala moiety and glycopeptide natural products . the x - ray crystal structure of the lys - dala - dala 
 peptide moiety bound to teicoplanin a2 - 2 ( 1 ) reported by loll and co - workers served 
 as a scaffold for the rational design of novel catalysts ( figure 2 ) . reaction 
 conditions : dpcp ( 6 equiv ) , 
 pemp ( 8 equiv ) , catalyst , thf , ch2cl2 , 23 c 
 unless noted otherwise . dpcp ( 3 equiv ) , pemp 
 ( 4 equiv ) , 
 catalyst , thf , ch2cl2 , 23 c . 7 was isolated in 23% 
 yield . pemp : 1,2,2,6,6-pentamethylpiperidine . predicted model of catalysts ( 3 and 5 ) and teicoplanin interaction based on the crystal 
 structure of the 
 lys - dala - dala and teicoplanin a2 - 2 complex 
 ( catalysts 3 and 5 were superposed to the 
 crystal structure adopted from pdb 3vfj ) . ( a ) predicted diphenylphosphorylated 
 z - lys(z)-dala - dpmh ( 3 , yellow - orange ) 
 and teicoplanin a2 - 2 ( 1 ) complex consistent 
 with the observed n - dodecylglucosamine (  top / red  
 sugar ) selectivity . ( b ) predicted diphenylphosphorylated boc - pmh - dpro - aib - dala - dala ( 5 , green ) 
 and teicoplanin a2 - 2 ( 1 ) complex consistent 
 with the observed n - acetylglucosamine (  left / green  
 sugar ) selectivity . while many experiments supported the strong correlation between 
 the hypothesis and the observed results , one element that was missing was any type of direct observation 
 of the interactions that we postulated to account for the selectivity . 
 a technique that we pursued was x - ray crystallographic analysis of 
 catalyst / substrate complexes . while the observation of a complex of 
 this nature is fraught with caveats , we 
 hoped to demonstrate at least plausibility of the models we proposed 
 by x - ray crystallographic analysis . we describe below our studies 
 that culminated in the direct observation of a crystal structure that 
 supported many features of the initial hypothesis presented above 
 for the catalyst targeted to selectively derivatize the most recalcitrant 
 sugar on the scaffold , the n - acetylglucosamine now 
 referred to as the  left / green  sugar . we initially 
 attempted the cocrystallization of catalyst 5 and teicoplanin 
 derivative 2 ( scheme 1 , eq 2 ) . however , despite numerous attempts with 
 various crystallization conditions , the formation of a single crystal 
 suitable for the x - ray crystallographic analysis remained elusive . hence , we envisioned incorporating a crystallization aid to the catalyst - teicoplanin 
 complex . loll and co - workers reported the carrier - protein strategy 
 for the x - ray crystal structure determinations of the complexes of 
 lys - dala - dala peptide residues and various glycopeptide 
 antibiotics . in their studies , the cys - lys - dala - dala peptide moiety was conjugated to a carrier 
 protein such as maltose - binding protein ( mbp ) , ubiquitin , or t4l using 
 expressed protein ligation . the resulting recombinant 
 protein construct was allowed to complex with glycopeptides such as 
 dalbavancin , ristocetin , vancomycin , and teicoplanin , which resulted 
 in crystals suitable for x - ray structure analysis . inspired by these 
 studies , we planned to covalently bind the peptide residues of catalyst 5 ( pmh - dpro - aib - dala - dala ) to the 
 carrier protein to obtain ligation product 10 and allow 
 it to interact with teicoplanin a2 - 2 ( 1 ) or 
 its derivative 2 to obtain single crystals suitable for 
 the x - ray structure determination ( scheme 1 , eq 3 ) . we envisioned coupling the carrier protein and the synthetic 
 peptide , 
 which contains the functional residues of catalyst 5 via 
 expressed protein ligation ( scheme 2 ) . the key peptide bond in the carrier protein  synthetic peptide 
 conjugate 10 would be formed by native chemical ligation 
 of the thioester of carrier protein 11 and the cysteine 
 residue at the n - terminus of the synthetic peptide 13 . cbd 
 ( chitin binding domain ) construct with a thiol reagent upon purification 
 on chitin beads . protein construct 12 was to be expressed from a recombinant plasmid , which utilized 
 the commercially available ptxb1 vector . peptide 13 would 
 be derived from the methyl ester derivative of  left / green  
 selective catalyst via two rounds of peptide coupling . based on our synthetic plan ( scheme 2 ) , various carrier protein  peptide conjugates with different linkers 
 were synthesized ( table 2 ) . the initial crystallization 
 attempt with ubiquitin as a carrier protein did not yield crystals 
 suitable for x - ray diffraction under the conditions examined ( table 2 , entries 1 and 2 ) . our first crystal was obtained 
 from the mbp - based construct in the absence of ligand ( table 2 , entry 3 ) . while this crystal provided us with 
 high resolution ( 1.3  ) structural information on the mbp and 
 the linker region , the catalytic peptide region ( pmh - dpro - aib - dala - dala ) was disordered ( table 2 , entry 3 ) . we reasoned that the longer linker was contributing to 
 the disorder of the peptide and therefore deleted two serine residues 
 from the linker . teicoplanin 
 a2 - 2 ( 1 ) complex remained elusive ( table 2 , entry 4 ) . in an attempt to lower the flexibility 
 of the linker , we point mutated the glycine residue to an alanine 
 residue and attempted crystallization with protected teicoplanin a2 - 2 ( 2 , table 2 , entry 
 5 ) and teicoplanin a2 - 2 ( 1 , table 2 , entry 6 ) . the insolubility of protected teicoplanin 
 derivative 2 in aqueous solution hampered the complexation 
 with the protein peptide conjugate . in spite of the high dilution 
 and the prolonged mixing time of 2 with the mbp peptide 
 construct , the crystal obtained from this sample showed analogous 
 unit cell dimensions to that without the ligand ( table 2 , entry 3 ) , and the peptide region was disordered , indicative 
 of the absence of the ligand in the crystal . on the other hand , mbp - aaaaac(carboxy 
 methylated cysteine)g - pmh - dpro - aib - dala - dala and teicoplanin a2 - 2 ( 1 ) complex formed 
 a homogeneous solution with high binding affinity ( kd 90 nm ) . cysteine was converted to s - carboxymethyl cysteine 
 to prevent oxidative byproduct 
 formation . screening was 
 conducted using mbclass , 
 mbclass ii , pegs , pegs ii suites , jcsg core suite i , jcsg core suite 
 ii , jcsg core suite iii , jcsg core suite iv from qiagen , index from 
 hampton research , and wizard classic 1 and 2 from emerald bio . o : 
 diffracting crystals , x : no crystals ,  : nondiffracting crystals . our first generation crystal structure including an ordered peptide 
 region was obtained from the t4l - peptide ligated product and teicoplanin 
 a2 - 2 ( 1 ) complex ( table 2 , entry 7 , pdb 4pjz ) . the data set obtained from this crystal was refined to an effective 
 resolution of 1.9  and unambiguously showed the electron density 
 map of t4l , teicoplanin a2 - 2 , and the dpro - aib - dala - dala peptide moiety ( figure 3 ) . we reasoned that the disorder resulted from a mismatch between the 
 spatial requirement to place eight amino acids ( from asn 163 to pmh 
 170 ) and the distance between lys 162 and dpro 171 residues , 
 which are 15.6  apart ( figure 3 ) . hence , 
 we generated the second - generation t4l construct deleting the gss 
 motif from the c - terminus of the protein . simultaneously , we mutated 
 the spacer glycine residue between the cysteine and pmh residues to 
 alanine ( g169a ) to provide additional structural rigidity ( table 2 , entry 8) . peptide 
 conjugate 
 and teicoplanin a2 - 2 ( 1 ) complex ( pdb 4pjz ) . the teicoplanin 
 a2 - 2 ligand is displayed as sticks , carbon atoms colored 
 gray . n - dodecylglucosamine ( top ) carbon atoms colored 
 in red , n - acetylglucosamine ( left ) carbon atoms colored 
 in green , mannose ( bottom ) carbon atoms colored in blue . the crystal obtained from the second - generation 
 t4l  peptide 
 construct provided an x - ray structure to 2.3  resolution with 
 all residues ordered ( figure 4 , pdb 4pk0 ) . interestingly , 
 the space group for both the first- and the second - generation crystal 
 structures is i222 , which has not been previously 
 observed for t4 lysozyme and is distinct from the more common p3221 symmetry in which the t4l - lys - dala - dala - vancomycin complex ( pdb 3run ) was determined . teicoplanin rests against the loop following 
 the first helix of t4l ( residues 1216 ) . the teicoplanin n - acetylglucosamine lies 6  from the active site residue 
 glu 11 of the molecule to which it is bound , while the first residue 
 of teicoplanin makes contact with glu 22 and arg 137 of a symmetry - related 
 molecule . ribbon diagram of the 2nd generation t4l - pmh - dpro - aib - dala - dala and teicoplanin a2 - 2 complex ( pdb 4pk0 ) . the c - terminus 
 appended by expressed protein ligation is displayed as sticks , carbon 
 atoms colored yellow . the teicoplanin a2 - 2 ligand is displayed 
 as sticks , carbon atoms colored gray . n - dodecylglucosamine 
 ( top ) carbon atoms colored in red , n - acetylglucosamine 
 ( left ) carbon atoms colored in green , mannose ( bottom ) carbon atoms 
 colored in blue . the aib - dala - dala residues are complexed to teicoplanin 
 a2 - 2 through five hydrogen bonds ( figure 5a ) , which is consistent with our catalyst design . the n - terminus of the dpro residue bound next to the 
 aib residue is pointing toward the left side of the molecule and places 
 the side chain of pmh in close proximity to the left / green  n - acetylglucosamine . on the basis of our crystal structure , 
 the distance from the nucleophilic nitrogen to the primary alcohol 
 of n - acetylglucosamine , n - dodecylglucosamine , 
 and mannose are 7.6 , 9.3 , and 20.3  , respectively ( figure 5a ) . notably , the close proximity of the nucleophilic 
 moiety of pmh to the  left / green  sugar is consistent 
 with our observed selectivity in the phosphorylation of teicoplanin 
 derivative 2 with catalyst 5 . ( a ) crystal structure of the second - generation pmh - dpro - aib - dala - dala and teicoplanin a2 - 2 ( 1 ) complex ( ( b ) 
 superposition of 
 the first - generation ( orange ) and the second - generation ( yellow ) of 
 crystal structures of c - terminus peptide region and teicoplanin a2 - 2 ( 1 ) complex . the teicoplanin a2 - 2 coordinate is adopted from the first - generation crystal structure . representative electron density is displayed for the refined model 
 of the first - generation peptide . 2f0-fc map ( blue ) contoured at 1.1 and f0-fc map ( green ) 
 contoured at 3.0 for the unmodeled pmh residue . interestingly , our crystal structure reveals that 
 the pmh - dpro - aib - dala - dala peptide moiety 
 complexed to teicoplanin 
 a2 - 2 ( 1 ) does not adopt a -hairpin 
 structure . literature precedents suggested that boc - pmh - dpro - aib - dala - dala - onbu4 ( 5 ) could adopt a short -turn , often associated with a dpro in the i+1 position of a tetrapeptide sequence . however , in the presence of teicoplanin ligand , and while affixed 
 to the carrier protein , dpro and aib adopt  and  
 values of an extended -sheet ( 170 , 130 ) and a left - handed 
 -helix ( 57 , 39 ) . the carboxymethylcysteine - alanine linker between 
 the final residue of t4l residue leu 164 and the catalytic pmh adopt 
  and  values consistent with a -strand . the superposition of the first and the second - generation 
 crystal structures of the t4l  peptide conjugate and teicoplanin 
 complex illustrates the conformational similarity between these two 
 crystal structures ( figure 5b ) . furthermore , 
 the location of f0-fc density of the first - generation crystal structure is consistent 
 with the refined position of pmh in the second - generation crystal 
 structure . these observations suggest the structural arrangement of 
 catalyst peptide residues  teicoplanin complex is independent 
 of the length and composition of the linker ( table 2 , entries 7 and 8) , at least in the context of the observed 
 space group and packing within the unit cell . a number of critical 
 differences between the actual reaction conditions 
 and the nature of the observed teicoplanin - catalytic sequence complex 
 must be noted . the active catalytic species in eq 1 ( table 1 ) is presumed to be the highly reactive phosphorylated 
 peptide catalyst , reminiscent of the phosphohistidine moiety implicated 
 as an intermediate in the catalytic cycle carried out by histidine - dependent 
 kinases . because of its high electrophilicity , we could not 
 obtain a crystal structure of phosphorylated peptide and teicoplanin 
 complex using a carrier protein strategy , which involves crystallization 
 in aqueous solution . notably , our crystal structure shows that the 
 nucleophilic nitrogen in the n - methyl imidazole moiety 
 of the peptide is pointing away from the  top / red  sugar 
 and parallel to the  left / green  sugar . hence , we surmise 
 that the electrophilic phosphoryl group bound to the active catalytic 
 species would be even further away ( > 9.3  ) from the primary 
 alcohol in the  top / red  sugar . furthermore , the c6 
 of the n - acetylglucosamine residue is 4.2  
 from the c of the pmh amino acid . the pmh side chain adopts 
 a common outward - facing rotamer , but a 120 rotation around the 
 cc bond would position the nucleophilic nitrogen 
 of pmh in closer proximity of the hydroxyl group it selectively derivatizes . this conformational flexibility of the pmh side chain ( figure 5a , presented with rounded arrow ) in conjunction 
 with the free rotation involving the methylene of the  left / green  
 and  top / red  sugars , suggest potentially closer proximity 
 of the imidazole moiety to the  left / green  sugar but 
 further distance to the  top / red  sugar . it is 
 also of note that we obtained the crystal structure of the 
 catalytic peptide sequence bound to teicoplanin a2 - 2 , without 
 any adornment with protecting groups , which were necessary to achieve 
 the site - selective phosphoryl transfer reactions . attempted cocrystallization 
 with the  allylyated  teicoplanin 2 has 
 as yet been unsuccessful . yet , it is striking that both the actual 
 asymmetric reactions and the crystal structure of the nominal catalyst 
 substrate complex give a self - consistent result . in 
 summary , a carrier protein strategy was effectively applied 
 for the x - ray crystal structure determination of an intriguing site - selective 
 catalyst that is effective for the modification of a teicoplanin a2 - 2 derivative . crystallographic analysis revealed a complex 
 between the catalytically competent peptide sequence and teicoplanin 
 itself that proved to be quite consistent with the original hypothesis 
 that led to its study . a catalytic peptide t4l conjugate based 
 on the  left / green - selective catalyst  was prepared 
 using expressed protein ligation . the resulting x - ray crystal structure 
 reveals that the n - methylimidazole moiety , which 
 serves to transfer the phosphoryl group in the o  p bond - forming 
 reaction , is in closer proximity to the n - acetylglucosamine 
 than the other sugar units , consistent with the observed selectivity 
 in the phosphorylation event of our prior studies . while consideration 
 of transition state structures based on analysis of intrinsically 
 ground - state crystallographic complexes must always be exercised with 
 caution , structural insight obtained 
 from this catalyst scaffold is expected to serve as a platform for 
 the design of novel catalysts capable of selectively derivatizing 
 glycopeptide antibiotics . furthermore , we speculate that the use of 
 a carrier protein strategy could be a broadly useful tool to obtain 
 structural information in the field of asymmetric catalysis in situations 
 where information on catalyst  substrate complexes are difficult 
 to obtain otherwise . structures 
 of s1s6 are shown in supplementary figure 1 , supporting information ) . to a flask charged with a known 
 compound 14(25 ) ( 253 mg , 0.416 
 mmol , 1 equiv ) was added a 4 m solution of hydrochloric acid in 1,4-dioxane 
 ( 4.6 ml ) and methanol ( 2.2 ml ) at 23 c . after 3 h , the reaction 
 mixture was concentrated under reduced pressure ( caution : hcl gas 
 release ) and exposed to high vacuum until residual solvent and hcl 
 were removed to yield s1 as a white solid . s1 was moved to the next step without further purification . to 
 a flask charged with protonated amine s1 ( 226 mg , 
 0.416 mmol , 1 equiv ) , alanine derivative s2 ( 78.7 mg , 
 0.416 mmol , 1.00 equiv ) , 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide 
 hydrogen chloride ( edchcl , 87.8 g , 0.458 mmol , 1.10 equiv ) , 
 and hydroxybenzotriazole hydrate ( hobth2o , 70.14 mg , 0.458 mmol , 1.10 equiv ) was added dichloromethane ( 4.8 ml ) at 
 23 c to yield a heterogeneous reaction suspension . after 3 min , n , n - diisopropylethylamine ( 165 l , 
 0.915 mmol , 2.2 equiv ) was added to the heterogeneous reaction mixture , 
 and within 1 min , the reaction mixture became homogeneous . after 12 
 h , the reaction mixture was diluted with dichloromethane ( 4 ml ) . subsequently , 
 saturated aqueous sodium bicarbonate solution ( 4 ml ) was added , and 
 the layers were separated . the organic layer was dried over anhydrous 
 sodium sulfate , filtered , and concentrated under reduced pressure 
 to give s3 as a white solid mixture . the sample of the 
 crude residue of s3 was purified by mplc ( snap - c18 120-g 
 column with a 12-g samplet : solvent a = water , solvent b = acetonitrile , 
 flow = 40 ml / min , = 210 , 254 nm , loaded crude s3 in methanol ; equilibrated at 5% b for 3 column volumes ( cv ) , then 
 ramped to 95% b over 10 column volumes ( cv ) ) to give methyl ester s3 ( 208 mg , 74% ) as a white solid . to a flask charged 
 with s3 ( 208 mg , 0.306 mmol , 1 
 equiv ) was added 4 m solution of hydrochloric acid in 1,4-dioxane 
 ( 4 ml ) and methanol ( 2 ml ) at 23 c . after 1.5 h , the reaction 
 mixture was concentrated under reduced pressure ( caution : hcl gas 
 release ) and exposed to high vacuum until residual solvent and hcl 
 were removed to yield s4 as a white foam . s4 was moved to the next step without further purification . to 
 a flask charged with clnh3-ala - pmh - dpro - aib - dala - dala - ome ( s4 , 199 mg , 0.306 mmol , 
 2.00 equiv ) , s5 ( 67.4 mg , 0.153 mmol , 1.00 equiv ) , 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide 
 hydrogen chloride ( edchcl , 64.6 mg , 0.337 mmol , 2.20 equiv ) , 
 and hydroxybenzotriazole hydrate ( hobth2o , 51.6 mg , 
 0.337 mmol , 2.20 equiv ) was added dichloromethane ( 4 ml ) at 23 c 
 to yield a heterogeneous reaction suspension . after 3 min , n , n - diisopropylethylamine ( 118 l , 
 0.673 mmol , 4.4 equiv ) was added to the heterogeneous reaction mixture , 
 and within 1 min , the reaction mixture turned homogeneous . after 24 
 h , the reaction mixture was diluted with dichloromethane ( 6 ml ) , saturated 
 aqueous sodium bicarbonate solution ( 6 ml ) was added , and the layers 
 were separated . the organic layer was dried over anhydrous sodium 
 sulfate , filtered , and concentrated under reduced pressure to give s6 as a yellow solid mixture . the sample of the crude residue 
 of s6 was purified by mplc ( snap - c18 120-g column with 
 a 12-g samplet : solvent a = water , solvent b = acetonitrile , flow 
 = 40 ml / min ,  = 210 , 254 nm , loaded crude s6 in 
 methanol ; equilibrated at 5% b for 3 column volumes ( cv ) , then ramped 
 to 95% b over 10 column volumes ( cv ) ) to give methyl ester s6 ( 150.5 mg , 63% ) as a white solid . h nmr ( 500 mhz , cdcl3 , 21 c ) :  8.18 ( app - d , j = 38.9 
 hz , 4h ) , 7.52 ( s , 2h ) , 7.46 ( d , j = 7.4 hz , 2h ) , 
 7.38 ( d , j = 7.6 hz , 2h ) , 7.18 ( s , 2h ) , 6.86 ( s , 
 2h ) , 5.95 ( s , 2h ) , 4.544.26 ( m , 10h ) , 4.14 ( app - s , 2h ) , 3.813.62 
 ( m , 4h ) , 3.62 ( s , 6h ) , 3.57 ( s , 6h ) , 3.21 ( br - s , 2h ) , 3.04 ( br - s , 
 4h ) , 2.96 ( br - s , 2h ) , 2.09 ( br - s , 2h ) , 1.95 ( br - s , 4h ) , 1.78 ( br - s , 
 2h ) , 1.49 ( s , 6h ) , 1.44 ( s , 6h ) , 1.38 ( app - s , 30h ) , 1.31 ( d , j = 7.4 hz , 6h ) . c nmr ( 151 mhz , cdcl3 , 21 c ) :  174.7 , 173.4 , 173.3 , 173.1 , 172.0 , 171.0 , 
 170.8 , 155.8 , 138.2 , 127.3 , 127.0 , 80.4 , 62.4 , 57.3 , 54.1 , 52.4 , 52.2 , 
 49.6 , 49.4 , 48.4 , 48.0 , 42.1 , 31.8 , 29.1 , 26.5 , 25.2 , 24.8 , 18.0 , 
 17.6 , 17.2 . ftir ( neat ) cm : 3326 ( w ) , 1651 ( s ) , 
 1528 ( m ) , 1450 ( w ) , 1162 ( m ) . hrms ( esi ) ( m / z ) : calcd for c68h108n18o20s2 , [ m + h ] 1561.7507 , found 
 1561.7487 . structures of s7 and s8 are shown in 
 supplementary figure 2 , supporting information ) . to a vial charged with s6 ( 79.7 mg , 51.1 mol , 
 1 equiv ) was added a 4 m solution of hydrochloric acid in 1,4-dioxane 
 ( 1.0 ml ) and methanol ( 1.0 ml ) at 23 c . after 1 h , the reaction 
 mixture was concentrated under reduced pressure ( caution : hcl gas 
 release ) and exposed to high vacuum until residual solvent and hcl 
 were removed to yield s7 as a white solid . sodium 
 hydroxide solution ( 1.0 m ) in water ( 300 l , 0.3 mmol , 
 6 equiv ) was added via syringe to a solution of s7 ( 76.9 
 mg , 51.1 mol , 1 equiv ) in methanol ( 1 ml ) at 23 c . after 
 2 h , 10% aqueous solution of hydrochloric acid ( 1 ml ) was added to 
 the reaction mixture , and the resulting mixture was concentrated under 
 reduced pressure . the sample of the crude residue of s8 was purified by mplc ( snap - c18 60-g column with a 12-g samplet : 
 solvent a = water , solvent b = acetonitrile , flow = 30 ml / min ,  
 = 210 , 254 nm , loaded crude s8 in 10% aqueous solution 
 of hydrochloric acid ; equilibrated at 1% b for 3 column volumes ( cv ) , 
 then run at 1% b for 11.9 column volumes ( cv ) , then ramped to 95% 
 b over 12 column volume ( cv ) ) to product s8 ( 51.4 mg , 
 68% ) as a pale yellow solid . h nmr ( 600 mhz , d2o , 21 c ) :  8.64 ( s , 2h ) , 
 7.32 ( s , 2h ) , 5.00 ( dd , j = 8.8 , 5.8 hz , 2h ) , 4.41 
 ( dd , j = 9.2 , 4.7 hz , 2h ) , 4.364.29 ( m , 5h ) , 
 4.294.24 ( m , 3h ) , 3.85 ( s , 6h ) , 3.603.56 ( m , 4h ) , h 3.47 ( dd , j = 15.2 , 4.6 hz , 2h ) , 3.21 ( dd , j = 16.1 , 5.8 hz , 2h ) , 3.09 ( td , j = 16.0 , 
 8.9 hz , 4h ) , 2.302.22 ( m , 2h ) , 2.01 ( p , j = 5.7 hz , 2h ) , 1.961.85 ( m , 4h ) , 1.471.44 ( s , 6h ) , 
 1.441.39 ( m , 12h ) , 1.391.35 ( m , 12h ) . c nmr ( 151 mhz , d2o , 21 c ) :  176.5 , 176.0 , 
 174.6 , 173.8 , 173.2 , 169.0 , 167.5 , 135.1 , 130.0 , 118.1 , 60.9 , 56.5 , 
 51.3 , 49.8 , 49.6 , 48.5 , 48.0 , 37.2 , 33.2 , 29.0 , 24.7 , 24.6 , 24.4 , 
 23.8 , 16.7 , 16.6 , 16.1 . ms ( esi ) ( m / z ) : calcd for c56h89n18o16s2 , [ m + h ] 1333.6145 , found 1333.7275 . the gene encoding 
 cysteine - free t4 lysozyme ( t4l ) was pcr - amplified from plasmid pet11b - t4l * 
 with 5 ndei/3 sapi restriction sites ( table s1 , supporting information ) . the pcr product was 
 digested , gel purified , and ligated into ndei / sapi - digested ptxb1 . the 
 gss sequence was subsequently deleted by site - directed mutagenesis 
 using asymmetric primers . recombinant plasmids were confirmed by restriction 
 digest and dna sequencing . both t4l - gss 
 ( first - generation t4 lysozyme ) and t4l ( second - generation t4 lysozyme ) 
 were expressed in rosetta ( de3 ) cells in terrific broth ( tb ) for 24 
 h at 20 c postinduction . the cell pellet 
 was thawed in buffer a ( 500 mm nacl , 25 mm tris ph 7.5 ) and lysed 
 by sonication , and the lysate was clarified by centrifugation at 100000 g for 30 min . the supernatant was loaded onto a chitin column 
 equilibrated with buffer a and washed until od280 < 
 0.01 . intein - cleavage was initiated by adding buffer b ( buffer a with 
 500 mm mesna ) to the column overnight at room temperature . t4l protein 
 was eluted , concentrated , and exchanged against buffer c ( 100 mm hepes 
 ph 8.0 , 500 mm nacl , 500 mm mesna ) . peptide 
 ligation was carried out with a 4-fold molar excess of synthesized 
 peptide s8 . the ligation 
 products were monitored by mass spectrometry to confirm the complete 
 conversion ( second - generation t4l : calculated mass , 19250.6 ; observed 
 mass , 19251 ) . the reaction was then loaded into a desalting column 
 twice using buffer d ( 100 mm sodium borate ph 8.0 , 150 mm nacl , 5 
 mm edta ) . peptide fusion was 
 protected by adding a 5-fold molar excess of iodoacetic acid for 2 
 h at room temperature . the protein peptide chimera was further 
 purified by size - exclusion chromatography using a hiload 16/60 superdex 
 75 column with buffer e ( 20 mm hepes ph 7.5 , 25 mm nacl ) . the purified protein the sample was concentrated 
 to 20 mg / ml ( t4l ) and screened in 96-well format with commercial sparse 
 matrix screens using an automated liquid handling system . initial 
 crystallization conditions of both complexes were found in the peg 
 i screen with 20% peg 3350 and 0.2 m dibasic potassium and sodium 
 phosphate . diffraction - quality crystals grew as thin plates ( supplementary figure 
 4 , supporting information ) at 19% peg 3350 
 and 0.2 m dibasic potassium phosphate solution . crystals were cryoprotected 
 by transfer to 20% glycerol in mother liquor and flash - frozen in liquid 
 nitrogen .
we 
 report the x - ray crystal structure of a site - selective peptide 
 catalyst moiety and teicoplanin a2 - 2 complex . 
 the expressed 
 protein ligation technique was used to couple t4 lysozyme ( t4l ) and 
 a synthetic peptide catalyst responsible for the selective phosphorylation 
 of the n - acetylglucosamine sugar in a teicoplanin 
 a2 - 2 derivative . 
 the t4l - pmh - dpro - aib - dala - dala construct was crystallized in the presence of teicoplanin 
 a2 - 2 . the resulting 2.3  resolution protein  peptide 
 teicoplanin 
 complex crystal structure revealed that the nucleophilic nitrogen 
 of n - methylimidazole in the pmh residue is in closer 
 proximity ( 7.6  ) to the n - acetylglucosamine 
 than the two other sugar rings present in teicoplanin ( 9.3 and 20.3 
  , respectively ) . 
 this molecular arrangement is consistent with 
 the observed selectivity afforded by the peptide - based catalyst when 
 it is applied to a site - selective phosphorylation reaction involving 
 a teicoplanin a2 - 2 derivative .