over 
 500  000 gene sequences have been discovered encoding 
 glycoside hydrolases that are grouped into more than 130 families 
 according to the carbohydrate active enzyme classification ( cazy : www.cazy.org ) .
endo--1,2-mannosidases 
 are eukaryotic proteins involved in n - linked glycan maturation , folding , 
 and quality control and are of clinical significance as they provide a means for viruses 
 and cancer to evade the effect of exo - glucosidase 
 inhibitors.endo--1,2-mannanases 
 are produced by bacteroides spp . , bacterial residents 
 of the gut microbiota .
they facilitate 
 the degradation of dietary yeast mannan consumed in bread and fermented 
 foods , facilitating the breakdown of these complex carbohydrates , 
 with beneficial effects on the gastrointestinal tract and , possibly , 
 mitigating the symptoms of crohn s disease . given the importance of family gh99 enzymes in n - linked 
 glycan maturation and carbohydrate processing by the microbiota , the 
 development of inhibitors has been of particular importance to allow 
 assessment and manipulation of their roles in these complex processes . 
 in this work ,
we investigate several mechanism - inspired inhibitor 
 design concepts for family gh99 endo--1,2-mannanases 
 from the gut microbiota constituents bacteroides thetaiotaomicron and bacteroides xylanisolvens ; btgh99 and bxgh99 , respectively .
our results cast light 
 on the importance of structural mimicry of shape and charge of species 
 along the reaction coordinate for achieving potent inhibition of this 
 enzyme family .
glycosidases that cleave their substrates with 
 retention of anomeric 
 configuration typically operate through a two - step mechanism that 
 proceeds via a covalent glycosyl - enzyme intermediate .
such enzymes 
 utilize enzymatic amino acid residues that in the first step act as 
 general acid and nucleophile to assist in departure of the anomeric 
 substituent while simultaneously substituting the anomeric group ; 
 in the second step the first carboxylate acts as a general base to 
 deprotonate a nucleophilic water molecule that hydrolyzes the covalent 
 glycosyl enzyme intermediate ( figure 1a ) .
important exceptions include 
 a range of -hexosaminidases that perform catalysis through 
 mechanisms involving neighboring group participation by the 2-acetamido 
 group of the substrate ( figure 1b ) .
these enzymes also operate 
 through a two - step mechanism : in the first step an amino acid residue 
 acts as a general acid to assist in departure of the leaving group 
 while the 2-acetamido group performs a nucleophilic attack on the 
 anomeric center , forming a bicyclic oxazoline / oxazolinium ion intermediate . 
 in
the second step the same amino acid residue acts as a general base , 
 assisting nucleophilic attack by a water molecule that opens the oxazolinium 
 ion ring , reforming the 2-acetamido group and completing the hydrolysis 
 reaction .
( a ) mechanism for a canonical retaining -glycosidase that 
 proceeds through a covalent glycosyl - enzyme intermediate .
( b ) mechanism 
 for a retaining -hexosaminidase involving neighboring group 
 participation by the 2-acetamido group , via an oxazolinium ion intermediate . 

( c ) proposed mechanism for family gh99 -mannosidases involving 
 neighboring group participation by the 2-oh group , via a 1,2-anhydro 
 sugar ( epoxide ) .
x - ray structures 
 are available for gh99 enzymes in complex with a variety of ligands 
 based on sugar - shaped heterocycles . however , in x - ray structures of b. xylanisolvens 
 bxgh99 with various substrate - mimicking ligands , it was not 
 possible to identify an appropriately positioned enzymatic nucleophile 
 within the typical <3  distance from the reactive anomeric 
 center , leading to the proposal of a nonclassical mechanism .
in particular , in structures of glcifg ( 2 ) and manifg ( 3 ) with bxgh99 , 
 there were no close contacts with a likely candidate enzymatic nucleophile , at odds with the usual observation of a carboxylate situated typically 
 2.62.7  away in classical retaining glycosidases .
moreover , 
 in complexes of glcdmj ( 1 ) with bxgh99 
 a conserved carboxyl residue ( e333 ; numbering refers to bxgh99 ) was located 2.7  from the 2-oh group ; similar observations extend to the binding of a substrate 
 ( -man-1,3--manmu ) to the carboxamide mutant bxgh99 e333q .
collectively these 
 data supported the proposal of a two - step reaction involving in the 
 first step the formation of a bicyclic 1,2-anhydro sugar intermediate , 
 through e333 acting as a general base residue to deprotonate the 2-oh 
 and facilitating a nucleophilic substitution at c1 coincident with 
 departure of the leaving group , assisted by e336 acting as general 
 acid ( figure 1c ) . in the second step of this proposed mechanism , 
 e333 acts as a general acid , assisting ring opening of the epoxide , 
 while e336 acts as a general base , promoting nucleophilic attack by 
 a water molecule .
while such a mechanism lacks precedent in enzymes , 
 there is strong evidence that the base - catalyzed solvolysis of 4-nitrophenyl 
 -d - mannoside and -mannosyl fluoride proceed 
 through similar neighboring group participation mechanisms .
the most stable conformation of 1,2-anhydro--d - mannose 
 is a h5 half - chair ; applying 
 the principle of least nuclear motion , a c1  e  h5 conformational 
 itinerary has been proposed for the first step of the family gh99 
 reaction coordinate .
intensive 
 efforts have been invested in the rational development 
 of glycosidase inhibitors , and many fundamental principles have been 
 articulated inspired by our deep mechanistic understanding of this 
 class of enzyme .
based on pioneering insights from pauling and wolfenden , it 
 is recognized that a common principle underpinning catalysis is the 
 selective affinity of an enzyme for a reaction transition state , relative 
 to the ground state .
accordingly , inhibitor design by transition state 
 mimicry , which can take advantage of the high transition state affinity 
 of a glycosidase , has proven a useful guiding strategy . while it is widely appreciated that perfect 
 transition state mimics are chemically unstable and thus unattainable , 

a general design principle is to develop analogues incorporating features 
 that mimic the shape and charge of the transition state .
three features have been highlighted for consideration 
 in the development of effective glycosidase inhibitors : configuration , 
 conformation and charge .
configuration 
 is the simplest to address and not surprisingly it is usually found 
 that glycosidases are normally best targeted by inhibitors with stereochemistry 
 matching that of the substrate . in the case of bxgh99 , an enzyme that has the ability to cleave both -glc-1,3--man - or 
 and -man-1,3--man - or configured substrates ( with a preference 
 for the latter ) , optimal inhibition is 
 achieved by inhibitors matching the preferred substrate configuration . 

glycosidases typically operate through transition states with substantial 
 oxocarbenium ion character , and partial double bond development between 
 o5 and c1 , leading to a flattened conformation at the transition state . 

consequently , mimicry of the flattened conformation expected at the 
 transition state has proven a second effective strategy , with inhibitors 
 bearing sp - hybridized atoms at the anomeric or endocyclic 
 oxygen positions , such as glyconolactones and -lactams , identified 
 as fairly broad spectrum glycosidase inhibitors . finally , partial 
 charge development at c1 and the endocyclic oxygen at the transition 
 state can be mimicked by the protonated forms of nitrogen - containing 
 heterocycles , exemplified by deoxymannojirimycin ( dmj ) with a nitrogen 
 in place of the endocyclic oxygen , and isofagomine with a nitrogen 
 in place of c1 .
motivated by the unusual mechanism proposed 
 for family gh99 enzymes 
 and the complexity of their biochemical roles , our understanding of 
 which should benefit from the development of potent inhibitors , we 
 undertook a study of several inhibitor designs inspired by the concepts 
 of charge and shape mimicry .
glcdmj ( 1 ) , an inhibitor 
 reported by spiro and co - workers , was 
 the first effective inhibitor of mammalian endo--1,2-mannosidase , and was subsequently shown to bind to and inhibit bxgh99 and btgh99 ( table 1 ) .
more potent 
 inhibition was achieved by glcifg ( 2 ) , which also proved to be a more effective inhibitor than 
 glcdmj of mammalian endo--1,2-mannosidase 
 in cell - based studies , demonstrating 
 that varying the position of charge can provide improvements in potency . 

enzymes as preferential endo--1,2-mannanases , we were able to configurationally match the substrate and develop 
 the inhibitor manifg ( 3 ) , the most potent inhibitor yet 
 reported for any gh99 enzyme .
however , 
 manifg and glcifg lack the 2-oh group of the substrate and thus can not 
 benefit from specific interactions with the putative acid / base e333 . 

separately , spiro and co - workers reported that two other neutral compounds 
 were almost as effective as glcdmj in the inhibition of mammalian endo--1,2-mannosidase , namely , glcddman ( 4 ) and glcglucal ( 6 ) .
we were intrigued 
 by these observations and sought to investigate whether the equivalent 
 configurationally matched species , manddman ( 5 ) and manglucal 
 ( 7 ) , and the related cyclohexene derivative ( 8) were inhibitors of bacterial gh99 enzymes and to understand how 
 they bind to the enzyme .
manddman ( 5 ) and manglucal ( 7 ) were prepared from -1,3-mannobiose 
 ( 10 ) ( scheme 1 ) .
acetylation , followed by bromination afforded mannobiosyl 
 bromide ( 12 ) , which was converted to the protected glucal 
 ( 13 ) using zn in meoh .
alternatively , reduction of 13 using h2/pd  c , followed by zempln 
 transesterification afforded manddman ( 5 ) . the preparation 
 of glcchex ( 8) will be described elsewhere .
dissociation constants for the binding of 
 compounds 5 and 7 to btgh99 and bxgh99 were determined by nmr spectroscopy 
 ( figure s1 ) . the 2d nmr sofast - hmqc spectra 
 of n - labeled enzymes determined in the presence or absence 
 of a saturating amount of the ligands revealed several h 
n 
 peaks that displayed significant chemical shift perturbations . for 
 instance , new signals for an arginine residue ( assigned as r295 in bxgh99 and r291 in btgh99 on the basis 
 of analysis of inter - residue noes from the 3d - hsqc - noesy spectra ; 
 see annotation to figure 4a ) appeared during the titration experiments , which were in 
 slow exchange with the initial ones in the chemical shift time scale 
 ( figure 2 )
. therefore , 
 since the relative intensities of these signals are proportional to 
 the populations of the bound and unbound forms ( see experimental section and supporting information ( si ) ) , the dissociation constants ( kd ) were readily calculated .
no evidence for 
 binding of glcchex could be obtained by either nmr or itc .
excerpt of 
 the side chain hn -arginine region in the 2d 
 sofast - hmqc nmr spectra of n - labeled btgh99 in the absence ( blue ) or presence of an excess ( top ) of manglucal 
 ( 7 ; red ) or ( bottom ) manddman ( 5 ; green ) . 

the arrow highlights the chemical shift perturbation observed for 
 the hn- signal corresponding to r295 . in order to analyze the mode of binding of the conformationally 
 restricted compounds ,
3-d structures of complexes of bxgh99 with 7 and 8 were determined by x - ray 
 crystallography at near atomic ( approximately 1.0  ) resolutions 
 ( table 2 , figure 3a , b ) .
manglucal 7 ( kd 111 m ) binds to bxgh99 in the 2 and 1 subsites , with the 
 1 glucal ring intact in a h5 conformation . for reasons most likely related to its poor 
 affinity for the enzyme , we were unable to obtain a complex of glcchex 
 with wildtype enzyme , but were serendipitously successful in obtaining 
 a complex with the catalytically inactive bxgh99 
 e333q mutant . in this complex glcchex 8
also bound in 
 the 2/1 subsite with the cyclohexene ring in a h5 conformation ( figure 3b ) .
relative to manglucal , 
 glcchex suffers both by replacement of the endocyclic oxygen with 
 methylene and by the presence of a nonreducing - end glucosyl moiety , 
 the latter of which is known to reduce binding to the bacteroides spp .
owing 
 to the unmeasurable binding of glcchex , the synthesis of the mannose 
 analogue was not pursued .
the lack of oxygen atoms within the glcchex 
 ring means it can not form hydrogen bonds with active site residues 
 y252 , e333 or e336 .
ms experiments indicated that the compound is 
 not affected by the enzyme or its variants so the reason for binding 
 to only the inactive variant is unclear .
( a ) bxgh99 with manglucal 
 ( 7 ) , ( b ) bxgh99-e333q with glcchex ( 8) , and ( c ) bxgh99 with manddman ( 5 ) .
depicted electron density maps are refmac maximum - likelihood/a weighted 2f0  fc syntheses contoured at 1.5 
 ( 0.57 , 0.59 , and 0.62 e , respectively ) .
glycals are often effective inhibitors of classical 
 retaining glycosidases . 
 inhibition is typically found to be time - dependent , owing to a chemical 
 reaction in which the conjugate acid of the nucleophile protonates 
 the enol ether of the glycal , and forms a 2-deoxy - glycosyl enzyme .
this mode of reactivity has been exploited to allow the identification 
 of the catalytic nucleophile by peptide sequencing , and the 2-deoxyglycosyl - enzymes are sufficiently stable 
 to be studied by x - ray crystallography . to date , only two classes of retaining glycosidases have been identified 
 upon which glycals bind as competitive inhibitors without exhibiting 
 this mode of reactivity .
these are n - acetylhexosaminidases 
 that use neighboring group participation and retaining sialidases , 
 and in both cases these enzymes lack a typical carboxylate nucleophile . 

retaining n - acetylhexosaminidases use a 2-acetamido 
 group , which is able to catalyze the slow hydration of the enol ether . retaining sialidases
lack a carboxylate nucleophile 
 capable of protonating the glycal , and instead use a less acidic tyrosine 
 residue as the catalytic nucleophile . in the case of sialidases , the corresponding glycal 2,3-didehydro-2-deoxy - n - acetylneuraminic acid has been elaborated into the extraordinarily 
 potent inhibitor zanamivir , a clinically 
 used antiinfluenza drug . despite its potency , quantitative examination 
 of transition state mimicry by zanamivir reveals it to be a poor transition 
 state mimic .
the observation that manglucal 
 binds to bxgh99 without a chemical reaction despite 
 the retaining mechanism of the enzyme provides further evidence in 
 favor of the unique neighboring group participation mechanism proposed 
 for this family . in order to harness the tighter binding 
 of the bacterial enzymes with a mannoside in the 2 subsite manddman ( 5 )
nmr titration 
 revealed manddman to bind to bxgh99 and btgh99 with kd values of 221 and 53 m , 
 respectively ( table 1 ) . by comparison , glcddman ( 4 )
is an inhibitor for rat 
 endomannosidase with an ic50 value of 3.8 m for inhibition of cleavage of c - labeled glcman9glcnac , only slightly worse than that 
 of glcdmj ( 1 ) ( ic50 = 1.7 m ) . the structure 
 of bxgh99 in complex with manddman was determined 
 at 1.03  resolution .
compound 5 binds in the 2/1 
 subsites of the enzyme in an undistorted c1 conformation .
this conformation matches that of the 
 ground state of the substrate , and while this is consistent with the 
 modest dissociation constant , it is noteworthy that the tight - binding 
 inhibitor manifg 3 also binds in a c1 chair . as manifg ( 3 ) , manddman ( 5 ) , and manglucal ( 7 ) all lack an o2 group , in considering 
 the contribution of shape and charge to inhibition , it would appear 
 that the cationic nature of manifg contributes most significantly 
 to inhibition , in spite of its conformational resemblance to the ground 
 state .
we therefore decided that it would be appropriate to investigate 
 a charged inhibitor based on manifg that was able to make the correct 
 o2 interactions , in particular with e333 .
inspired by the work of 
 bols and co - workers on the development of noeuromycin , a 2-hydroxy 
 analogue of isofagomine that binds 24000 times more tightly 
 than isofagomine to various glycosidases , we therefore synthesized the noeuromycin derivative , mannoe .
this 
 inhibitor was synthesized by the regioselective mannosylation of the 
 nitrile diol 16(35 ) by trichloroacetimidate 15scheme 2 .
the sole acetate group of the glycoside 17 was cleaved by treatment with hcl / meoh to afford alcohol 18 , and the nitrile group was reduced using bh3me2s , followed by protection as the boc derivative 19 .
hydrogenolysis of the benzyl ethers of 19 using h2/pd  c , and then cleavage of the boc group 
 with hcl , afforded mannoe.hcl ( 9 ) [ as a mixture of -hydroxypiperidine 
 and pyranose isomers ( not drawn ) ; see the si ] .
isothermal titration calorimetry revealed mannoe ( 9 ) to bind to bxgh99 with kd = 13 nm , and to btgh99 with a kd = 30 nm , 17- and 5-fold more tightly than 
 manifg ( 3 ) to the respective enzymes , commensurate with 
 improvements 
 seen for binding of ifg versus noe for other enzymes , and demonstrating that better matching of the substrate by reinstatement 
 of the 2-oh group absent in the latter compound provides more effective 
 inhibition ( table 1 , figure s4 ) .
3-d structures were solved 
 of a binary complex of bxgh99mannoe , and 
 a ternary complex of bxgh99mannoe-1,2-mannobiose 
 at resolutions of 1.14 and 1.05  , respectively ( figure 4 ) .
the poses of mannoe in both complexes were essentially 
 identical and the more informative ternary complex , with mannoe in 
 the 2/1 subsites , and -1,2-mannobiose in the 
 + 1/+2 subsites will therefore be discussed .
the noe heterocycle binds 
 in a c1 conformation , similar 
 to that seen for manifg with the same enzyme .
a close contact with 
 e333 oo2 of 2.58  is evident , similar 
 to that seen in the complex of bxgh99 with glcdmj 
 ( 2.54  , pdb 4ad3 ) .
( a ) x - ray structure of ternary complex of bxgh99 
 with mannoe ( above ) and -1,2-mannobiose ( below ) .
the + 1 subsite mannose residue electron density is best modeled by 
 two mannose conformations with 0.6/0.4 occupancy , rotated by about 
 30 with respect to + 2 mannose .
assembled using ccp4 mg . in order to understand the 
 intrinsic conformational preferences of 
 the d - glucal , 1,2-dideoxymannose ( ddman ) , chex , and noeuromycin ( noe ) 
 inhibitor warheads , so as to help rationalize the conformations observed 
 on - enzyme , we calculated their conformational free energy landscapes 
 ( fels ) .
fels were computed by ab initio metadynamics ( see experimental section ) , and the cremer 
pople 
 puckering coordinates  and  were used as collective 
 variables , yielding a mercator representation for each inhibitor fel ( figure 5 ) .
the same procedure has been previously used to analyze 
 the conformational preferences of related gh inhibitors ( mannoimidazole , 
 glucoimidazole , and ifg ) .
conformational free - energy 
 landscapes ( fels , mercator projection ) 
 of isolated d - glucal ( a ) , chex ( b ) , ddman ( c ) , and noe ( d ) , contoured 
 at 1 kcal mol .
fels have been annotated ( yellow 
 star ) with the inhibitor conformations of manglucal 7 ( for a ) , glcchex 8 ( for b ) , manddman 5 ( for c ) , and mannoe 9 ( for d ) that have been observed 
 on - enzyme in this work .
the fels of both d - glucal and chex ( figure 5a , b ) exhibit a main energy minimum centered 
 at h5 .
h5e5 , with an  arm  toward b3,o , indicating substantial conformational freedom around h5 .
there is also a local minimum 
 in the southern hemisphere , centered at h4 .
however , this is 3 kcal mol higher 
 in energy and thus less populated at room temperature .
interconversion 
 between the two minima is hindered by an 8 kcal mol energy barrier .
therefore , both d - glucal and chex display only one 
 main accessible conformation at room temperature , h5 , but can readily adopt the nearby e conformation predicted for the transition states 
 leading to and from the proposed 1,2-anhydrosugar intermediate .
these 
 data suggest that d - glucal and chex provide good shape mimicry of 
 the transition state or intermediate for the gh99 catalyzed reaction . that both d - glucal and chex adopt a h5 conformation when complexed to bxgh99 
 ( table 2 and yellow 
 star in figure 5a , b ) 

indicates that the conformational preference of the isolated molecules 
 are not significantly perturbed on - enzyme .
this is also the case for 
 ddman , for which the fel ( figure 5c ) is strongly biased toward the c1 chair ( the local minima on the equator are 
 8 kcal / mol higher in energy ) , as observed in the x - ray structure 
 on - enzyme .
however , the fel of ddman does not exhibit any stable minimum 
 around h5 , thus it can not 
 be considered a gh99 transition state shape mimic , and should instead 
 be considered a mimic of the substrate conformation in the michaelis 
 complex .
noe differs from the other inhibitors considered here 
 as it is 
 a basic molecule and was therefore considered as both the neutral 
 ( figure 5d ) and protonated 
 species ( figure s5 ) . while the topographies 
 of the fels for the two protonation states are broadly similar , they 
 differ greatly in relative energies , most importantly for the global 
 and local minima , such that the most stable state in noe , a c1 chair , becomes the  ring - flipped  c4 conformer in protonated noe . 
 in that case , the two most stable species , the c4 and s5 states , are characterized by the presence of transannular hydrogen 
 bonds between nh2 and o6 or o3 , respectively . 

what then is the most appropriate fel to consider in relation to the 
 enzyme - bound state , which is expected to be protonated , but which 
 in a c1 conformation lacks 
 a transannular hydrogen bond ?
it is known that in the absence of solvation 
 ( in the gas phase ) flexible molecules in low charge states tend to 
 compensate charge effects by forming stabilizing intramolecular interactions 
 that do not take place in other environments ( e.g. , in solution ) . 

for example , low - charge state proteins in the gas phase tend to adopt 
 more compact structures owing to increased intramolecular interactions . on balance
, we consider the fel of neutral noe 
 to be a more relevant representation of the enzyme - bound conformations , 
 because interactions of the inhibitor with active site residues prevent 
 the formation of intramolecular hydrogen bonds that dominate the conformations 
 of charged species .
accordingly , the fel displays a wide main minimum 
 situated close to c1 ( figure 5d ) , and is thus similar 
 to that of ddman .
the noe fel is also reminiscent of the one previously 
 computed for the closely related neutral ifg inhibitor ( which differs from noe by the absence of the 
 2-hydroxyl group ) .
interestingly , the transition state region between 
 the north pole and the equator in noe ( h3/e ) is shifted by 60 
 in  in ddman .
this is likely due to a vicinal intramolecular 
 hydrogen bond formed between the 2-oh and the 3-oh , which stabilizes 
 the h3/e conformations in noe ; this interaction is not present 
 in ddman or ifg as they both lack a 2-oh .
overall , noe most closely 
 resembles the conformation of the substrate in the michaelis complex 
 and in its protonated state on - enzyme provides mimicry of an oxocarbenium - ion - like 
 transition state ; it is therefore best considered a  charge-mimicking 
 inhibitor with no significant shape mimicry of the transition state .
the proposed gh family 99 neighboring group 
 participation mechanism , 
 via a 1,2-anhydro sugar , allows prediction of a c1  e  h5 conformational itinerary for the first step of the reaction coordinate . 

the fels for glucal and chex suggest that when these inhibitor warheads 
 are extended to manglucal and glcchex , their flattened conformations 
 provide mimicry of the e transition state 
 and h5 intermediate conformations . 

x - ray structures of these compounds in complex with bxgh99 revealed them to bind in a h5 conformation , most closely matching the proposed intermediate 
 conformation .
the modest dissociation constant of manglucal , and lack 
 of detectable binding for glcchex , suggests that shape mimicry of 
 the intermediate or transition state provides only weak affinity for 
 the enzyme . on the other hand
the fel for the neutral sugar ddman 
 reveals a preference for a c1 conformation , and the corresponding complex of manddman with bxgh99 revealed this compound to bind in the same conformation , 
 albeit with an affinity relative to manglucal 8- or 18-fold worse 
 for bx or btgh99 enzymes , respectively . 
 as manglucal , glcchex and manddman all
lack a 2-oh group , these ratios 
 provide an estimate for the contribution of shape - mimicry of the ts 
 or intermediate to enzyme binding . based on the above analysis , 
 and combined with the previous discovery 
 that the best inhibitor for gh99 endo--mannanases 
 is manifg , which provides charge mimicry , 
 but poor transition state shape mimicry , we were inspired to reinstate 
 the 2-hydroxyl group missing in this compound .
mannoe was synthesized 
 and shown to bind to bx and btgh99 
 with kd values of 30 and 13 nm , the most 
 tightly binding ligand for these enzymes yet reported , and a 20-fold 
 enhancement of affinity relative to manifg .
the x - ray structure of 
 mannoe in complex with bxgh99 reveals a c1 ground - state conformation mimicking the michaelis complex , 
 and we conclude that this inhibitor acts primarily to mimic the charge 
 of the transition state .
data set hkl index was matched to a previous solution using 
 aimless software and freer set was generated from bxgh99manddman data , and then used for every other 
 data set .
initial polypeptide chain model was obtained from the same 
 previous solution , refined against bxgh99manddman 
 data and used as starting model for other structure solutions .
refmac5 with prosmart was used for restrained refinement 
 and coot for real - space refinement . during 

fcalc difference map was examined at 3. 
 validation was performed using coot and edstats .
mannoe binding to bt and bxgh99 
 was measured using a microcal 
 autoitc200 instrument ( malvern instruments , formerly ge healthcare ) 
 at 25 c in 25 mm hepes ph 7.0 , 100 mm nacl buffer .
protein concentration 
 was kept at 5 m and ligand concentration at 50 m . as 
 mannoe exists as a 5:2 ratio of d - gluco and d - manno 
 noe isomers ;
n sofast - hmqc spectra were recorded 
 at 298 k for 1 h using n - labeled btgh99 
 and bxgh99 on a bruker avance iii 800 mhz spectrometer 
 with cryoprobe . upon binding of manglucal or manddman , chemical shift 
 perturbations
a signal corresponding 
 to n-h of r295 ( numbering based on bxgh99 ) , and which is close to the enzyme active site , was chosen as 
 binding reporter . in the case of bxgh99 , this signal 
 shifts from  7.33 , 87.5 ppm ( h , n ) 
 in the free state to  7.41 , 87.2 ppm ( h , n ) in the bound state . bound and free protein populations at different 
 protein / ligand ratios were calculated from peak intensities .
nmr measurements 
 were made in 50 mm potassium phosphate ph 7.0 , 50 mm kcl with 5% d2o added .
protein concentration was determined by measuring 
 280 nm uv absorbance after denaturing the solution in 6 m guanidinium 
 chloride ( average protein concentration : 58 m ) .
ligand concentrations 
 were cross verified by integrating the h peaks against 
 the internal standard tsp - d4 ( sigma - aldrich ) . 

the dissociation constants ( kd values ) were estimated using in - house matlab 2015b scripts , 
 using the following equation : where l is the 
 free ligand concentration and 
 ( [ pl]/pt ) is the ligand - bound protein fraction .
errors were 
 propagated using a monte carlo algorithm to estimate the uncertainty 
 in the kd values .
a distribution of kd values ( n = 10  000 ) 
 were obtained from data sets randomly varying within the error bars , 
 and the standard deviation of was used for the kd error estimation .
to 
 obtain the conformational 
 free energy landscapes of ddman , glucal , chex , and noe , quantum mechanical 
 calculations were performed using density functional theory based 
 molecular dynamics ( md ) , according to the car  parrinello ( cp ) 
 method .
each molecule was enclosed in 
 an isolated cubic box of 12.0   12.0   12.0 
  .
a fictitious electron mass of 700 au was used for the cp lagrangian 
 and a time step of 0.12 fs was used in all cpmd simulations . the kohn 
sham 
 orbitals were expanded in a plane wave ( pw ) basis set with a kinetic 
 energy cutoff of 70 ry .
the perdew , burke , and ernzerhoff generalized gradient - corrected 
 approximation ( pbe ) was selected in view 
 of its good performance in previous work on isolated sugars , glycosidases , and 
 glycosyltransferases .
the metadynamics 
 algorithm , provided by the plumed 2 plugin , was used to explore the conformational free 
 energy landscape of the systems , taking as collective variables  
 and  of the puckering coordinates of cremer and pople , in the spirit of the pioneering work by dowd , 
 french , and reilly .
initially , the height 
 of these gaussian terms was set at 0.6 kcal mol and a new gaussian - like potential was added every 250 md steps . 

once the whole free energy space was explored , the height of the gaussian 
 terms was reduced to half of its initial value ( 0.3 kcalmol ) and a new gaussian - like potential was added every 
 500 md steps .
the simulations were stopped when energy differences among wells remain 
 constant , which was further confirmed by a time - independent free energy 
 estimator .
for all molecules , the phase 
 space was fully explored in less than 60 ps and the simulations were 
 further extended up to 140 ps for chex and glucal , 160 ps for ddman , 
 and 240 ps for noe .
the errors in the principal minima , taken as a 
 standard deviation ( sd ) from the last 60 ps , are below 0.6 kcal mol ( figure s6 ) .
 inhibitor 
 design incorporating features of the reaction coordinate 
 and transition - state structure has emerged as a powerful approach 
 for the development of enzyme inhibitors . 
 such inhibitors find use 
 as mechanistic probes , chemical biology tools , and therapeutics . 
 endo--1,2-mannosidases and endo--1,2-mannanases , 
 members of glycoside hydrolase family 99 ( gh99 ) , are interesting targets 
 for inhibitor development as they play key roles in n - glycan maturation 
 and microbiotal yeast mannan degradation , respectively . 
 these enzymes 
 are proposed to act via a 1,2-anhydrosugar  epoxide  
 mechanism that proceeds through an unusual conformational itinerary . 
 here 
 , we explore how shape and charge contribute to binding of diverse 
 inhibitors of these enzymes . 
 we report the synthesis of neutral dideoxy , 
 glucal and cyclohexenyl disaccharide inhibitors , their binding to 
 gh99 endo--1,2-mannanases , and their structural 
 analysis by x - ray crystallography . 
 quantum mechanical calculations 
 of the free energy landscapes reveal how the neutral inhibitors provide 
 shape but not charge mimicry of the proposed intermediate and transition 
 state structures . building upon the knowledge of shape and charge 
 contributions to inhibition of family gh99 enzymes , we design and 
 
 synthesize -man-1,3-noeuromycin , which is revealed to be the 
 most potent inhibitor ( kd 13 nm for bacteroides xylanisolvens gh99 enzyme ) of these enzymes 
 yet reported . 
 this work reveals how shape and charge mimicry of transition 
 state features can enable the rational design of potent inhibitors . 
Introduction
Results and Discussion
Conclusions
Experimental Section