tuberculosis ( tb ) is a dreadful infectious disease whose causative 
 agent is mycobacterium tuberculosis ( mtb ) . streptomycin was first used to cure tb in the 
 late 1940s . for a short time
however , tb has reemerged as a 
 major global health threat due to poverty , co - infection with hiv , 
 long treatment times , and poor drug compliance practices .
this has 
 led to the emergence of extensively drug - resistant tb ( xdr - tb ) , which 
 is considered almost untreatable .
trehalose ( -d - glucopyranosyl-(1  1)--d - glucopyranoside ) is a pivotal metabolite in mycobacteria used 
 for carrying mycolic acid during biosynthesis of the mycolyl arabinogalactan 
 and trehalose-6,6-dimycolate ( tdm ) . following the mycolyl transfer reaction ,
trehalose is transferred 
 back into the cytoplasm from the periplamic space and is modified 
 in the production of various trehalolipids and sulfolipids .
the 
 conversion process involves four enzymes : trehalose synthase ( tres ) , 
 maltokinase ( pep2 ) , glge , and glgb .
the process involves the isomerization 
 of trehalose to maltose ( 4-o--d - glucopyranosyl - d - glucose ) by tres , and then -maltose-1-phosphate ( m1p ) 
 is produced by phosphorylation of maltose by pep2 .
reported that absence of glge led to self - poisoning 
 by the accumulation of phosphosugar m1p , directed by a self - amplifying 
 feedback response leading to cell death .
cell death was not due to absence of glucan in the cell wall and 
 capsule , but rather it was due to accumulation of m1p to a lethal 
 concentration , which caused cell death .
glge is essential for survival 
 of the pathogen , and the absence of a human homologue substantiates 
 glge as a new drug target .
syson et al . reported the structure of streptomyces coelicolor ( sco ) glge isoform - i , which indicated that glge 
 belongs to the glycosyl hydrolase family gh13 .
recently , syson et al . trapped sco glge with 2-deoxy-2-fluoro--maltosyl 
 fluoride , which provided additional evidence for a double - displacement 
 mechanism proceeding through a transition state as shown in scheme 1 .
the first step of 
 the reaction is nucleophilic attack of asp418 on the anomeric center 
 of m1p to generate a -maltosyl enzyme intermediate .
it is predicted 
 that the anomeric oxygen on the phosphate moiety is protonated by 
 acid / base glu447 .
literature supports the mechanism showing retention of configuration 
 of m1p and its conversion to -1,4-glycosidic 
 linkage .
however , until now attempts to 
 prepare a transition - state inhibitor have not been reported .
the replacement of the endocyclic oxygen atom in carbohydrates 
 with a nitrogen atom has been found in natural products that inhibit 
 glycosyl hydrolases in the micromolar range .
nojirimycin ( 5-amino-5-deoxy - d - glucose , 1 ) 
 and 1-deoxynojirimycin ( 1 , 5-dideoxy-1 , 5-imino - d - glucitol , 2 ) ( figure 1 ) are classical examples 
 of such compounds .
poly - hydroxypyrolidines 3 and 4 represent 5-membered ring compounds related to monosaccharides 
 that also act as potent inhibitors for many glucosidases or glycoside 
 hydrolases .
the hydroxyl groups present 
 on these compounds resemble the glycan moiety of the substrates . in 
 all of these examples ,
the nitrogen atom has the ability to become 
 protonated to maintain a positive charge at physiological ph that 
 permits strong interactions with a carboxylate group found in the 
 active site of this enzyme class .
the carboxylate is predicted to 
 stabilize the positive charge that develops in the transition state , 
 and it is notable that the 5-membered aza - sugars 3 and 4 are more potent inhibitors , with ki = 0.18 m and ki = 0.33 
 m , respectively , than 6-membered aza - sugars 1 and 2 , ki = 6.3 m and ki = 12.6 m , respectively , against yeast 
 -glucosidase . according to wong 
 et al .
, nojirimycin - type azasugars can mimic only the transitory charge 
 generated during the hydrolysis of glucosides , while the pyrolidine - type 
 aza sugars also mimic the shape of the postulated flattened half - chair 
 transition state ( figure 1 ) .
wong et al . also show that five - membered aza sugars were potent 
 inhibitors of -l - rhamnosidase ( glycoside 
 hydrolase family ) from penicillium decumbens .
for example , compounds 58 are good inhibitors of pyranose - based -l - rhamnosidase with ki values 
 0.14 , 5.5 , 11.5 , and 46 m , respectively . keeping in mind that 
 five - membered ring poly - hydroxypyrolidine have been shown to exhibit 
 very good activity against -glucosidases , we became interested 
 in other modified analogues of these molecules as transition - state 
 inhibitors for glge and designed inhibitor 9 .
poly - hydroxypyrrolidine - based inhibitor 9 and illustration 
 of expected binding interactions in the enzyme active site .
we synthesized the 
 compounds p - methylphenyl 2,3,4,6-tetra - o - benzyl-1-thio--d - glucopyranoside ( 14 ) ( scheme 2 ) and 5-azido-3-o - benzyl-5-deoxy-1,2-o - isopropylidene--d - fructopyranose ( 23 ) ( scheme 3 ) separately .
subsequently , the two glycosides 14 and 23 were coupled together to afford disaccharide 24 , which was subjected to global deprotection , rearrangement , 
 and reductive amination to afford poly - hydroxypyrolidine 9 as shown in ( scheme 4 ) . to synthesize the 
 thioglycosyl donor 14 , d - glucose ( 10 )
was peracetylated with acetic anhydride followed by glycosylation 
 with p - thiocresol and boron trifluoride diethyl etherate 
 to generate a peracetylated thioglycosyl donor 11 with 
 85% yield ( scheme 2 ) .
subsequent deacetylation 
 under zempln conditions afforded tetraol 13 , 
 which was then benzylated to afford the desired p - methylphenyl-2,3,4,6-tetra - o - benzyl-1-thio--d - glucopyranoside ( 14 ) .
have reported the generation of 
 key intermediate 23 by following a protection and deprotection 
 strategy on d-(+)-fructose 15.d - fructose ( 15 ) was protected with acetone 
 to give 1,2:4,5-di - o - acetonide ( 16 ) . 

alcohol 16 was benzylated at the oh-3 position to afford 
 protected d-(+)-fructose ( 17 ) .
selective deprotection 
 of the 4,5-o - isopropylidene group was achieved with 
 60% acetic acid to produce diol 18 .
initially , controlled 
 garegg conditions were employed on 18 to give selective 
 iodination at oh-5 as reported by rollin et al . however , the major 
 product was eliminated product as shown in the cited literature . therefore , as reported by yez 
 and co - workers , 18 was subjected to selective benzoylation 
 using dibutyltin oxide to afford products 19 and 20 in 38% and 52% yields , respectively .
the reaction of 20 with modified garegg s conditions caused the substitution 
 of the hydroxyl group at c-5 with iodine by inversion of configuration 
 to give the corresponding 4-o - benzoyl-3-o - benzyl-5-deoxy-5-iodo-1,2-o - isopropylidene--l - sorbopyranose ( 21 ) .
compound 21 was subjected to an sn2 substitution 
 reaction with sodium azide in n , n - dimethylformamide to give product 22 with 90% yield . 

finally , the deprotection of the benzoyl group at oh-4 was achieved 
 using zempln conditions to afford 23 ( scheme 3 ) .
coupling of compound 14 and 23 using classical glycosylation conditions with n - iodosuccinamide and trimethylsilyl trifluoromethanesulfonate 
 yielded disaccharide 24 with 76% yield with the -anomer 
 as a single product . the deprotection 
 of 1,2-o - acetonide was achieved using 60% trifluoroacetic 
 acid to afford diol 25 .
compound 25 was 
 first subjected to reduction and intramolecular reductive amination 
 using methanol , ethanol , and ethyl acetate as solvents in 1 n hydrochloric 
 acid ( hcl ) and yielded the desired product mixed with methyl- and 
 ethyl - substituted products , respectively , as confirmed by esi - ms . 
 presumably , aldehydes formed from trace oxidation of the respective 
 solvents participated in a tandem reductive amination process to produce 
 the observed masses .
these undesired reductive - amination products 
 were avoided by replacing the alcoholic solvents with a solution of 
 toluene / water / hcl ( 1:1:1 ) .
the use of h2 gas at 100 psi 
 also helped to afford a polyhydroxypyrolidine 9 in a 
 clean transformation ( scheme 4 ) . to access 
 m1p for working on inhibition studies we followed our reported procedure .
two different glge homologues were 
 studied using the enzchek phosphate assay kit ( molecular probes ) . 

this assay couples the phosphate produced from glge activity to the 
 phosphorylase activity of purine nucleoside phosphorylase , which is 
 based on work originally described by webb .
briefly , purine nucleoside phosphorylase ( pnp ) converts the substrate 
 2-amino-6-mercapto-7-methylpurine riboside ( mesg ) to ribose-1-phosphate 
 and 2-amino-6-mercapto-7-methylpurine ( figure 2 ) .
conversion of mesg to 2-amino-6-mercapto-7-methylpurine 
 shifts absorbance from 330 to 360 nm , respectively .
glge - coupled enzyme assay utilizing pnp ; ( a ) m1p and glycogen ( n = a number of maltose units ) serve as substrates for glge ; 
 ( b ) pnp consumes pi and mesg to form 2-amino-6-mercapto-7-methylpurine . using this assay , we determined a ki for compound 9 for mtb glge and the sco glgei - v279s variant , which was designed using multiple 
 sequence alignments to better emulate the mtb glge 
 maltosyl donor site ( figures 3 and 4 ) .
assays were performed under atmospheric pressure 
 at 22 c in a 96 well format on a spectra max 340pc ( molecular 
 devices ) .
all 1 mm mesg stock solutions were prepared and stored in 
 dh2o at 20 c .
phosphate release , catalyzed 
 by mtb glge , was monitored by the production of 2-amino-6-mercapto-7-methylpurine 
 in a coupled assay catalyzed by purine nucleoside phosphorylase .
the 
 reaction was monitored at 5 s intervals for 30 min . each reaction 
 consisted of 1 mm mesg , 0.2 u of pnp , 20x reaction buffer ( 1.0 m tris  hcl , 
 20 mm mgcl2 , ph 7.5 , containing 2 mm sodium azide ) , glycogen 
 as maltosyl acceptor , 50 nm glge ( 0.01 u ) , 250 m m1p , and varied 
 concentration of inhibitor 9 .
inhibition was determined 
 by comparing the relative rate of the reaction performed with inhibitor 
 against a reaction that contained no inhibitor ( vi/ vi ) , where vi and vi are steady - state rates with and without inhibitor , respectively . 

the equilibrium dissociation constant ( ki ) for inhibitor 9 for mtb glge found 
 to be 237 m  27 and sco glgei - v279s 
 found to be 102 m  7.52 .
these values are near the reported mtb glge km of 250 m 
  5 .
we have initiated efforts to develop transition - state inhibitors 
 for glge by replacing of the endocyclic oxygen atom of glycosides 
 with a nitrogen atom by considering the structures of natural products 
 that inhibit glycoside hydrolases .
we anticipate the resulting secondary 
 ammonium moiety will mimic that partial positive charge accumulation 
 at the anomeric carbon of the true substrate and form a strong interaction 
 with the asp418 nucleophile conserved in this enzyme class .
the confluence 
 of these interactions would therefore promote reasonable inhibition 
 of all glge homologues .
we were able to synthesize 2,5-dideoxy-3-o--d - glucopyranosyl-2,5-imino - d - mannitol ( 9 ) by following a convergent glycosylation 
 strategy followed by in situ deprotection and intramolecular reductive 
 amination .
the h , c nmr and optical rotation 
 data are in accordance with the literature values .
we evaluated the ki for both mtb glge and sco glgei - v279s using a pnp - based 
 coupled enzyme assay we developed .
we also tested whether 2,5-dideoxy-3-o--d - glucopyranosyl-2,5-imino - d - mannitol ( 9 ) would inhibit the coupling enzyme pnp 
 and no inhibition was observed ( supporting information , figure s-21 ) . since compound 9 inhibited both mtb glge and sco glgei - v279s to a similar 
 degree , this confirms that sco glgei - v279s can be 
 used as a surrogate for mtb glge due to its similarity 
 to the mtb glge maltosyl donor site , enzymatic activity , 
 laboratory stability and ease of crystallization .
we are currently 
 in the process of addressing the detailed mode of interaction of 2,5-dideoxy-3-o--d - glucopyranosyl-2,5-imino - d - mannitol ( 9 ) with sco glgei - v279s 
 via x - ray crystallography .
the data from those ongoing studies are 
 expected to provide valuable insight concerning the development of 
 more potent transition - state inhibitors for mtb glge .
we evaluated the inhibitory activity of 9 against mtb glge and sco glgei - v279s .
the ki values were 237  27 m for mtb glge and ki 102  7.52 
 m for sco glgei - v279s , respectively .
compound 9 has been successfully cocrystallized with sco glgei - v279s and the x - ray diffraction data will be published 
 elsewhere .
the data will help us develop an improved class of transition 
 state - based glge inhibitors which may lead to the discovery of new 
 therapies to treat tb .
all fine chemicals such as d-(+)-glucose monohydrate , benzyl bromide , acetic anhydride , p - thiocresol , benzoyl chloride , dibutyltin oxide , 5% palladium 
 on activated carbon , trifluoroacetic acid , sodium azide , and anhydrous 
 solvents such as anhydrous methanol and n , n - dimethylformamide were purchased and used without purification . 

tlc ( silica gel 60 , f254 ) was visualized under uv light 
 or by charring ( 5% h2so4meoh ) .
flash 
 column chromatography was performed on silica gel ( 230400 
 mesh ) using solvents as received .
h nmr were recorded 
 either on 600 mhz spectrometer in cdcl3 using residual 
 chcl3 as internal references , respectively .
c nmr were recorded on the 600 mhz in cdcl3 using the 
 triplet centered at  77.27 as internal reference .
high resolution mass 
 spectrometry ( hrms ) was performed on a micro mass q - tof2 instrument . 

d-(+)-glucose 
 ( 10 ) ( 3.00 g , 16.6 mmol ) was suspended in dry pyridine 
 ( 6.71 ml , 83.2 mmol ) and acetic anhydride ( 7.8 ml , 83.0 mmol ) , and 
 a catalytic amount of 4-(dimethylamino)pyridine was added .
the solution 
 was stirred at ambient temperature for 16 h. the reaction mixture 
 was diluted with ethyl acetate and washed successively with 1 n hcl 
 ( 30 ml ) and saturated aq nahco3 ( 60 ml ) .
the resulting 
 organic phase was dried ( anhydrous na2so4 ) and 
 filtered , and the filtrate concentrated under reduced pressure to 
 give product 11 : yield 86% ( 5.6 g ) ; silica gel tlc rf = 0.5 ( 1:1 acetone  hexane ) ; h nmr ( cdcl3 , 600 mhz )  2.02 ( d , j = 12 hz , 9h ) , 2.10 ( d , j = 12 hz , 6h ) , 
 3.83 ( dd , j = 1.8 hz , 6 hz , 1h ) , 4.10 ( d , j = 6 hz , 1h ) , 4.28 ( d , j = 12 hz , 1h ) , 
 5.115.26 ( m , 3h ) , 5.70 ( d , j = 12 hz , 1h ) 
 ppm . peracetylated glucose ( 11 ) ( 2.0 g , 5.1 mmol ) 
 was dissolved in dichloromethane ( 20 ml ) . to the solution were added 
 by p - thiocresol ( 955 mg , 7.60 mmol ) and boron trifluoride  diethyl 
 etherate ( 0.7 ml , 5.6 mmol ) at 0 c .
the reaction 
 was monitored by tlc and appeared to be complete after 16 h. the reaction 
 mixture was diluted with dichloromethane ( 14 ml ) and successively 
 washed with saturated aq nahco3 ( 16 ml ) , 10% aq nacl ( 8 
 ml ) , and water ( 16 ml ) .
the resulting organic phase was dried ( anhydrous 
 na2so4 ) and filtered , and the filtrate was concentrated 
 under reduced pressure to give product 12 .
the product 
 was purified by silica gel flash column chromatography by eluting 
 with 1:9 acetone  hexane .
the product fractions were combined , 
 concentrated , and dried in vacuum to afford a white crystalline product 
 to give product 12 : yield 85% ( 1.9 g ) ; silica gel tlc rf = 0.61 ( 2:8 acetone  hexane ) ; h nmr ( cdcl3 , 600 mhz )  2.0 ( d , j = 12 hz , 6h ) , 2.09 ( s , 6h ) , 2.35 ( s , 3h ) , 3.69 ( dd , j = 1.8 hz , 6 hz , 1h ) , 4.19 ( m , 2h ) , 4.63 ( d , j = 6 hz , 1h ) , 4.925.03 ( m , 2h ) , 5.21 ( d , j = 12 hz , 1h ) , 7.127.40 ( m , 4h ) ppm .
compound 12 ( 800 mg , 1.76 mmol ) was deacetylated by dissolving in dry methanol 
 followed by addition of a catalytic amount of sodium metal until the 
 solution reached ph 9 .
the reaction was neutralized by adding amberlite ira-118h h resin until the ph reached 7 .
then resin was filtered away 
 and the filtrate was concentrated under reduced pressure to afford 
 the compound 13 : yield 95% ( 0.48 g ) ; h nmr 
 ( cdcl3 , 600 mhz ) :  2.42 ( s , 3h ) , 3.313.52(m , 
 4h ) , 3.79 ( dd , j = 6 hz , 12 hz , 1h ) , 3.98 ( d , j = 12 hz , 1h ) , 4.64 ( d , j = 10.2 hz , 1h ) , 
 7.237.59 ( m , 4h ) ppm .
a solution of compound 13 ( 462 mg , 1.61 mmol ) in dry n , n - dimethylformamide ( 10 ml ) was cooled to 0 c . the solution 
 was treated dropwise with a suspension of sodium hydride ( 60% dispersion 
 in mineral oil ) ( 452 g , 11.3 mmol ) .
benzyl bromide ( 1.14 ml , 9.66 
 mmol ) was added dropwise over 30 min and the solution stirred at room 
 temperature for 16 h. the reaction was poured over ice and extracted 
 with diethyl ether ( 10 ml ) .
the resulting organic phase was dried ( anhydrous na2so4 ) and filtered , and the filtrate concentrated 
 under reduced pressure to obtain a product .
the product was purified 
 by silica gel flash column chromatography by eluting with 1:9 acetone  hexane . 

the product fractions were combined , concentrated , and dried in vacuum 
 to afford a yellow solid product 14 : yield 85% ( 1.87 g ) ; 
 silica gel tlc rf = 0.61 
 ( 8:2 hexane  ethyl acetate ) ; h nmr ( cdcl3 , 600 mhz )  2.32 ( s , 3h ) , 3.50 ( t , j = 12 
 hz , 2h ) , 3.643.81 ( m , 4h ) , 4.554.92 ( m , 8h ) , 7.047.52 
 ( m , 20h ) ppm .
to a suspension of d-(+)-fructose ( 15 ) ( 10.0 
 g , 55.5 mmol ) and acetone ( 200 ml ) was added concentrated sulfuric 
 acid ( 0.97 ml ) .
the reaction was cooled to 0 c and was quenched with naoh ( 3.0 
 g ) in h2o ( 28 ml ) .
the organic phase was 
 separated , washed with water and brine , and then dried over anhydrous 
 mgso4 .
after filtration and removal of solvents under reduced 
 pressure , the residue was recrystallized with ether / hexane to afford 
 the product as white solid 16 : yield 59% ( 8.5 g ) ; silica 
 gel tlc rf = 0.41 ( 1:1 
 ethyl acetate  hexane ) ; h nmr ( cdcl3 , 
 600 mhz )  1.36 ( s , 3h ) , 1.41 ( s , 3h ) , 1.49 ( s , 3h ) , 1.56 ( s , 
 3h ) , 2.11 ( d , j = 6.0 hz , 1h ) , 3.71 ( dd , j = 6.0 , 18 hz , 2h ) , 3.79 ( d , j = 12 hz , 
 1h ) , 3.92 ( dd , j = 1.8 hz,13.2 hz , 1h ) , 4.25 ( dd , j = 1.2 hz , 7.8 hz , 1h ) , 4.35 ( d , j = 2.4 
 hz , 1h ) , 4.62 ( dd , j = 3 hz , 8.4 hz , 1h ) ppm . to a solution of 16 ( 4.0 g , 15.7 mmol ) in 
 dry n , n - dimethylformamide ( 10 ml ) 
 was added sodium hydride ( 524 mg , 31.5 mmol ) at room temperature . 

the solution was stirred for 0.5 h , and then benzyl bromide ( 1.3 ml , 
 31.5 mmol ) was added .
the reaction was further stirred for 1 h at 
 room temperature , diluted with water , and then extracted with diethyl 
 ether .
the combined extracts were washed with water and brine , dried 
 over anhydrous mgso4 , and concentrated under reduced pressure . 

the residue was purified by flash chromatography on silica gel with 
 1:9 acetone  hexane to give 3-o - benzyl-1,2-o - isopropylidene--d - fructopyranose ( 17 ) as an oily product : yield 86% ( 4.76 g ) ; silica gel tlc rf = 0.34 ( 3:7 acetone  hexane ) ; h nmr ( cdcl3 , 600 mhz )  1.33 ( s , 3h ) , 1.41 
 ( s , 3h ) , 1.43 ( s , 3h ) , 1.55 ( s , 3h ) , 3.61 ( dd , j = 
 12.0 hz , 30 hz , 2h ) , 3.74 ( d , j = 12 hz , 1h ) , 3.91 
 ( d , j = 6 hz , 1h ) , 4.25 ( d , j = 
 6 hz , 1h ) , 4.454.68 ( m , 2h ) , 7.287.34 ( m , 5h ) ppm .
a solution of benzyl ether 17 ( 1.0 g , 3.2 
 mmol ) in 60% acetic acid in water was stirred overnight at 55 c . 

after evaporation of solvent , the residue was purified by flash chromatography 
 on silica gel with 1:9 acetone  hexane to give the diol 18 as a white solid : yield 76% ( 675 mg ) ; silica gel tlc rf = 0.25 ( 3:7 ethyl acetate 
hexane ) ; h nmr ( cdcl3 , 600 mhz )  1.30 ( s , 3h ) , 1.51 
 ( s , 3h ) , 3.18 ( d , j = 6 hz , 1h ) , 3.48 ( d , j = 6 hz , 1h ) , 3.72 ( d , j = 12.0 hz , 1h ) , 
 3.734.05 ( m , 2h ) , 4.57 ( d , j = 12 hz,1h ) , 
 4.67 ( d , j = 12 hz , 1h ) , 7.317.38 ( m , 5h ) 
 ppm . to a stirred solution of 18 ( 500 mg , 1.61 mmol ) in anhydrous methanol ( 5 ml ) was added dibutyltin 
 oxide ( 441 mg , 1.77 mmol ) .
the suspension was heated for 7 h under 
 reflux and then concentrated to afford the 3,4-dibutylstannylene derivative 
 as a solid foam .
the 
 foam was taken up in triethylamine ( 0.24 ml , 1.77 mmol ) in anhydrous 
 dioxane ( 5 ml ) and cooled ( 0 c ) .
to the solution was added a 
 solution of benzoyl chloride ( 0.21 ml , 1.77 mmol ) in the same solvent 
 ( 1 ml ) by dropwise addition .
the reaction was allowed to warm to room 
 temperature and was stirred for an additional 4 h. tlc revealed the 
 presence of two new compounds of higher mobility .
methanol ( 5 ml ) 
 was added , and after 30 min the mixture was concentrated and the residue 
 was subjected to a flash chromatography with 1:9 acetone - hexane to 
 afford first pure 19 : yield 38% ( 253 mg ) ; silica gel 
 tlc rf = 0.26 ( 3:7 acetone  hexane ) ; h nmr ( cdcl3 , 600 mhz )  1.47 ( s , 3h ) , 1.51 
 ( s , 3h ) , 3.79 ( d , j = 12.0 hz , 1h ) , 3.90 ( dd , j = 1.8 hz , 12 hz , 1h ) , 4.0 ( dd , j = 1.8 
 hz , 13 hz , 1h ) , 4.124.14 ( m , 2h ) , 4.32 ( dd , j = 6 hz , 12 hz , 1h ) , 4.78 ( d , j = 12 hz , 1h ) , 4.93 
 ( d , j = 12 hz , 1h ) , 5.44 ( m , 1h ) , 7.368.08 
 ( m , 10h ) ppm ; a second elution gave 20 ; yield : 52% ( 350 
 mg ) ; silica gel tlc rf = 0.36 ( 3:7 acetone - hexane ) ; h nmr ( cdcl3 , 600 mhz )  1.44 ( s , 3h ) , 1.54 ( s , 3h ) , 3.81 ( dd , j = 2 hz , 12.0 hz , 1h ) , 4.01 ( m , 2h ) , 4.13 ( m , 1h ) , 4.66 
 ( d , j = 12 hz , 1h ) , 4.87 ( d , j = 
 12 hz , 1h ) , 5.54 ( d , j = 6 hz , 1h ) , 7.298.11 
 ( m , 10h ) ppm . to a solution of 
 triphenylphosphine ( 430 mg , 1.64 mmol ) ,
imidazole ( 223 mg , 3.28 mmol ) , 
 and iodine ( 416 mg , 1.64 mmol ) in dry toluene was added compound 20 ( 340 mg , 0.82 mmol ) .
the solution was heated at 110 c 
 for 2 h. tlc revealed a compound higher mobility than starting material . 

the reaction was cooled , washed successively with 10% aq sodium thiosulfate 
 and brine , and then concentrated .
the concentrated residue was subjected 
 to a flash chromatography with 1:9 acetone  hexane to afford 
 compound 21 : yield 90% ( 0.38 g ) ; silica gel tlc rf = 0.5 ( 3:7 acetone  hexane ) ; h nmr ( cdcl3 , 600 mhz )  1.42 ( s , 3h ) , 1.52 
 ( s , 3h ) , 3.61 ( d , j = 6 hz , 1h ) , 3.86 ( d , j = 12 hz , 1h ) , 3.903.94 ( m , 2h ) , 4.11 ( m , 2h ) , 
 4.47 ( d , j = 12 hz , 1h ) , 4.65 ( d , j = 6 hz , 1h ) , 5.83 ( m , j = 9 hz , 1h ) , 7.138.11 
 ( m , 10h ) ppm . a stirred solution 
 of 21 ( 275 mg , 0.52 mmol ) and sodium azide ( 171 mg , 2.62 
 mmol ) in dry n , n - dimethylformamide 
 ( 10 ml ) was heated at 100 c for 24 h. tlc revealed a slightly 
 slower running compound in comparison to starting material .
the reaction 
 was cooled , diluted with water , and then extracted with diethyl ether . 

the combined extracts were washed with water and brine , dried over 
 anhydrous mgso4 , and concentrated under reduced pressure . 

the residue was purified by flash chromatography on silica gel with 
 1:9 acetone  hexane to give compound 22 as a crystalline 
 product : yield 91% ( 0.23 g ) ; silica gel tlc rf = 0.73 ( 3:7 acetone  hexanes ) ; h nmr ( cdcl3 , 600 mhz )  1.44 ( s , 3h ) , 1.53 
 ( s , 3h ) , 3.78 ( dd , j = 6 hz , 12 hz , 1h ) , 3.99 ( dd , j = 12 hz , 18 hz , 2h ) , 4.05 ( d , j = 6 hz , 
 1h ) , 4.15 ( dd , j = 6 hz , 12 hz , 1h ) , 4.27 ( m , 1h ) , 
 4.67 ( d , j = 12 hz , 1h ) , 4.88 ( d , j = 12 hz , 1h ) , 5.66 ( dd , j = 6 hz , 12 hz , 1h ) , 7.298.14 
 ( m , 10h ) ppm .
the compound 22 ( 0.21 g , 0.47 mmol ) was debenzoylated by being dissolved in dry 
 methanol followed by addition of a catalytic amount of sodium metal 
 until the solution reached ph 9 .
the reaction was neutralized by adding amberlite ira-118h 
 h resin until the ph reached 7 .
the resin was filtered 
 away , and the filtrate concentrated under reduced pressure and purified 
 by flash chromatography on silica gel with 1:9 acetone 
hexane 
 to afford the compound 23 : yield 98% ( 0.15 g ) ; silica 
 gel tlc rf = 0.42 ( 3:7 
 acetone  hexanes ) ; h nmr ( cdcl3 , 600 
 mhz )  1.43 ( s , 3h ) , 1.48 ( s , 3h ) , 3.67 ( d , j = 12 hz , 1h ) , 3.944.05 ( m , 5h ) , 4.19 ( dd , j = 6 hz , 12 hz , 1h ) , 4.76 ( d , j = 12 hz , 1h ) , 4.84 
 ( d , j = 12 hz , 1h ) , 7.317.38 ( m , 5h ) ppm . a mixture of p - methylphenyl 
 2,3,4,6-tetra - o - benzyl-1-thio--d - glucopyranoside ( 14 ) ( 185 mg , 0.280 mmol ) and 5-azido-3-o - benzyl-5-deoxy-1,2,-o - isopropylidene--d - fructopyranose ( 23 ) ( 80.0 mg , 0.23 mmol ) was 
 dissolved in dichloromethane ( 5 ml ) . powdered 4  molecular sieves 

were added ( 200 mg ) to the solution , and the mixture was stirred under 
 n2 at 0 c 1 h. n - iodosuccinamide 
 ( 80.3 mg , 0.350 mmol ) and trimethylsilyl triflate ( 0.017 ml , 0.097 
 mmol ) were added . after being stirred for 10 min ,
the reaction mixture 
 was neutralized with triethylamine , diluted with dichloromethane , 
 and filtered through celite .
the filtrate was washed successively 
 with saturated sodium thiosulfate , water , and brine .
the filtrate 
 was dried over sodium sulfate ( anhydrous ) , filtered , concentrated 
 to dryness , and purified by flash chromatography by eluting with 1:9 
 acetone  hexane to give compound 24 : yield 76% 
 ( 0.35 g ) ; [ ]d = 14.0 ( c = 1 , chcl3 ) ; silica gel tlc rf = 0.45 ( 3:7 ethyl acetate 
hexanes ) ; h nmr ( cdcl3 , 600 mhz )  1.44 ( s , 3h , ch3 ) , 1.48 ( s , 3h , ch3 ) , 3.49 ( dd , 1h , j = 3.6 , 9.6 hz , h-2 ) , 3.57 ( t , 1h , j = 9.6 
 hz , h-6a ) , 3.67 ( m , 1h , h-4 ) , 3.86 ( dd , 2h , j = 9.0 , 29.0 hz , ha , hb ) , 3.97 ( dd , 
 2h , j = 12 , 84 hz , h-6a , b ) , 4.06 ( m , 2h , 
 h-3 , h-6b ) , 4.14 ( m , 1h , h-5 ) , 4.445.15 
 ( m , 10h , ch2ph ) , 4.97 ( d , 1h , j = 3.6 hz , h-1 ) , 
 7.157.31 ( m , 25h , aromatic h s ) , 4.854.37 ( m , 
 14h , ch2ph ) , 5.40 ( d , j = 3.6 
 hz , h-1 ) , 7.32- 7.39 ( m , 35h , aromatic h s )
; c nmr ( 150 mhz , cdcl3 )  26.2 , 27.2 , 61.4 , 61.9 , 
 69.0 , 71.6 , 71.9 , 73.5 , 73.7 , 74.3 , 75.3 , 75.8 , 79.9 , 79.9 , 80.7 , 
 81.9 , 100.4 , 106.0 , 112.3 , 127.5 , 127.6 , 128.1 , 128.5 , 138.0 , 138.3 , 
 138.7 ppm ; mass spectrum ( hrms ) , m / z = 880.3793 ( m + na ) , c50h55n3o10 requires 880.3785 .
a solution of compound 24 in 60% aqueous trifluoroacetic acid was stirred at room 
 temperature for 30 min .
the residue was purified by silica gel 
 flash column chromatography by eluting with 1:9 ethyl acetate
the product fractions were combined , concentrated , and dried in vacuum 
 to afford a yellow solid product 25 : yield 85% ( 1.87 
 g ) ; [ ]d = 10.1 ( c = 
 1 , chcl3 ) ; silica gel tlc rf = 0.61 ( 8:2 hexanes  ethyl acetate ) ; h nmr ( cdcl3 , 600 mhz )  3.36 ( dd , 2h , j = 6 , 72 hz , h-6a , h-6b ) , 3.51 ( dd , 1h , j = 3.6 , 7.2 hz , h-2 ) , 3.62 ( m , 4h s , h-5 , h-4 , h - a , 
 h-6 ) , 3.70 ( d , 1h , j = 18 hz , h-3 ) , 
 3.96 ( d , 1h , j = 18 hz , ha ) , 4.05 ( m , 3h , h-3 , 
 h-4 , h-6 ) , 4.12 ( m , 1h , h-5 ) , 4.45.0 ( m , 10h s , 
 ch2ph ) , 5.01 ( d , 1h , j = 3.6 hz , 

h-1 ) , 7.07- 7.33 ( m , 25h , aromatic h s ) ; c nmr ( 150 mhz , cdcl3 )  55.3 , 57.1 , 60.9 , 61.5 , 
 65.2 , 65.2 , 68.1 , 69.0 , 71.5 , 71.6 , 73.1 , 73.4 , 73.6 , 73.7 , 74.2 , 
 74.9 , 75.2 , 76.0 , 77.0 , 77.2 , 77.9 , 78.9 , 79.7 , 80.2 , 81.8 , 82.3 , 
 96.9 , 98.1 , 100.4 , 127.9 , 128.5 , 128.8 , 137.0 , 137.3 , 137.8 , 137.9 , 
 138.5 , 138.7 ppm ; mass spectrum ( hrms ) , m / z = 840.3472 ( m + na ) , c47h51n3o10 requires 840.3469 .
palladium ( 5% ) on carbon ( catalytic amount ) was added to a solution 
 of compound 25 ( 90.0 mg , 0.08 mmol ) in toluene ( 0.5 ml ) 
 and water ( 0.5 ml ) .
the reaction mixture was stirred at ambient temperature 
 under hydrogen atmosphere ( 98 psi ) for 24 h. the catalyst was filtered 
 away , and reaction mixture was washed with methanol . the filtrate 
 was concentrated and the residue purified by gel permeation column 
 chromatography on sephadex lh-20 ( h2o ) to obtain pure compound 9 as a crystalline solid : yield quantitative ( 27 mg ) ; [ ]d = + 48.5 ( c = 0.1 , h2o ) [ lit .
[ ]d = + 49.5 ( c = 0.1 , h2o ) ] ; h nmr ( cdcl3 , 600 mhz ) 
3.323.93 
 ( m , 12 h , ha , hb , h-1 , h-4 , ha , hb , h-2 , h-3 , h-4 , h-5 , 
 h-6a , b ) , 4.20 ( t , 1h , j = 6 hz , h-2 ) , 
 4.30 ( t , 1h , j = 6 hz , h-3 ) , 5.19 ( d , 1h , j = 3.84 hz , h-1 ) ; c nmr ( 150 mhz , 
 cdcl3 )  57.1 , 58.3 , 60.3 , 62.0 , 63.1 , 69.2 , 70.8 , 
 72.4 , 72.5 , 73.5 , 80.4 , 98.1 ppm ; mass spectrum ( hrms ) , m / z = 326.1463 ( m + h ) , c12h23no9 requires 326.1451 .
the glge ( rv1327c ) gene from m. tuberculosis strain h37rv was pcr amplified using the following primers : 5- 
 cac cat atg agt ggc cgg gca at-3 and 5- aaa gga tcc 
 tca cct gcg cag ca- 3. the product was placed between the 
 ndei and bamhi cut sites of a modified pet-28 plasmid 
 ( emd biosciences ) .
the resulting pdr28-glge encodes a recombinant 
 glge enzyme possessing an n - terminal polyhistidine tag .
the gene encoding 
 the sco glgei - v279s variant was placed between the ndei and xhoi cut 
 sites of pet32 ( emd biosciences ) resulting in plasmid pet32-sco - v279s .
the recombinant protein expressed by this plasmid 
 possesses a c - terminal polyhistidine tag .
the pdr28-glge or pet32-sco - v279s 
 plasmid was used to transform t7 rosetta cells .
the bacterial cells 
 were cultured at 37 c in luria broth to an o.d .
protein expression was induced by the addition of iptg 
 to a final concentration of 1 mm .
pelleted cells were resuspended 
 in buffer a containing 5 mm imidazole , 500 mm nacl , 20 mm tris ph 
 7.5 , 10% glycerol , and 5 mm -mercaptoethanol .
lysozyme ( 10 
 m ) and dnasei ( 100 m ) were added to the cell resuspension 
 and incubated for 1 h on ice prior to lysis by sonication .
the supernatant was applied to a 5 ml hitrap talon crude column ( ge 
 healthcare ) previously equilibrated with buffer a. proteins were eluted 
 from the column by applying a linear gradient of imidizole from 5500 
 mm over 20 column volumes .
fractions containing glge were pooled and 
 applied to a hi - load superdex 200 size - exclusion column for further 
 purification .
glge was eluted from the column isocratically with a 
 buffer containing 150 mm nacl , 20 mm tris ph 7.5 , and 0.3 mm tcep . 

the purity 
 of the protein was confirmed using sds - page and concentrated using 
 ultrafiltration .
assays were performed under atmospheric 
 pressure at 22 c in a 96-well format on a spectra max 340pc 
 ( molecular devices ) .
all 1 mm mesg stock solutions were prepared and 
 stored in dh2o at 20 c .
phosphate release 
 catalyzed by mtb glge was monitored using a coupled 
 assay utilizing purine nucleoside phosphorylase at 5 s intervals for 
 10 min . for each reaction
, a master mix was made consisting of 1 mm 
 mesg ( 20 l ) , 0.2 u of pnp ( 1 l ) , 20 reaction buffer 
 ( 1.0 m tris  hcl , 20 mm mgcl2 , ph 7.5 , containing 
 2 mm sodium azide ) ( 5 l ) , glycogen ( 0.5 l ) , 50 nm glge 
 ( 5 l ) , 250 m m1p ( 3.125 l ) , and 40 mm stock solution 
 of 2,5-dideoxy-3-o--d - glucopyranosyl-2,5-imino - d - mannitol ( 9 ) inhibitor in dh2o and 
 was added for each corresponding stock solution to reach the desired 
 inhibitor concentration ( 0.01.5 mm ) and varied volumes of 
 dh2o for an overall reaction volume of 100 l .
inhibitor 9 was not added to the positive control , and the negative 
 control lacked glge .
inhibition was determined by comparing the relative 
 rate of the reaction performed with inhibitor against a reaction that 
 contained no inhibitor ( vo/vo ) , where vo 
 and vo are steady - state rates with and 
 without inhibitor , respectively .
the equilibrium dissociation constant 
 ( ki ) for inhibitor was obtained by fitting 
 the data into the following equation using prismwhere km is the 
 michaelis  menten constant for m1p and [ s ] and [ i ] are the concentrations 
 of m1p and inhibitor , respectively .
 long treatment times , poor drug compliance , and natural selection 
 during treatment of mycobacterium tuberculosis ( mtb ) have given rise to extensively drug - resistant tuberculosis 
 ( xdr - tb ) . as a result 
 , there is a need to identify new antituberculosis 
 drug targets . 
 mtb glge is a maltosyl transferase 
 involved in -glucan biosynthesis . 
 mutation of glge in mtb increases the concentration of maltose-1-phosphate ( m1p ) , 
 one substrate for glge , causing rapid cell death . 
 we have designed 
 2,5-dideoxy-3-o--d - glucopyranosyl-2,5-imino - d - mannitol ( 9 ) to act as an inhibitor of glge . 
 
 compound 9 was synthesized using a convergent synthesis 
 by coupling thioglycosyl donor 14 and 5-azido-3-o - benzyl-5-deoxy-1,2-o - isopropylidene--d - fructopyranose ( 23 ) to form disaccharide 24 . 
 a reduction and intramolecular reductive amination transformed 
 the intermediate disaccharide 24 to the desired pyrolidine 9 . 
 compound 9 inhibited both mtb glge and a variant of streptomyces coelicolor ( sco ) glgei with ki = 237  
 27 m and ki = 102  7.52 m , 
 respectively . 
 the results confirm that a sco glge - v279s 
 variant can be used as a model for mtb glge . in conclusion 
 , 
 we designed a lead transition state inhibitor of glge , which will 
 be instrumental in further elucidation of the enzymatic mechanism 
 of mtb glge . 
Introduction
Results
Discussion
Conclusion
Experimental Section