selection pressure 
 caused by frequent use of -lactam antibiotics , 
 including penicillins , cephalosporins , carbapenems , and monobactams , 
 has resulted in the emergence and dissemination of highly efficient 
 resistance mechanisms in clinically relevant bacterial pathogens . this rapid adaptation of bacterial strains to commonly used antibiotics 
 is an increasing global threat to public health care . an important mechanism of resistance to -lactam antibiotics , 
 including clinically challenging gram - negative organisms , involves 
 -lactamase catalysis . -lactamases can be classified 
 into those employing a nucleophilic serinyl residue at their active 
 site ( serine--lactamases , sbls , classes a , c , and d ) and those 
 employing one or two zinc ions to promote hydrolysis ( metallo--lactamases , 
 mbls , class b ) . the combination of a penicillin 
 antibiotic and an sbl inhibitor has substantially extended the utility 
 of the former , as , for example , in the case of amoxicillin and clavulanic 
 acid ( augmentin ) . in addition to clavulanic 
 acid , two other class a ( penicillinase ) -lactamase inhibitors 
 are used , tazobactam and sulbactam ; all three inhibitors are themselves 
 -lactams , though in contrast to penicillins they react with 
 sbls to form relatively stable acyl - enzyme complexes . -lactam antibiotics have 
 been developed that possess resistance to sbls , or as in the case 
 of carbapenems , they have been developed to be inhibitors or relatively 
 poor substrates . however , since the pioneering 
 introduction of the sbl - targeting inhibitors , progress in the development 
 of clinically useful , -lactamase - specific inhibitors has been 
 limited . in the case of sbls , at least one compound , avibactam , is 
 currently in clinical trials as a combined class a and class c sbl 
 inhibitor . mbls , which , unlike the 
 sbls , are not structurally or mechanistically 
 related to the penicillin - binding proteins ( pbps ) , were first identified 
 approximately 50 years ago . however , as 
 a result of their apparently restricted distribution to the chromosomes 
 of less pathogenic species , they were long not considered to represent 
 a significant threat to the clinical effectiveness of -lactams . the emergence of horizontally acquired mbls , where horizontal gene 
 transfer permits genetic material to be transferred between individual 
 bacteria of the same species or between different species , has led 
 to their extensive dissemination across geographical and species boundaries 
 and now threatens the effectiveness of almost all -lactams , 
 including carbapenems ( i.e. ,  last resort  antibiotics ) ; the monobactam aztreonam is a current exception . mbls identified on transferrable plasmids ( e.g. , the ndm- , imp- , vim- , 
 gim- , and spm - type enzymes ) are considered a prevalent and immediate 
 clinical problem . various structurally 
 diverse types of mbl inhibitors have been described , including carboxylic / succinic 
 acids , triazoles / tetrazoles , thiols , trifluoromethyl 
 ketones , and others . to date , no clinically useful mbl inhibitor has been reported . this , in part , may reflect the technical and scientific challenges 
 in the development of an mbl-lactam - based combination 
 therapy . there is also extensive structural diversity in the mbl family , 
 particularly in the proposed mobile loop regions around the active 
 site . for an mbl inhibitor to be clinically 
 useful , it is possible that more than one mbl will need to be inhibited . 
 in an effort to help enable work that will lead to the development 
 of useful mbl inhibitors , we here describe an assay platform for clinically 
 relevant mbls , including protein production procedures and assay conditions 
 using both chromogenic and fluorogenic substrates several screening methods for the in vitro and in vivo detection 
 of -lactamases have been reported . representative substrates currently in use for -lactamases 
 include chromogenic cephalosporin - based substrates , such as centa , padac , and nitrocefin , cephalosporin - based fluorogenic substrates , bioluminescent probes , and fluorescence resonance energy transfer ( fret)-based substrates . these substrates have mainly been applied in 
 research centered around sbls and only in one case on mbls ( l1 and 
 ccra ) . the large - scale application of 
 these type of compounds in automated high - throughput screening ( hts ) 
 for mbl inhibitors is , however , hampered by their lengthy and often 
 difficult synthesis and the high costs associated with these substrates . more importantly , these substrates suffer from poor substrate recognition 
 by some mbls as a result of the high diversity of this enzyme family 
 whose members vary significantly in sequence , structure , and substrate 
 specificity , thus making it hard to use a single substrate for broad 
 mbl activity screening . the development of new assays for broad - range 
 mbl activity screening , based on hydrolysis of chromogenic or fluorogenic 
 -lactams , would , however , significantly facilitate inhibitor 
 identification . not unimportantly , large - scale inhibitor screening 
 against different mbls needs an inexpensive substrate , thus requiring 
 a simple , scalable , and cost - efficient synthetic route . to develop 
 assay procedures suitable for a range of mbls , we targeted four clinically 
 relevant enzymes as well as the mbl from bacillus cereus ( bcii ) , which has been extensively used as a model enzyme . the clinically 
 relevant enzymes chosen were ndm-1 ( new delhi mbl ) , imp-1 ( imipenemase ) , 
 spm-1 ( so paulo mbl ) , and vim-2 ( verona integron - encoded mbl ) . 
 to identify mbl substrates useful for efficient inhibitor screening , 
 we prepared and tested substrates on the above - mentioned panel of 
 mbls . these substrates included three chromogenic substrates , i.e. , 
 imipenem , nitrocefin , and centa , and five coumarin - linked substrates , 
 i.e. , fc1fc5 ( figure 1 ) . fluorescent 
 substrates fc1 and fc2 release 7-mercapto-4-methylcoumarin upon hydrolysis , 
 resulting in a decrease of fluorescence . conversely , fluorogenic substrates 
 fc3fc5 liberate 7-hydroxycoumarin upon hydrolysis of the lactam 
 ring , producing an increased fluorescence signal ( figure 2 ) . substrates for metallo--lactamase activity measurements . hydrolysis of substrates by mbls , resulting 
 in either an increase 
 or a decrease of fluorescence . details are provided in the supporting information . for the production of 
 recombinant bcii , imp-1 , and ndm-1 mbls in escherichia 
 coli , vectors that have been previously described 
 were used , i.e. , the pet9a - bcii , pet-26b imp-1 , and popinf ndm-1 plasmids . in the case of the mbls vim-2 and spm-1 , these vectors encode the mature vim-2 and spm-1 sequences ( i.e. , 
 with the periplasmic export sequences removed ) fused to n - terminal hexahistidine tags , cleavable with 3c protease . the vim-2 
 and spm-1 genes were codon optimized for e. coli ( genscript , piscataway , nj ) and inserted into the popinf vector . all the mbls were produced using in - house - constructed e. coli bl21(de3 ) plys cells , which were grown in 
 2ty medium . the purity of the mbl proteins was determined to be > 95% 
 by 
 sds  page analysis ( see the supporting information , figure si_4 ) . cleavage 
 of 5,5-dithiobis(2-nitrobenzoic acid ) by dithiothreitol ( dtt ) 
 yielded 3-carboxyl-4-nitrothiophenol , which was subsequently used 
 to convert commercially available cephalothin into centa by heating 
 ( 65 c , 3 h ) . use of chromatographically purified 3-carboxyl-4-nitrothiophenol 
 gave centa in high purity and satisfactory yield after a single acid / base 
 extraction . fluorogenic cephalosporin 
 1 ( fc1 ) was prepared from commercially available p - methoxybenzyl ( pmb ) ester - protected chlorocephalosporin ( cc ) via 
 substitution with 7-mercapto-4-methylcoumarin ( mmc ) , followed by acid - mediated 
 pmb deprotection ( scheme 1a ) . treatment of 
 cc with mmc resulted in the formation of both the 2- and 3-alkene isomers of compound 1 . subjecting the 23 isomer 
 mixture to acid - mediated pmb deprotection conditions led to complete 
 deprotection within 1 h , generating fc1 ( 23 mix ) . to circumvent the 23 isomerization issue , oxidation of the thiazine sulfur atom 
 with mcpba was performed prior to alkylation ( scheme 1a ) . the 3-alkene ( s)-sulfoxide 2 was 
 obtained in good yield ( 72% ) ; subsequent alkylation with mmc in the 
 presence of n , n-diisopropylethylamine 
 gave 3 as a single isolate isomer in reasonable yield 
 ( 49% ) . notably , mmc appears to be nonfluorescent in its free form , whereas 
 the thioether cephalosporin derivatives ( i.e. , 1 and 3 ) showed significant fluorescence . while this was not our 
 objective , fc2 has potential as an  inverse fluorogenic  
 substrate ( for spectroscopic data see the supporting 
 information , figure si_1 ) . reagents and conditions : ( a ) 
 mmc , dipea , dmf , rt , 2 h ; ( b ) tfa / anisole ( 5:1 ) , 0 c , 30 min ; 
 ( c ) mcpba ( 1 equiv ) , ch2cl2 , 0 c , 1 h ; ( d ) nai ( 10 equiv ) , acetone , rt , 2 h , ( e ) 7-hc , 
 k2co3 , mecn , rt , 4 h ; ( f ) mcpba ( 2 equiv ) , ch2cl2 , 0 c , 3 h. with the aim of developing a fluorogenic mbl substrate , 
 we focused 
 on 7-hydroxycoumarin ( 7-hc ) cephalosporin derivatives , with the knowledge 
 that umbelliferone shows strong fluorescence in its free form while 
 being nearly nonfluorescent when ether - derivatized . during the course of our investigations , rao and co - workers 
 reported the synthesis and application of fc3 and fc4 as sbl fluorogenic 
 substrates ( specific for balc over tem-1 ) . their reported procedure ( with minimal 23 isomerization ) for the synthesis of fc3 and fc4 proved to 
 be efficient and yielded the desired fluorogenic probes ( fc3 and fc4 ) 
 in satisfactory yields ( scheme 1b ) . besides 
 substrates fc3 ( sulfide ) and fc4 ( ( s)-sulfoxide ) , 
 we also prepared substrate fc5 ( sulfone ) . treatment of compound 4 with 2 equiv of mcpba gave clean conversion 
 to compound 6 , which upon pmb deprotection yielded substrate 
 fc5 in good yield . initially time - dependent 
 changes in absorbance and/or emission spectra upon hydrolysis of the 
 potential mbl substrates were recorded to investigate the optimal 
 wavelengths for analysis ( see the supporting information , figures si_2 and si_3 ) . for the frequently used chromogenic substrates , 
 i.e. , imipenem ( carbapenem ) and nitrocefin and centa ( cephalosporins ) , 
 this resulted in preferred readout absorption wavelengths of 300 , 
 405 , and 492 nm , respectively . for fluorogenic substrates fc3fc5 the absorption and emission spectra of umbelliferon in hepes assay 
 buffer ( ph 7.5 ) were measured and their corresponding optimal wavelengths 
 applied in the fluorescence - based assay ( ex = 380 
 nm and em = 460 nm ) . substrate 
 combinations . the data for the chromogenic substrates ( table 1 ) are generally in good agreement with previously 
 reported data , with the only substantial dissimilarity 
 being the lower km values obtained by 
 us in the case of centa , possibly because of increased purity of the 
 substrate . direct quantitative comparison of the obtained data with 
 reported values is , however , difficult as a result of variations in 
 protein production conditions , concentration of metal ions added , 
 etc . the improved stability of centa compared to imipenem renders 
 it an attractive substrate for inhibitor identification . however , 
 mbl inhibition by the hydrolyzed cephalosporin products ( for bcii , cpha , and sfh - i ) or the liberated thiophenol from centa ( for 
 imp-1 ) could complicate interpretation of the results . examination 
 of the data in table 1 reveals the high sensitivity 
 of the fluorogenic mbl substrates , which enable the use of enzyme 
 concentrations ( generally 1 nm ) well below those used with 
 chromogenic substrates ( generally 150 nm ) . the enzyme concentrations of the 
 purified proteins were determined using a nanodrop spectrometer ; concentrations 
 of diluted solutions used in the assay were calculated from the original 
 concentration . the following apparent ki value for substrate / product inhibition was 
 determined : 
 36.6  15.2 . in the 
 case of the combination of ndm-1 and fc3fc5 , up 
 to 520 times lower enzyme concentration was used as compared 
 to enzyme concentrations required for imipenem or nitrocefin . further 
 testing of ndm-1 with the three fluorogenic substrates fc3fc5 , 
 with different sulfur oxidation states , disclosed significantly different kcat / km values ( up 
 to 20-fold ) , implying catalytic efficiency is altered by the oxidation 
 state of the thiazine sulfur atom . we observed that the stability 
 of fc3 in aqueous solution is substantially lower than that of fc4 
 and fc5 . autohydrolysis studies in buffer at room temperature showed 
 substantial hydrolysis of fc3 after 3 h , while both fc4 and fc5 were 
 found to be stable for at least 8 h. solutions of fc4 and fc5 could 
 be used for 12 days when stored on ice . moreover , dmso stock 
 solutions , stored at 20 c , could be used several months 
 after preparation without any detectable decomposition . storage of 
 compounds fc4 and fc5 as solids at 20 c enabled use 
 of these compounds over a prolonged period of time ( i.e. , months ) . 
 as a result , fc4 and fc5 were tested on the other mbls . for 
 nearly all the clinically important mbls , fc4 and fc5 showed 
 higher kcat / km values than the chromogenic substrates ( with vim-2 being the only 
 exception ) . on analysis of the vim-2 results , we found that fc5 is 
 preferred over fc4 and performs equally well as nitrocefin , having 
 similar kcat values though slightly different km values ( 7.2 m vs 15.2 m , respectively ) 
 and consequently different kcat / km values ( 31.2 ms vs 19.1 ms ) . the kinetic data of the five substrates for imp-1 reveal 
 that fc4 
 outperforms all other substrates mainly as a result of its high kcat value ( i.e. , 807 ms ) . as depicted in figure 2a substrate concentrations up to 100 m could be applied 
 without perturbing the measurement . although fc4 has a low km compared to the other substrates tested on 
 imp-1 , its high sensitivity allows for accurate measurement as indicted 
 by the low interexperimental error ( figure 3a , experiment performed in triplicate ) . as a result of the high sensitivity of both fc4 and fc5 , a 20-fold 
 lower imp-1 concentration could be used ( i.e. , picomolar enzyme concentration 
 range ) while retaining a km value in the 
 micromolar range , thus allowing for the detection of weak binding 
 fragments or slow - binding inhibitors . errors are reported as standard errors , n = 3 . in the case of spm-1 , 
 both fc4 and fc5 outperform all the tested 
 chromogenic substrates . interestingly , we found that spm-1 is inhibited 
 by nitrocefin / nitrocefin - derived species at a concentration of 36 
 m ( see figure 3b and table 1 ) . although -lactams can act as inhibitors 
 of mbls , as is apparent for nitrocefin and imipenem at high concentrations 
 ( e.g. , nitrocefin for spm-1 , this paper , and for cpha and sfh - i , refs ( 45 ) and ( 42 ) , respectively ) ; the high 
 sensitivity of the here reported fluorogenic substrates ( fc4 and fc5 ) 
 allows for the use of extremely low substrate concentrations . in an attempt to investigate the 
 different substrate binding constants of nitrocefin , imipenem , centa , 
 and fc4 for the different mbls , we performed in silico docking studies . docking of nitrocefin , imipenem , centa , and fc4 in imp-1 , ndm-1 , and 
 vim-2 ( pdb ids 1jjt,3q6x , and 1ko3 , respectively ) was performed using autodock 4 and sprout software . after analysis of the obtained unhydrolyzed enzyme models 
 ( see the supporting information ) , the different 
 docking poses , calculated by autodock 4 and sprout , were scored and 
 compared to the substrate binding constants ( km ) ( table si_1 , supporting information ) . assessment of the sprout docking scores generally reveals a better 
 correlation with the kinetic parameters obtained in vitro ( specifically km values in the cases of ndm-1 and vim-2 ) compared 
 to the docking scores generated by autodock 4 . however , for all three 
 enzymes , the highest ranking substrate in terms of the sprout and 
 autodock scores is fc4 , which was also measured to have the highest 
 binding affinity for all three enzymes . although in - depth structural 
 analyses are required , these modeling results suggest that the cephalosporins 
 may bind to the active site in more than one orientation , including 
 catalytically nonproductive ones . substrate 
 combinations and the need for more structural analyses on mbl  substrate 
 complexes . to achieve clinical 
 utility , this is 
 potentially challenging because of the generally low sequence similarity 
 between mbls of different subfamilies ( b1 , b2 , b3 ) . most clinically 
 relevant mbls are from the b1 subfamily ; however , spm-1 presents a 
 high similarity with both the b1 and b2 subfamilies and hence was used as a starting point for proof of principle 
 inhibitor screening . to validate our screening method , we screened 
 a series of 4-chloroisoquinolinol derivatives ( 7a7 g ) against spm-1 using fc4 as 
 a reporter substrate ( table 2 ) . this set of 
 compounds was selected as a result of their previously reported inhibition 
 of metal - binding oxygenases . screening 
 for residual activity of compounds 7a7 g , at concentrations of 200 m and 1 mm , revealed that the 4-chloroisoquinolinols 
 bearing a phe- , tyr- , or val - based side chain were poor inhibitors . in contrast , the compounds derivatized with a trp , leu , asp , or glu 
 residue inhibited spm-1 , with the tryptophan - functionalized chloroisoquinolinols 
 ( s)-7d and ( r)-7d giving the most promising results ( 3% and 8% residual activity 
 ( ra ) at 200 m , respectively ) . for the 
 remaining activity was measured 
 by applying a 10 min preincubation time used in previous mbl work ; 
 see refs ( 18a ) and ( 44 ) . following from the positive screening results obtained 
 with the 
 spm-1/fc4 pair , ic50 values were determined for ( s)-7d and ( r)-7d against our mbl panel using fc4 and the conditions reported in table 1 ; the fc4 concentrations correspond to the experimentally 
 determined km values ( table 1 ) . the ic50 values ( table 3 ) reveal that both ( s)-7d and ( r)-7d are relatively weak , but broad - spectrum 
 mbl inhibitors with ic50 values ranging from 20 to 130 
 m . since the inhibitor activity of these compounds is in the 
 low micromolar range , they may serve as good starting points for further 
 optimization . ic50 determinations were 
 performed in duplicate over a range of 0.25200 m . to further validate our assay 
 platform , we measured the ic50 value of the reported inhibitor l - captopril against ndm-1 . measuring 
 at the km value of fc4 on ndm-1 , we obtained 
 an ic50 value of 10.0  1.9 m corresponding 
 to a ki of 5.0 m for l - captopril . using imipenem as the reported 
 substrate , an 
 ic50 of 13.2  1.5 m corresponding to a ki of 6.6 m were determined . thus , under 
 the same assay conditions , we found that the fluorogenic substrate 
 ( fc4 ) and the chromogenic substrate ( imipenem ) produced similar ic50 and ki values . however , our 
 values are lower than the previously reported ic50 and ki values for l - captopril on ndm-1 using 
 imipenem as the reported substrate ( 202 m and 39 m , respectively ) , 
 possibly reflecting differences in assay conditions , protein constructs , 
 and enzyme production procedures . the 
 development of broad - screen mbl inhibitors has been hampered 
 by the lack of an appropriate , efficient screening platform for multiple 
 mbls . the development of such a screening platform has been hindered 
 by the lack of suitable detection substrates . the results of our comparative 
 study , employing five substrates and five mbls , reveal striking differences 
 in the kinetic parameters for different enzyme  substrate combinations . fluorogenic substrates fc4 and fc5 are highly sensitive and efficient 
 substrates for the clinically relevant mbls , with one or both of them 
 having kcat / km values of > 10 ms . the suitability of fc4 for inhibitor screening was demonstrated 
 by testing a set of 4-chloroisoquinolinols as mbl inhibitors . this 
 work yielded two compounds ( ( s)-7d and 
 ( r)-7d ) that displayed inhibitory activity 
 against our panel of mbls , albeit at moderate inhibition concentrations 
 ( m ) ; nonetheless , the results reveal the feasibility of broad - spectrum 
 mbl inhibition . the supporting information contains 
 a complete general experimental section , including all procedures 
 and equipment used . in general , chemicals were purchased from commonly 
 used suppliers ( aldrich , acros , alfa aesar , and tci ) and were used 
 without further purification . ( 6r,7r)-4-methoxybenzyl 3-(chloromethyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-aza - bicyclo[4.2.0]oct-2-ene-2-carboxylate 
 ( pmb - protected cep - cl ) was obtained from activate scientific ( prien , 
 germany ) . to a suspension of cl - cep - opmb ester ( 680 
 mg , 1.4 mmol ) in acetone ( 10 ml ) was added nai ( 2.10 g , 14.0 mmol , 
 10 equiv ) . the reaction was stirred at rt for 2 h , after which the 
 solvent was removed in vacuo . the crude mixture was partitioned between 
 h2o ( 10 ml ) and etoac ( 10 ml ) , and the layers were separated . 
 the h2o layer was extracted with etoac ( 2  15 ml ) , 
 after which the combined organic layers were washed with a 5% nas2o3 aq solution ( 2  20 ml ) and brine ( 20 ml ) . the organic layer was dried over na2so4 , filtered , 
 and concentrated in vacuo . the crude product was dissolved in mecn 
 ( 15 ml ) , and 7-hydroxycoumarin ( 454 mg , 2.8 mmol , 2 equiv ) and k2co3 ( 4.2 mmol , 4 equiv ) were added . the conversion 
 of the reaction was monitored by tlc analysis ( chex / etoac , 1:1 ) , and 
 completion was reached after 4 h. the solvent was evaporated in vacuo , 
 and the resulting crude mixture was partitioned between h2o ( 10 ml ) and etoac ( 10 ml ) . after separation of the layers , the 
 h2o layer was extracted with etoac ( 2  15 ml ) , after 
 which the combined organic layers were washed with a 5% nas2o3 aq solution ( 2  20 ml ) and brine ( 20 ml ) . the 
 organic layer was dried over na2so4 , filtered , 
 and concentrated in vacuo . the crude product was purified by column 
 chromatography ( chex / etoac , 1:1 ) to yield compound 4 as 
 a light brown solid ( 392 mg , 46% ) . h nmr ( 400 
 mhz , dmso - d6 ) :  = 9.20 ( d , j = 8.0 hz , 1h ) , 7.98 ( d , j = 9.5 hz , 1h ) , 
 7.59 ( d , j = 8.5 hz , 1h ) , 7.17 - 7.33 ( m , 8h ) , 6.92 
 ( d , j = 1.5 hz , 2h ) , 6.87 ( dd , j = 8.5 , 2.5 hz , 1h ) , 6.79 ( d , j = 8.5 hz , 2h ) , 6.30 
 ( d , j = 9.5 hz , 1h ) , 5.44 ( dd , j = 7.5 , 4.0 hz , 1h ) , 5.115.19 ( m , 2h ) , 5.015.10 ( m , 
 app d , 2h ) , 4.644.76 ( m , app q , 2h ) , 3.69 ( s , 3h ) , 3.473.57 
 ( m , 2h ) ppm . c nmr ( 101 mhz , dmso - d6 ) :  = 170.9 , 167.0 , 163.8 , 160.9 , 160.3 , 159.3 , 155.2 , 
 144.3 , 135.7 , 130.0 ( 2c ) , 129.5 , 129.1 ( 2c ) , 128.2 ( 2c ) , 126.9 , 126.5 , 
 122.4 , 118.8 , 113.7 ( 2c ) , 112.9 , 112.7 ( 2c ) , 101.4 , 69.8 , 67.2 , 60.8 , 
 55.0 , 52.9 , 49.7 , 41.6 ppm . lrms : mass calcd for c33h27n2o8s ( m  h ) 611.15 , mass found 
 ( m  h ) 611.0 . to a suspension of compound 4 ( 122 mg , 0.2 mmol ) in dry ch2cl2 ( 5 
 ml ) cooled to 0 c was added mcpba ( 45 mg , 0.2 
 mmol , 1 equiv ) . the reaction was stirred at 0 c for 30 min followed 
 by an additional 1 h at rt ( a white precipitate was formed ) . the crude 
 product was dry - loaded onto silica and purified by column chromatography 
 ( ch2cl2/etoac , 8:2 ) to yield compound 5 as a cream white solid ( 60 mg , 48% ) . h nmr ( 400 mhz cdcl3 ) :  = 7.64 
 ( d , j = 9.5 hz , 1h ) , 7.277.40 ( m , 8h ) , 6.91 
 ( d , j = 8.5 hz , 2h ) , 6.746.78 ( m , 2h ) , 6.67 
 ( d , j = 10.0 hz , 1h ) , 6.30 ( d , j = 9.5 hz , 1h ) , 6.10 ( dd , j = 10.0 , 5.0 hz , 1h ) , 
 5.225.35 ( m , app mix of d and q , 3h ) , 4.80 ( d , j = 13.5 hz , 1h ) , 4.45 ( dd , j = 5.0 , 1.5 hz , 1h ) , 
 3.99 ( d , j = 19.0 hz , 1h ) , 3.81 ( s , 3h ) 3.65 ( m , 
 app d , j = 5.5 hz , 2h ) , 3.28 ( d , j = 19.0 hz , 1h ) ppm . c nmr ( 101 mhz , dmso - d6 ) :  = 171.1 , 164.5 , 160.7 ( 2c ) , 160.2 , 159.4 , 
 155.2 , 144.3 , 135.8 , 130.4 ( 2c ) , 129.5 , 129.1 ( 2c ) , 128.3 ( 2c ) , 126.8 , 
 126.6 , 125.0 , 119.6 , 113.7 ( 2c ) , 112.8 , 101.6 , 67.5 , 67.3 , 66.5 , 58.4 , 
 55.1 , 45.4 , 41.4 ppm . lrms : mass calcd for c33h27n2o9s ( m  h ) 627.14 , mass found ( m 
  h ) 627.0 . to a suspension of compound 4 ( 100 mg , 0.16 mmol ) in dry ch2cl2 ( 10 
 ml ) cooled to 0 c was added mcpba ( 74 mg , 0.35 
 mmol , 2 equiv ) . the reaction was stirred at 0 c for 30 min ( formation 
 of sulfoxide observed ) , after which the reaction was warmed to rt 
 and stirred overnight . upon completion of the reaction , the crude 
 product was dry - loaded onto silica and purified by column chromatography 
 ( ch2cl2/meoh , 9:1 ) to yield the desired product 
 as a white solid ( 35 mg , 34% ) . h nmr ( 400 mhz , dmso - d6 ) ( small amount 
 of mcba present ) :  = 8.93 ( d , j = 8.5 hz , 1h ) , 8.01 ( d , j = 9.5 hz , 1h ) , 7.877.93 
 ( m , 1h ) , 7.63 ( d , j = 8.5 hz , 1h ) , 7.187.34 
 ( m , 7h ) , 6.946.88 ( m , 2h , 2 signals overlapping ) , 6.816.86 
 ( m , app d , 2h ) , 6.33 ( d , j = 9.5 hz , 1h ) , 6.01 ( dd , j = 8.5 , 5.0 hz , 1h ) , 5.43 ( d , j = 4.5 
 hz , 1h ) , 5.22 ( s , 2h ) , 4.894.84 ( m , app d , 2h ) , 4.46 ( d , j = 18.5 hz , 1h ) ( part of ab system ) , 4.23 ( d , j = 18.5 hz , 1h ) ( part of ab system ) , 3.70 ( s , 3h ) , 3.60 ( d , j = 5.0 hz , 2h ) ppm . c nmr ( 101 mhz , dmso - d6 ) :  = 170.9 , 164.5 , 160.6 ( 2c ) , 160.2 , 
 159.4 , 155.2 , 144.3 , 135.6 , 130.4 ( 2c ) , 129.5 , 129.2 ( 2c ) , 128.2 ( 2c ) , 
 126.6 , 126.5 , 124.6 , 124.0 , 113.7 ( 2c ) , 112.9 , 112.8 , 101.6 , 67.8 , 
 66.8 , 66.2 , 58.4 , 55.1 , 50.9 , 41.2 ppm . lrms : mass calcd for c33h27n2o10s ( m  h ) 
 643.14 , mass found ( m  h ) 643.0 . compound 5 ( 50 mg , 81.6 mol ) 
 was cooled to 0 c prior to the addition of tfa / anisole ( 5:1 , 
 3 ml ) . the resulting reaction mixture was stirred at 0 c for 
 30 min with constant monitoring of the conversion by tlc analysis 
 ( ch2cl2/etoac , 8:2 ) . upon completion of the 
 reaction , cold et2o ( 5 ml ) the solid material was filtered off , 
 washed with et2o ( 2  5 ml ) , and subsequently dried 
 under high vacuum to yield the desired product as an off - white solid 
 ( 15 mg , 37% ) . h nmr 
 ( 400 mhz , dmso - d6 ) :  = 8.45 ( d , j = 8.5 hz , 1h ) , 8.00 ( d , j = 9.5 hz , 1h ) , 
 7.65 ( d , j = 8.5 hz , 1h ) , 7.277.32 ( m , 4h ) , 
 7.24 ( td , j = 8.5 , 4.0 hz , 1h ) , 6.937.01 
 ( m , 1h ) , 6.31 ( d , j = 9.5 hz , 1h ) , 5.81 ( dd , j = 8.0 , 4.5 hz , 1h ) , 5.14 ( d , j = 12.5 
 hz , 1h ) , 4.874.93 ( m , 1h ) , 3.98 ( d , j = 18.0 
 hz , 1h ) , 3.70 ( d , j = 14.0 hz , 1h ) ( part of ab system ) , 
 3.62 ( d , j = 18.5 hz , 1h ) , 3.54 ( d , j = 14.0 hz , 1h ) ( part of ab system ) ppm . c nmr ( 101 
 mhz , dmso - d6 ) :  = 171.1 , 164.2 , 
 162.2 , 161.1 , 155.3 , 144.3 , 135.8 , 129.6 , 129.1 ( 2c ) , 128.3 ( 2c ) , 
 126.6 , 112.8 ( 2c ) , 101.6 , 67.5 , 66.3 , 58.3 , 45.3 , 41.5 ppm . lrms : 
 mass calcd for c25h19n2o8s ( m  h ) 507.09 , mass found ( m  h ) 507.0 . compound 6 ( 20 mg , 0.03 
 mmol ) was cooled to 0 c prior to the addition of tfa / anisole 
 ( 5:1 , 1.2 ml ) . the reaction was stirred at 0 c for 30 min followed 
 by 30 min at rt with constant monitoring of the conversion by tlc 
 analysis ( ch2cl2/meoh , 9:1 ) . upon completion 
 of the reaction , cold et2o ( 5 ml ) the solid material was filtered 
 off , washed with et2o ( 2  3 ml ) , and subsequently 
 dried under high vacuum to yield the desired product as an off - white 
 solid ( 10 mg , 63% ) . h nmr ( 400 mhz , dmso - d6 ) :  = 8.92 
 ( d , j = 9.0 hz , 1h ) , 8.01 ( d , j = 
 9.5 hz , 1h ) , 7.66 ( d , j = 8.5 hz , 1h ) , 7.197.31 
 ( m , 5h ) 7.04 ( d , j = 2.5 hz , 1h ) , 6.99 ( dd , j = 8.5 , 2.5 hz , 1h ) , 6.32 ( d , j = 9.5 
 hz , 1h ) , 5.97 ( dd , j = 8.5 , 4.5 hz , 1h ) , 5.42 ( d , j = 4.5 hz , 1h ) , 4.905.00 ( m , app q , 2h ) , 4.41 ( d , j = 18.0 hz , 1h ) , 4.19 ( d , j = 18.0 hz , 
 1h ) , 3.543.65 ( m , app q , 2h ) ppm . c nmr ( 101 
 mhz , dmso - d6 ) :  = 170.9 , 164.3 , 
 162.1 , 160.8 , 160.2 , 155.2 , 144.3 , 135.6 , 129.6 , 129.2 ( 2c ) , 128.2 
 ( 2c ) , 126.5 , 125.2 , 123.8 , 112.9 , 112.9 , 101.7 , 66.8 , 66.3 , 58.3 , 
 50.8 , 41.2 ppm . lrms : mass calcd for c25h19n2o8s ( m  h ) 507.09 , mass found ( m  
 h ) 507.0 .
metallo--lactamases 
 ( mbls ) are a growing threat to the use 
 of almost all clinically used -lactam antibiotics . 
 the identification 
 of broad - spectrum mbl inhibitors is hampered by the lack of a suitable 
 screening platform , consisting of appropriate substrates and a set 
 of clinically relevant mbls . 
 we report procedures for the preparation 
 of a set of clinically relevant metallo--lactamases ( i.e. , 
 ndm-1 ( new delhi mbl ) , imp-1 ( imipenemase ) , spm-1 ( so paulo 
 mbl ) , and vim-2 ( verona integron - encoded mbl ) ) and the identification 
 of suitable fluorogenic substrates ( umbelliferone - derived cephalosporins ) . 
 the fluorogenic substrates were compared to chromogenic substrates 
 ( centa , nitrocefin , and imipenem ) , showing improved sensitivity and 
 kinetic parameters . 
 the efficiency of the fluorogenic substrates was 
 exemplified by inhibitor screening , identifying 4-chloroisoquinolinols 
 as potential pan mbl inhibitors .