bacterial infections 
 as well as the emergence and spread of antibiotic 
 resistance in human pathogens are serious public health problems in 
 hospital and community settings across the globe .
new strategies to prevent and treat bacterial infections 
 are needed , including methods to overcome antibacterial resistance 
 that results from the outer membrane permeability barrier in gram - negative 
 organisms and targeting approaches that afford species- or pathogen - specific 
 therapeutics .
almost 
 all 
 bacterial species have a metabolic iron requirement and therefore 
 employ various strategies to acquire this metal ion when colonizing .
siderophores are high - affinity fe(iii ) chelators that are 
 produced by bacteria under conditions of iron limitation , such as 
 those encountered in the vertebrate host , to scavenge this metal ion 
 from the environment .
siderophore producers also 
 express dedicated ferric siderophore import machinery and employ 
 various mechanisms to release siderophore - bound iron following 
 cellular uptake ( e.g. , reductive and/or hydrolytic release mediated 
 by reductases and/or esterases , respectively ) .
numerous studies support the importance of siderophore - based 
 iron acquisition during bacterial infections .
thus , the potential of using siderophores , or targeting siderophore biosynthetic and transport machineries , in therapeutic development continues to attract significant interest . 
 of particular relevance to the advances described herein are prior 
 investigations pertaining to the development of siderophore 
this strategy has received particular attention for the delivery 
 of antibiotics into gram - negative bacteria because these organisms 
 are inherently less sensitive to many antibiotics used in the clinic 
 as a result of the outer membrane permeability barrier .
both native siderophores and synthetic siderophore mimics 
 have been evaluated as platforms for therapeutic development . in the clinic , the native siderophore desferrioxamine 
 b is used for iron - chelation therapy in patients with iron overload . 

several antibiotic small molecules found in nature called  sideromycins  
 provide inspiration for synthetic siderophore 
the sideromycins are secondary 
 metabolites comprised of a siderophore moiety and a toxic cargo ; 
 the siderophore portion targets sideromycins to bacterial strains 
 expressing the appropriate siderophore receptor .
antibiotic conjugate produced by a clinical 
 isolate of klebsiella pneumoniae , is an 84-residue 
 antibacterial peptide with a glucosylated enterobactin ( ent , figure 1 ) derivative attached to its c - terminus that exhibits 
 enhanced antibacterial activity against strains expressing the enterobactin 
 receptor fepa . from the standpoints of 
 antibacterial activity and therapeutic potential , studies of synthetic 
 siderophore 
antibiotic conjugates have provided the 
 community with mixed results , causing some skepticism about the potential 
 of siderophore - based approaches despite the successful utilization 
 of such molecules by nature .
structures of enterobactin ( 1 , 
 ent ) and a generalized 
 enterobactin  cargo conjugate .
many of the failures encountered with early and recent studies 
 of siderophore - based antibiotic delivery may be attributed , 
 at least in part , to ( i ) use of non - native siderophores with 
 relatively low fe(iii ) affinities and/or compromised receptor recognition ; ( ii ) modification of antibiotics such that the 
 antibacterial activity is attenuated or lost completely ; ( iii ) bridging the siderophores and antibiotics with problematic 
 linkers , including linkers designed for drug release that are either 
 too stable or too labile , the latter of which promotes premature release ; and ( iv ) antibiotic resistance .
nevertheless , 
 the lessons of many unsuccessful studies highlight the complexity 
 of siderophore - based therapeutic development and provide a foundation 
 for inventing improved next - generation approaches .
many of the issues 
 described above may be overcome by careful molecular design and biological 
 evaluation .
in particular , the selection of appropriate native siderophore 
 platforms and modification of these platforms in ways that do not 
 compromise iron binding or receptor recognition , installation of an 
 antibacterial cargo in such a manner that antibacterial activity is 
 retained , and the development and application of assays that afford 
 insight into the fate of siderophore  antibiotic conjugates 
 are critical to the overall success of this approach .
along such lines , 
 a recent and insightful study by pfizer addressed complications associated 
 with using relatively low - molecular - weight siderophore mimics in vivo .
their results indicate 
 that competition between the siderophore - conjugated monobactam 
 mb-1 and native siderophores resulted in poor in vivo efficacy against pseudomonas aeruginosa and provide 
 support for designing and evaluating siderophore 
one recent 
 and successful example based on a native siderophore platform 
 is a mycobactin 
artemisinin conjugate that exhibits enhanced 
 antibacterial activity against mycobacterium tuberculosis compared to unmodified artemisinin .
enterobactin ( ent , figure 1 ) is a triscatecholate 
 siderophore biosynthesized by enteric bacteria and used for 
 iron acquisition in the vertebrate host . motivated by the importance of ent in the host / microbe interaction 
 as well as the decades of investigations pertaining to its ( bio)synthesis , 
 coordination chemistry , and biology , in prior work we reported a synthetic 
 route to monofunctionalized ent platforms .
moreover , we established that the native ent platform , when monofunctionalized 
 at the c5 position of one catecholate ring ( figure 1 ) , affords delivery of nontoxic small - molecule cargo across 
 the outer membrane of gram - negative organisms that express ent uptake 
 machinery ( e.g. , fepabcdg of escherichia coli ) .
as described herein , this proof - of - concept study 
 motivated us to demonstrate that ent effectively delivers antibacterial 
 cargo to organisms that utilize ent for iron acquisition , thereby 
 providing antibiotic targeting to specific sub - populations and a means 
 to address antibiotic resistance that results from the gram - negative 
 outer membrane permeability barrier . in this work
, we present 
 the syntheses and characterization of 
 siderophore  antibiotic conjugates based on the native 
 ent platform that harbor the clinically relevant -lactam antibiotics 
 ampicillin ( amp ) and amoxicillin ( amx ) .
these antibiotics block cell 
 wall biosynthesis by inhibiting transpeptidases , also named 
 penicillin binding proteins ( pbps ) , located in the periplasm of e. coli .
we report that the ent--lactam conjugates 
 exhibit significantly enhanced antibacterial activity ( up to 1000-fold ) 
 against pathogenic e. coli and provide more rapid 
 cell - killing than the parent -lactams as a result of ent - mediated 
 delivery to the periplasm . moreover ,
in proof - of - concept studies for 
 species - specific killing , these conjugates selectively kill e. coli in the presence of staphylococcus aureus , a gram - positive organism that is more susceptible to the parent 
 -lactams .
these studies support the notion that native siderophore 
 platforms provide an effective means to target molecular cargo to 
 siderophore - utilizing organisms and to hijack siderophore 
 uptake machinery to deliver cargos , including antibiotics , across 
 the outer membrane permeability barrier of gram - negative microbes .
dimethylformamide ( dmf ) and dichloromethane 
 ( ch2cl2 ) were obtained from a vac solvent purification 
 system ( vacuum atmospheres ) .
anhydrous dimethyl sulfoxide ( dmso ) was 
 purchased from sigma - aldrich and used as received .
l - ent 1 , the d - enantiomer of benzyl - protected 
 ent - co2h 2 , and the benzyl - protected ent - peg3-n33 were synthesized according to 
 previously reported procedures .
11-azido-3,6,9-trioxaundecan-1-amine 
 was purchased from fluka . all other chemicals and solvents were purchased 
 from sigma - aldrich or alfa aesar in the highest available purity and 
 used as received .
benzyl - protected ent - azide 3 ( 80 mg , 55 mol ) and pentamethylbenzene ( pmb , 147 mg , 
 990 mol ) were dissolved in 5 ml of anhydrous ch2cl2 to give a light yellow solution .
this solution was 
 cooled to 78 c in an acetone / dry ice bath under n2 , and bcl3 ( 660 l of 1 m solution in ch2cl2 , 660 mol ) was added slowly along the 
 flask wall .
after the solution was stirred for 1.5 h , dipea ( 300 l , 
 1.73 mmol ) was added to the flask , followed by meoh ( 2 ml ) to quench 
 the reaction . the reaction was then warmed to room temperature , and 
 the solvents were removed under reduced pressure . the resulting white 
 solid was dissolved in 5:3 meoh/1,4-dioxane and purified by preparative 
 hplc ( 33% b for 5 min and 3360% b over 20 min , 10 ml / min ) . 

the product eluted at 17 min and was lyophilized to yield compound 4 as white solid ( 13.9 mg , 28% ) .
h nmr ( dmso - d6 , 500 mhz ) :  3.353.57 ( 16h , 
 m ) , 4.384.41 ( 3h , m ) , 4.634.69 ( 3h , m ) , 4.894.96 
 ( 3h , m ) , 6.74 ( 2h , dd , j = 7.5 , 8.0 hz ) , 6.97 ( 2h , 
 d , j = 7.5 hz ) , 7.35 ( 2h , d , j = 
 8.0 hz ) , 7.46 ( 1h , s ) , 7.94 ( 1h , s ) , 8.338.35 ( 1h , m ) , 9.12 
 ( 2h , d , j = 6.0 hz ) , 9.29 ( 1h , d , j = 6.0 hz ) , 9.44 ( 2h , bs ) , 9.76 ( 1h , bs ) , 11.6 ( 2h , bs ) , 11.9 ( 1h , 
 bs ) .
c nmr ( cdcl3 , 125 mhz ) :  50.1 , 
 51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 , 115.3 , 115.4 , 115.4 , 
 117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 , 148.7 , 148.7 , 150.8 , 
 166.0 , 168.4 , 169.1 , 169.6 , 169.7 .
ir ( kbr disk , cm ) : 3389 , 2954 , 2928 , 2868 , 2111 , 1754 , 1645 , 1589 , 1535 , 1460 , 1384 , 
 1329 , 1266 , 1176 , 1132 , 1074 , 992 , 846 .
11-azido-3,6,9-trioxaundecan-1-amine 
 ( 36 l , 181 mol ) and d - bn6ent - cooh 
 ( 2 , 177 mg , 142 mol ) were dissolved in 5 ml of 
 dry ch2cl2 .
pyaop ( 147 mg , 283 mol ) and 
 dipea ( 98.5 l , 568 mol ) were added to give a light yellow 
 solution .
the reaction was stirred for 4 h at room temperature and 
 concentrated , and the crude product was purified by preparative tlc 
 ( 50% etoac / ch2cl2 ) to afford 5 as 
 white foam ( 159 mg , 77% ) .
h nmr ( dmso - d6 , 500 mhz ) :  3.33 
 ( 2h , j = 5.2 hz ) , 3.623.69 ( 14h , m ) , 4.024.06 
 ( 3h , m ) , 4.154.18 ( 3h , m ) , 4.914.94 ( 3h , m ) , 5.045.21 
 ( 12h , m ) , 6.96 ( 1h , s ) , 7.117.45 ( 36h , m ) , 7.657.67 
 ( 2h , m ) , 7.857.85 ( 1h ,
m ) , 7.977.97 ( 1h , m ) , 8.508.54 
 ( 3h , m ) .
c nmr ( cdcl3 , 125 mhz ) :  25.6 , 
 29.5 , 38.8 , 40.0 , 45.3 , 51.3 , 51.4 , 63.9 , 64.1 , 69.8 , 39.8 , 70.0 , 
 70.3 , 70.4 , 70.4 , 71.2 , 71.2 , 76.3 , 76.3 , 116.8 , 117.5 , 120.4 , 123.0 , 
 124.3 , 125.4 , 126.1 , 126.2 , 127.6 , 127.6 , 127.9 , 128.2 , 128.3 , 128.4 , 
 128.4 , 128.5 , 128.5 , 128.6 , 128.6 , 128.8 , 128.8 , 128.9 , 129.0 , 130.1 , 
 135.4 , 135.7 , 135.9 , 136.0 , 136.1 , 146.8 , 146.9 , 149.1 , 151.6 , 151.8 , 
 164.3 , 164.9 , 164.9 , 165.8 , 168.9 , 169.0 , 169.1 , 176.2 . ir ( kbr disk , 
 cm ) : 3357 , 3062 , 3032 , 2958 , 2923 , 2859 , 2104 , 
 1751 , 1551 , 1576 , 1515 , 1455 , 1375 , 1345 , 1299 , 1264 , 1204 , 1126 , 
 1082 , 1040 , 1018 , 957 , 915 , 854 , 811 . hrms ( esi ) :
compound 6 was synthesized 
 from 5 ( 153 mg , 105 mol ) following the same procedure 
 as for compound 4 .
the crude reaction was purified by 
 preparative hplc ( 33% b for 5 min and 3360% b over 20 min , 
 10 ml / min ) .
the product eluted at 17.0 min and was lyophilized to 
 yield compound 6 as white solid ( 31 mg , 33% ) .
h nmr ( cdcl3 , 500 mhz ) :  3.343.56 ( 16h , 
 m ) , 4.394.41 ( 3h , m ) , 4.614.66 ( 3h , m ) , 4.884.94 
 ( 3h , m ) , 6.74 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h , 
 d , j = 7.8 hz ) , 7.33 ( 2h , d , j = 
 7.8 hz ) , 7.44 ( 1h , s ) , 7.91 ( 1h , s ) , 8.318.33 ( 1h , m ) , 9.129.13 
 ( 2h , m ) , 9.279.28 ( 1h , m ) , 9.50 ( 2h , bs ) , 9.84 ( 1h , bs ) , 11.6 
 ( 2h , bs ) , 11.9 ( 1h , bs ) .
c nmr ( cdcl3 , 125 
 mhz ) :  50.1 , 51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 , 
 115.3 , 115.4 , 115.4 , 117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 , 
 148.7 , 148.7 , 150.8 , 166.0 , 168.4 , 169.1 , 169.6 , 169.7 .
ir ( kbr disk , 
 cm ) : 3390 , 2958 , 2925 , 2863 , 2110 , 1754 , 1645 , 
 1589 , 1535 , 1460 , 1384 , 1342 , 1262 , 1176 , 1117 , 1074 , 841 , 800 .
hrms 
 ( esi ) : [ m+na]m / z calcd 
 936.2506 , found 936.2512 .
5-hexynoic acid ( 113 l , 1.00 
 mmol ) and thionyl chloride ( 1.00 ml , 13.8 mmol ) were combined and 
 refluxed for 1 h. the reaction was cooled to room temperature and 
 concentrated under reduced pressure , and the resulting crude acyl 
 chloride was dissolved in acetone ( 0.5 ml ) and carried on to the next 
 step without purification .
ampicillin sodium salt ( 186 mg , 0.500 mmol ) 
 was dissolved in a solution of nahco3 ( 210 mg , 2.5 mmol ) 
 in 4:1 water / acetone ( 2.5 ml ) and cooled on ice , to which the acyl 
 chloride was added slowly with stirring .
the reaction was subsequently 
 warmed to room temperature and stirred for 1 h. water ( 3 ml ) was added 
 to the reaction , and the aqueous phase was washed with etoac ( 2  
 10 ml ) , acidified to ph 2 by addition of hcl , and extracted with etoac 
 ( 20 ml ) .
the resulting organic phase was washed with cold water ( 2 
  5 ml ) , dried over na2so4 , and concentrated 
 under reduced pressure . the crude reaction was triturated with hexanes , 
 which afforded a yellow solid ( 180 mg , 77% ) .
h nmr ( dmso - d6 , 
 500 mhz ) :  1.41 ( 3h , s ) , 1.55 ( 3h , s ) , 1.641.69 ( 2h , 
 m ) , 2.132.16 ( 2h , m ) , 2.292.32 ( 2h , m ) , 2.772.78 
 ( 1h , m ) , 4.20 ( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) , 
 5.52 ( 1h , dd , j = 4.0 , 8.0 hz ) , 5.70 ( 1h , d , j = 8.0 hz ) , 7.257.43 ( 5h , m ) , 8.57 ( 1h , d , j = 8.0 hz ) , 9.11 ( 1h , d , j = 8.0 hz ) .
c nmr ( dmso - d6 , 125 mhz ) :  
 17.4 , 24.4 , 26.6 , 30.4 , 33.8 , 55.5 , 58.1 , 63.7 , 67.3 , 70.3 , 71.5 , 
 84.2 , 127.2 , 127.6 , 128.2 , 138.2 , 169.0 , 170.2 , 171.5 , 173.5 .
ir ( kbr 
 disk , cm ) : 3297 , 3058 , 3023 , 2970 , 2937 , 2863 , 
 2626 , 2526 , 2120 , 1780 , 1688 , 1518,1455 , 1437 , 1390 , 1373 , 1324 , 1295 , 
 1208 , 1139 , 1027 , 1001 , 843 .
compound 8 was synthesized 
 as described for compound 7 except that amoxicillin ( 760 
 mg , 2.1 mmol ) was used instead of ampicillin sodium salt .
compound 8 was obtained as light yellow solid ( 533 mg , 56% ) after trituration 
 and employed without further purification .
h nmr ( dmso - d6 , 500 mhz ) : 
  1.42 ( 3h , s ) , 1.56 ( 3h , s ) , 1.631.68 ( 2h , m ) , 2.122.15 
 ( 2h , m ) , 2.252.29 ( 2h , m ) , 2.762.78 ( 1h , m ) , 4.19 
 ( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) , 5.515.54 
 ( 2h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) , 7.19 ( 2h , d , j = 8.5 hz ) , 8.41 ( 1h , d , j = 8.0 hz ) , 
 8.93 ( 1h , d , j = 8.0 hz ) , 9.40 ( 1h , bs ) .
c nmr ( dmso - d6 , 125 mhz ) :  17.6 , 
 24.5 , 26.8 , 30.4 , 34.0 , 55.4 , 58.2 , 63.9 , 71.6 , 71.7 , 84.4 , 115.1 , 
 128.4 , 128.7 , 128.8 , 157.0 , 169.2 , 170.9 , 171.7 , 173.8 . ir ( kbr disk , 
 cm ) : 3356 , 3294 , 3045 , 2970 , 2928 , 1770 , 1738 , 
 1650 , 1615 , 1515 , 1457 , 1373 , 1208 , 1009 , 945 , 839 , 815 . hrms ( esi ) : 
 [ m+na]m / z calcd 460.1537 , 
 found 460.1534 .
compound 7 ( 60 mg , 0.13 
 mmol ) was dissolved in 1:1 h2o / mecn ( 5 ml ) , and tfa was 
 added to a final concentration of 1% .
the solution was incubated at 
 37 c for 24 h and purified by preparative hplc ( 2050% 
 b over 25 min , 10 ml / min ) , which afforded a white powder ( 12 mg , 25% ) . 

the white powder is a diastereomeric mixture of products , and no further 
 separation was performed .
h nmr ( dmso - d6 , 500 mhz ) :  ( mixture of two diastereomers ) 1.221.23 
 ( 3h , pair of s ) , 1.541.58 ( 3h , pair of s ) , 1.621.68 
 ( 2h , m ) , 2.122.14 ( 2h , m ) , 2.282.30 ( 2h , m ) , 2.77 
 ( 1h , s ) , 3.183.24 ( 0.5h , m ) , 3.303.36 ( 0.5h , m ) , 3.443.49 
 ( 0.5h , m ) , 3.553.60 ( 0.5h , m ) , 3.92 ( 0.5h , s ) , 4.01 ( 0.5h , 
 s ) , 4.67 ( 0.5h , dd , j = 6.7 , 6.5 hz ) , 4.78 ( 0.5h , 
 dd , j = 5.2 , 5.2 hz ) , 5.455.48 ( 1h , m ) , 7.277.40 
 ( 5h , m ) , 8.508.61 ( 2h , m ) .
c nmr ( dmso - d6 , 125 mhz ) :  ( mixture of two diastereomers ) 
 18.1 , 25.0 , 27.6 , 28.1 , 28.4 , 29.5 , 34.4 , 42.2 , 56.8 , 56.8 , 72.1 , 
 72.2 , 84.8 , 127.9 , 128.2 , 128.9 , 139.2 , 139.4 , 171.2 , 171.4 , 172.0 . 

ir ( kbr disk , cm ) : 3297 , 3071 , 3041 , 2967 , 2938 , 
 2535 , 2124 , 1734 , 1653 , 1527 , 1456 , 1427 , 1375 , 1299 , 1199 , 1137 , 
 1070 , 1027 , 836 . hrms ( esi ) :
compound 10 was synthesized 
 as described for compound 9 except that compound 8 was used instead of 7 ( 60 mg , 0.13 mmol ) .
the 
 product was purified by preparative hplc ( 2050% b over 25 
 min , 10 ml / min ) , and obtained as white powder ( 14.5 mg , 24% ) .
h nmr ( dmso - d6 , 500 mhz ) :  
 ( mixture of two diastereomers ) 1.26 ( 3h , s ) , 1.551.59 ( 3h , 
 pair of s ) , 1.611.67 ( 2h , m ) , 2.122.14 ( 2h , m ) , 2.242.27 
 ( 2h , m ) , 2.77 ( 1h , s ) , 3.203.26 ( 0.5h , m ) , 3.343.39 
 ( 0.5h , m ) , 3.423.47 ( 0.5h , m ) , 3.563.60 ( 0.5h , m ) , 
 4.03 ( 0.5h , s ) , 4.12 ( 0.5h , s ) , 4.68 ( 0.5h , dd , j = 6.5 , 6.5 hz ) , 4.79 ( 0.5h , dd , j = 5.5 , 5.5 hz ) , 
 5.285.31 ( 1h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) , 
 7.17 ( 2h , d , j = 8.5 hz ) , 8.368.50 ( 3h , m ) .
c nmr ( dmso - d6 , 125 mhz ) :  
 ( mixture of two diastereomers ) 17.5 , 24.3 , 26.9 , 27.5 , 27.7 , 28.8 , 
 33.8 , 41.4 , 55.7 , 55.8 , 71.4 , 71.6 , 84.2 , 114.8 , 115.0 , 128.5 , 128.7 , 
 128.8 , 156.9 , 158.3 , 158.6 , 171.2 , 171.3 .
ir ( kbr disk , cm ) : 3301 , 3071 , 3028 , 2973 , 2928 , 2548 , 2111 , 1737 , 1662 , 1606 , 1593 , 
 1515 , 1435 , 1377 , 1197 , 1139 , 837 . hrms ( esi ) :
ampicillin - alkyne 7 ( 120 l 
 of an 80 mm solution in dmso , 9.6 mol ) and 
 ent - peg3-n34 ( 250 l of 
 a 13 mm solution in 1,4-dioxane , 3.3 mol ) were combined , and 
 400 l of dmso was added .
cuso4 ( 100 l of 
 a 90 mm solution in water , 9.0 mol ) and tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta , 200 l of 
 a 50 mm solution in dmso , 10 mol ) were combined , and 100 l 
 of dmso was added to give a blue - green solution , to which naasc ( 400 
 l of a 90 mm solution in water , 36.0 mol ) was added . 
 this solution became light yellow and was immediately added to the 
 alkyne / azide solution .
the reaction was shaken on a benchtop rotator 
 for 2 h at room temperature , diluted by 3- to 4-fold with 1:1 mecn / water , 
 centrifuged ( 13,000 rpm  10 min , 4 c ) , and purified by 
 semi - preparative hplc ( 20% b for 5 min and 20%50% b over 11 
 min , 4 ml / min ; 0.005% tfa was used in the solvent system to prevent 
 decomposition of the -lactam ) .
the hplc fractions containing 11 were collected manually and flash frozen in liquid n2 immediately after collection to prevent -lactam decomposition . 

h nmr ( dmso - d6 , 500 mhz ) :  1.40 
 ( 3h , s ) , 1.54 ( 3h , s ) , 1.781.81 ( 2h , m ) , 2.27 ( 2h , t , j = 6.8 hz ) , 2.58 ( 2h , t , j = 6.5 hz ) , 
 3.48 ( 12h , m ) , 3.76 ( 2h , s ) , 4.19 ( 1h , s ) , 4.384.44 ( 5h , m ) , 
 4.644.66 ( 3h , m ) , 4.914.92 ( 3h , m ) , 5.39 ( 1h , d , j = 3.5 hz ) , 5.515.52 ( 1h , m ) , 5.72 ( 1h , d , j = 7.5 hz ) , 6.74 ( 2h , dd , j = 7.8 , 7.8 
 hz ) , 6.96 ( 2h , d , j = 7.5 hz ) , 7.267.35 ( 5h , 
 m ) , 7.427.45 ( 3h , m ) , 7.81 ( 1h , s ) , 7.92 ( 1h , s ) , 8.33 ( 1h , 
 s ) , 8.55 ( 1h , d , j = 7.5 hz ) , 9.12 ( 3h , d , j = 7.0 hz ) , 9.29 ( 1h , d , j = 6.5 hz ) , 
 9.42 ( 2h , bs ) , 9.74 ( 1h , s ) , 11.6 ( 2h , s ) , 11.9 ( 1h , bs ) , 13.35 ( 1h , 
 bs )
. hrms ( esi ) : [ m+na]m / z calcd 1379.4021 , found 1379.4046 .
compound 12 was 
 synthesized as described for 11 except that compound 8 was used instead of compound 7 .
h nmr ( dmso - d6 , 500 mhz ) :  1.40 ( 3h , s ) , 1.54 ( 3h , 
 s ) , 1.781.80 ( 2h , m ) , 2.23 ( 2h , t , j = 6.5 
 hz ) , 2.57 ( 2h , t , j = 6.5 hz ) , 3.47 ( 12h , m ) , 3.76 
 ( 2h , bs ) , 4.18 ( 1h , s ) , 4.394.43 ( 5h , m ) , 4.634.65 
 ( 3h , m ) , 4.90 ( 3h , bs ) , 5.38 ( 1h , s ) , 5.525.56 ( 2h , m ) , 6.68 
 ( 2h , d , j = 8.5 hz ) , 6.73 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h , d , j = 7.5 ) , 7.19 ( 2h , 
 d , j = 8.5 hz ) , 7.33 ( 2h , d , j = 
 7.5 hz ) , 7.44 ( 1h , s ) , 7.80 ( 1h , s ) , 7.92 ( 1h , s ) , 8.338.39 
 ( 2h , m ) , 8.94 ( 1h , d , j = 8.0 hz ) , 9.119.12 
 ( 2h , m ) , 9.29 ( 1h , bs ) , 9.389.43 ( 3h , m ) , 9.75 ( 1h , s ) , 11.6 
 ( 2h , bs ) , 11.9 ( 1h , bs ) .
compound 13 was 
 synthesized as described for 11 except 
 that compound 6 was used instead of compound 4 .
hrms ( esi ) : 
 [ m+na]m / z calcd 1379.4021 , 
 found 1379.4022 .
compound 14 was synthesized as described for 12 except 
 that compound 6 was used instead of compound 4 .
hrms ( esi ) : 
 [ m+na]m / z calcd 1395.3970 , 
 found 1395.3995 .
compound 15 was synthesized as described for 11 except that compound 9 was used instead of compound 7 .
hrms ( esi ) : [ m+h]m / z calcd 1331.4409 , found 
 1331.4389 .
compound 16 was synthesized as described for 12 except that compound 10 was used instead of compound 8 .
hrms ( esi ) : [ m+na]m / z calcd 1369.4177 , found 
 1369.4191 .
general microbiology materials and methods , including 
 details of 
 ent - amp / amx stock solution preparation and storage , are provided as supporting information .
overnight cultures of 
 the bacterial strains ( table s1 ) were prepared 
 in 15-ml polypropylene tubes by inoculating 5 ml of lb media with 
 the appropriate freezer stock .
the overnight cultures were incubated 
 at 37 c for 1618 h in a tabletop incubator shaker set 
 at 150 rpm and housing a beaker of water .
each overnight culture was 
 diluted 1:100 into 5 ml of fresh lb media containing 200 m 
 2,2-dipyridyl ( dp ) and incubated at 37 c with shaking 
 at 150 rpm until od600 reached 0.6 .
g / l ) with or without 200 m 
 dp to achieve an od600 value of 0.001 .
a 90-l aliquot 
 of the diluted culture was combined with a 10-l aliquot of 
 a 10 solution of the antibiotic or ent - antibiotic conjugate 
 in a 96-well plate , and the covered plate was wrapped in parafilm 
 and incubated at 30 c with shaking at 150 rpm for 19 h in a 
 tabletop incubator housing a beaker of water .
bacterial growth was 
 determined by measuring od600 ( end point analysis ) using 
 a biotek synergy ht plate reader .
each well condition was prepared 
 in duplicate and at least three independent replicates using two different 
 synthetic batches of each conjugate were conducted on different days . 

the resulting mean od600 values are reported , and the error 
 bars are the standard error of the mean ( sem ) obtained from the independent 
 replicates .
these assays were performed with e. coli atcc 
 35218 and k. pneumoniae atcc 13883 following the 
 general procedure except that sulbactam ( sb ) or potassium clavulanate 
 ( pc ) were mixed with ampicillin or amoxicillin and the ent - amp or 
 ent - amx conjugates , respectively .
the molar ratios of the inhibitor/-lactam 
 mixtures were sulbactam / amp or ent - amp , 1.5:1 , and potassium clavulanate / amx 
 or ent - amx , 0.9:1 .
these assays were 
 performed with e. coli k-12 and 
 cft073 following the general procedure except that varying concentrations 
 ( 1100 m ) of synthetic l - ent were mixed with 
 ent - amp / amx .
this assay is based on a published protocol and was conducted with e. coli cft073 . 

each overnight culture was serially diluted into 
 m9 minimal medium to provide 1010 cfu / ml . 

lipocalin 2 was diluted in pbs to a concentration of 10 m upon 
 arrival , aliquoted , and stored at 20 c until use .
a 
 90-l aliquot of the diluted culture was added to each well 
 of a 96-well plate that contained varying concentrations of lipocalin 
 2 , ent - amp , and ent , and the final volume was adjusted to 100 l 
 with sterile pbs .
the 96-well plate was incubated at 37 c for 
 24 h in a tabletop incubator set at 150 rpm , and bacterial growth 
 was determined by measuring od600 using a plate reader . 

each well condition was repeated at least three times independently 
 on different days and with different batches of lipocalin 2 .
the resulting 
 mean od600 is reported , and the error bars are the sem .
a 5-ml overnight culture of e. coli k-12 or cft073 was grown in lb ( vide supra ) and diluted 1:100 into 5 ml of fresh lb media containing 200 m 
 dp , and this culture was incubated at 37 c with shaking at 150 
 rpm in a tabletop incubator housing a beaker of water until od600 reached 0.3 .
the culture was centrifuged ( 3000 
 rpm  10 min , rt ) , and the resulting pellet was washed twice 
 by resuspension in 50% mhb and centrifugation ( 3000 rpm  10 
 min , rt ) .
the resulting pellet was resuspended in 50% mhb with or 
 without dp , and the od600 was adjusted to 0.3 .
a 90-l 
 aliquot of the resulting culture was mixed with a 10-l aliquot 
 of a 10 solution of amp / amx or the ent - amp / amx in a 96-well 
 plate , which was covered , wrapped in parafilm , and incubated at 37 
 c with shaking at 150 rpm .
the od600 values were 
 recorded at t = 0 , 1 , 2 , and 3 h by using a plate 
 reader . in a parallel experiment ,
a 10-l aliquot of the culture 
 was taken at t = 0 , 1 , 2 , and 3 h , serially diluted 
 by using sterile phosphate - buffered saline ( pbs ) , and plated on lb - agar 
 plates for colony counting ( cfu / ml ) .
the resulting 
 mean od600 or cfu / ml is reported , and the error bars are 
 the sem .
a 5 ml overnight culture of each 
 bacterial strain was grown in lb , diluted 1:100 into 5 ml of fresh 
 lb media containing 200 m dp , and incubated at 37 c with 
 shaking at 150 rpm in a tabletop incubator housing a beaker of water 
 until od600 reached 0.6 .
each mid - log - phase culture was 
 diluted to 10 cfu / ml in 50% mhb with or without 200 m 
 dp . for experiments requiring a mixture of two species ,
a 1:1 mixture 
 was prepared ( 10 cfu / ml for each strain ) in 50% mhb with 
 or without 200 m dp from the mid - log - phase cultures . to confirm 
 cfu / ml of each culture ,
the single- and double - species cultures were 
 serially diluted by using sterile pbs , and aliquots were plated on 
 a chrom - uti plate (  starter - culture plate  ) .
for each 
 cell - killing experiment , a 90-l aliquot of each culture was 
 combined with a 10-l aliquot of a 10 m solution of the 
 antibiotic or ent - antibiotic conjugate in a 96-well plate , which was 
 covered , wrapped in parafilm , and incubated at 30 c with shaking 
 at 150 rpm for 19 h. bacterial growth was assayed both by measuring 
 od600 using the plate reader and by plating on chrom - uti 
 plates after serial dilution (  assay plate  ) .
each well 
 condition was repeated at least three times independently on different 
 days .
the resulting mean od600 is reported , and the error 
 bars are the sem .
we aimed to harness our enterobactin - mediated 
 cargo delivery strategy to enable the transport of toxic cargo across 
 the outer membrane of e. coli . to address this goal , 
 we linked the -lactam antibiotics ampicillin ( amp ) and amoxicillin 
 ( amx ) to a monofunctionalized ent scaffold where ent is derivatized 
 at the c5 position of one catechol ring via a flexible and stable 
 peg3 linker .
we selected amp and amx as antibacterial cargo 
 for several reasons : these molecules are commercially available and 
 amenable to synthetic modification , retain antibacterial activity 
 when appropriately modified , possess periplasmic targets in gram - negative 
 bacteria and must cross the outer membrane to be active against these 
 species , and have relatively low molecular weights .
we selected low - molecular - weight 
 antibiotics because our prior studies of ent - mediated cargo transport 
 indicated that the ent transport machinery of e. coli k-12 imports ent - cargo conjugates harboring relatively small cargos 
 ( e.g. , cyclohexane , naphthalene , phenylmethylbenzene ) 
 to the cytosol readily , whereas large cargos ( e.g. , vancomycin ) are 
 not transported to the cytosol .
moreover , 
 in prior studies , various -lactams including amp and amx have 
 been linked to simple catechols and more complex catechol - containing siderophores 
 or mimics thereof , which provides the 
 opportunity to compare the outcomes obtained for different siderophore - inspired 
 design strategies . in scheme 1
, we present the syntheses of 
 ent - amp 11 and ent - amx 12 , which feature 
 installation of alkyne - modified -lactam warheads onto ent - azide 4 via copper - catalyzed azide - alkyne cycloaddition ( hereafter 
 click reaction ) in the final step .
the catechol moieties of benzyl - protected 
 ent - azide 3 were deprotected by using bcl3 at 78 c to achieve ent - azide 4 as a white 
 powder in 28% yield following purification by reverse - phase preparative 
 hplc . catalytic hydrogenation using hydrogen gas and a pd / c catalyst 
 is typically employed to deprotect ent catechols ; however , we observed that amp / amx decompose under these conditions 
 and poison the pd / c catalyst .
moreover , deprotection of the enterobactin 
 catechols prior to installing the -lactams requires preservation 
 of the azide moiety , and we therefore employed bcl3 for 
 this reaction .
initial attempts at assembling ent - amp / amx using standard 
 conditions for the copper - catalyzed click reactions with ent - azide 4 and the alkyne - modified -lactams 7/8 , prepared by thionyl chloride coupling of 5-hexynoic acid 
 to the amino group of amp / amx , failed because of copper - mediated -lactam 
 decomposition .
this problem was overcome 
 by including the metal - ion chelator tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta ) in the click reactions , and ent - amp and ent - amx were obtained as 
 white powders in high purity and yields of 66% and 76% , respectively , 
 following semi - preparative hplc purification .
it was necessary to 
 perform hplc purification with eluents containing only 0.005% tfa 
 to prevent decomposition of the acid - sensitive -lactam moieties . 

this synthetic route was likewise employed to prepare the d - enantiomers of ent - amp / amx 13 and 14 ( scheme 1 ) .
deprotected ent - azide 4 enables alkyne - functionalized 
 molecules to be covalently linked to ent via a click reaction , including 
 molecules that are incompatible with reaction conditions required 
 to deprotect the ent catechols .
moreover , the deprotected ent - azide 
 may be employed to append ent to surfaces , other materials , or biomolecules 
 harboring alkyne groups .
indeed , very 
 few examples employing the copper(i)-catalyzed click reaction with 
 fused -lactams are reported in the literature , and it is likely that this paucity stems from the fact that -lactams 
 are incompatible with standard conditions for copper - catalyzed azide - alkyne 
 cycloaddition .
the conditions defined 
 in this work employing tbta allow for copper - catalyzed triazole formation 
 and preserve the -lactam warhead . in the absence of fe(iii ) , 
 ent - amp / amx exhibit an absorption band centered at 316 nm resulting 
 from catecholate absorption and the solutions are colorless ( meoh , 
 rt ) .
addition of 1.0 equiv of fe(iii ) to methanolic solutions of ent - amp 
 and ent - amx causes the solution to immediately change from colorless 
 to purple - red , and a broad absorption feature in the 400700 
 nm range appears ( figure s1 ) , confirming 
 that both ent - amp / amx readily chelate fe(iii ) . to ascertain whether ent - amp / amx provide antibacterial activity 
 against e. coli , including pathogenic strains , we preformed antimicrobial activity assays using 
 six strains ( table s1 , figures 2 , s2s7 ) .
e. coli atcc 25922 is a laboratory susceptibility test strain 
 originally obtained as a clinical isolate .
coli uti89 and 
 cft073 are both pathogens of the human 
 urinary tract ( upec).e .
coli atcc 35401 ( serotype o78:h11 ) is an enterotoxigenic ( etec ) strain 
 that was isolated from human feces .
e. coli atcc 
 43895 ( serotype o157:h7 ) is an enterohemorrhagic ( ehec ) strain that 
 was isolated from raw hamburger meat implicated in a hemorrhagic colitis 
 outbreak .
both etec and ehec strains produce virulence factors and 
 toxins and cause diarrhea in humans .
all e. coli strains biosynthesize ent for iron acquisition and 
 express the ent receptor fepa .
some e. coli strains 
 employed in this work also have the capacity to produce and utilize 
 salmochelins , c - glucoyslated ent derivatives .
these molecules are produced by salmonella spp . 
 and pathogenic e. coli strains for iron acquisition . 

the iroa gene cluster ( irobcden ) 
 encodes proteins required for the biosynthesis and transport of salmochelins , 
 and iron is the outer membrane receptor for salmochelins encoded by 
 the iroa cluster .
studies with salmonella indicate that iron has the ability to transport 
 ent as well as its glucosylated forms . of the strains considered in this work , e. coli cft073 and uti89 harbor the iroa gene cluster .
e. coli h9049 does not produce salmochelins , and a blast search using available e. 
 coli genomes reveals that the e. coli 43985 
 genome does not contain the iroa cluster .
the genome 
 for e. coli atcc 25922 is unpublished ; however , this 
 strain is reported to be sensitive to lipocalin-2 ( vide infra ) , which suggests that its genome does 
 not encode the iroa cluster .
lastly , it should be noted that e. coli cft073 is 
 celebrated for having redundant iron import machineries , and this 
 strain also harbors the iha gene , which encodes the 
 outer membrane ent receptor iha that is distinct from fepa .
antibacterial activity of ent - amp / amx against various e. 
 coli strains that include human pathogens .
all assays 
 were performed in 50% mhb medium supplemented with 200 m dp 
 to provide iron - limiting conditions ( mean  sem , n  3 ) .
the data for assays performed in the absence of dp 
 are presented in figures s2s7 .
we performed antibacterial activity 
 assays using a 10-fold dilution 
 series to compare the abilities of ent - amp / amx and unmodified amp / amx 
 to kill e. coli ( figures 2 , s2s7 ) .
these assays were conducted 
 in 50% mhb and in the absence or presence of 200 m dp .
the 
 latter growth conditions provide iron limitation and result in expression 
 of the ent uptake machinery fepabcdg .
amp / amx exhibit minimum inhibitory 
 concentration ( mic ) values of 10 m against these e. 
 coli strains regardless of the presence of dp in the growth 
 medium ( figures 2 , s2s7 ) .
all six e. coli strains are more susceptible 
 to ent - amp / amx than amp / amx under conditions of iron limitation ( figure 2 ) .
based on the 10-fold dilution series , ent - amp / amx 
 are 100-fold more potent against e. coli 25922 , uti89 , 
 and h9049 under iron - limiting conditions .
although the mic values 
 for 35401 and 43895 are only ca . 10-fold higher than for amp / amx in 
 this assay , a significant reduction in growth is observed at 100 nm 
 ent - amp / amx , whereas this concentration of amp / amx affords no growth 
 inhibition .
the enhanced sensitivity of e. coli cft073 
 to ent - amp / amx is remarkable .
this strain exhibits the greatest sensitivity 
 to ent - amp / amx , providing a 1000-fold decreased mic value ( 10 nm ) , 
 and growth inhibition in the presence of 1 nm of the conjugate . 

moreover , in the absence of dp , e. coli cft073 exhibits 
 the greatest susceptibility to ent - amp / amx ( figures 
 s2s7 ) .
a noteworthy characteristic of cft073 is its 
 multiple mechanisms for iron acquisition , and we hypothesize that 
 the presence of multiple receptors that recognize and transport ent 
 ( fepa , iron , iha ) contributes to this enhanced sensitivity .
cft073 
 and uti89 both express iron , which may indicate that iha is responsible 
 for the enhanced susceptibility of cft073 ; however , we can not rule 
 out the possibility that the conserved receptors function differently 
 or exhibit different expression levels depending on the strain . moreover
, 
 other unappreciated mechanisms may contribute to the potent bactericidal 
 action exhibited by ent - amp / amx against cft073 and other strains . to gain further insight into the mechanism 
 of ent - amp / amx antibacterial action
, we performed a series of experiments 
 with the standard laboratory strain e. coli k-12 . 

we employed the same 10-fold dilution series to compare the activities 
 of ent - amp / amx and amp / amx against k-12 . in the absence of dp ,
ent - amp / amx 
 and amp / amx exhibit comparable mic values , with complete killing observed 
 at 10 m . at lower concentrations , ent - amp / amx exhibit 
 slightly greater antibacterial activity than unmodified amp / amx ( figure 3a ) .
this phenomenon is most evident at a conjugate / drug 
 concentration of 1 m , where ent - amp / amx inhibit e. 
 coli k-12 growth to varying degrees and amp / amx do not affect 
 bacterial growth . under conditions of iron limitation ,
a 100-fold 
 reduction in mic value ( 10 m to 100 nm ) for ent - amp / amx is 
 observed , and ca .
50% growth inhibition occurs at 10 nm of each conjugate 
 ( figure 3b ) .
these trends are comparable to 
 those observed for e. coli atcc 25922 , h9049 , and 
 uti89 ( figures 2 , s2 , s3 , 
 and s5 ) .
when the antibacterial activity assay was performed 
 using a 1:1 ratio of native l - ent and amp / amx , no reduction 
 of amp / amx mic values was observed ( figure 3c ) , which suggests the conjugation between ent and the -lactams 
 is required for the enhanced bactericidal action
. moreover , treatment 
 of e. coli k-12 with the iron - bound forms of ent - amp / amx , 
 obtained by pre - incubating each conjugate with 1 equiv of ferric chloride , 
 afforded the same mic values as observed for apo ent - amp / amx .
these 
 results indicate that the enhanced antibacterial activity does not 
 result from iron chelation in the growth media ( figure 3d ) . in total
, the data obtained for e. coli k-12 as well as the six other e. coli strains demonstrate 
 that the antibacterial activity of ent - amp / amx against e. 
 coli k-12 is enhanced under conditions of iron limitation 
 and support a model of ent - mediated delivery of antibacterial cargo 
 to the e. coli periplasm . antibacterial activity 
 of ent - amp / amx against wild - type and mutant e. coli k-12 .
( a , b ) growth inhibition of e. coli k-12 by 
 amp / amx and ent - amp / amx in the absence ( a ) and presence 
 ( b ) of dp .
( c ) growth inhibition of e. coli k-12 
 treated with a 1:1 molar ratio of ent / amp and ent / amx .
( d ) growth 
 inhibition of e. coli k-12 treated with ferric ent - amp / amx . 

( e  g ) growth inhibition of fepa- ( e ) , fepc- ( f ) , and fes- ( g ) by amp / amx and 
 ent - amp / amx .
( h , i ) growth of e. coli k-12 in the 
 presence of 1 m ent - amp / amx (  conjugate  ) and 
 mixtures of ent - amp / amx ( 1 m ) and 1 , 5 , or 20 equiv of exogenous 
 ent in the absence ( h ) and presence ( i ) of dp . the * * indicates od600 < 0.01 .
all assays were performed in 50% mhb medium 
 with or without 200 m dp ( see panels ) ( mean  sem ,
the data for additional assays performed 
 in the absence of dp are presented in figure s8 . to evaluate transport of ent - amp / amx 
 into e. coli , we investigated the antimicrobial activity 
 of ent - amp / amx and amp / amx against three single - gene knockout e. coli k-12 strains obtained from the keio collection , fepa- , fepc- , and fes- ( figures 3e 
we selected these mutants to ascertain 
 how components of the enterobactin transport and processing machinery 
 contribute to ent - amp / amx antibacterial activity .
e. colifepa- lacks the outer membrane ent receptor fepa 
 that allows periplasmic delivery , fepc- lacks the 
 atpase component of the inner membrane ent permease that transports 
 ent into the cytosol , and e. colifes- lacks the cytoplasmic esterase fes responsible for hydrolysis of 
 the ent macrolactone for iron release . on the basis of our studies 
 with wild - type k-12 and other e. coli strains , we 
 hypothesized that the activity of ent - amp / amx would be attenuated 
 for the fepa- mutant .
moreover , we questioned whether 
 loss of fepc or fes would modulate the antimicrobial activity .
overnight cultures of fepa- and fepc- reached od600 values ( 0.15 ) similar to that observed 
 for wild - type k-12 when grown in 50% mhb supplemented with 200 m 
 dp .
in contrast , the fes- strain exhibited a severe 
 growth defect under these conditions ( od600  0.04 ) . 

treatment of fepa- with ent - amp / amx afforded the 
 same mic values as for amp / amx ( figure 3e ) 
 and hence a 100-fold reduction in activity as compared to wild - type 
 k-12 .
iron deprivation is deleterious to e. coli , 
 and we contend that the growth inhibition observed at 10 m 
 ent - amp / amx results from iron starvation rather than an antibacterial 
 activity of the amp / amx cargo .
analysis of 50% mhb by inductively 
 coupled plasma optical emission spectroscopy ( icp - oes ) revealed a 
 total iron concentration of ca .
4 m ( table 
 s2 ) , which can be compared to 10 m of a high - affinity 
 extracellular iron chelator .
indeed , we previously observed similar 
 growth inhibition of fepa- with 10 m of an 
 ent - vancomycin conjugate ( extracellular iron chelation ) and also 10 
 m d - ent ( iron chelator that can not be used for iron 
 acquisition ) under these growth conditions .
these data confirm that fepa is essential for the potent antibacterial 
 activity of ent - amp / amx against e. coli k-12 .
in contrast to fepa- , the growth of fepc- and fes- was completely inhibited with 100 nm ent - amp / amx 
 under iron - limiting conditions ( figure 3f , g ) , 
 comparable to what was observed for the wild - type strain . the targets 
 of -lactam antibiotics are penicillin binding proteins ( pbps ) , 
 which are located in the periplasm of gram - negative bacteria .
after 
 crossing the outer membrane through fepa , ent - amp / amx enter the periplasm 
 where covalent capture by the pbps presumably occurs .
thus , it is 
 reasonable that the downstream ent transport and processing steps 
 involving fepcdg and fes do not affect the antimicrobial activity 
 of the conjugates if they are trapped in the periplasm as a result 
 of pbp binding .
although it is possible that the ent uptake machinery 
 ( e.g. , periplasmic binding protein fepb ) competes with the pbps for 
 ent - amp / amx , no improved antibacterial activities were observed for 
 the fepc- and fes- mutants compared 
 to wild - type k-12 .
this observation indicates that ent - amp / amx bind 
 to pbps and are trapped in the periplasm .
it should be noted that 
 the fepb- mutant , which lacks the periplasmic binding 
 protein , was also considered in this work ; however , this strain exhibited 
 a severe growth defect and afforded inconsistent results . to probe 
 interaction between fepa and ent - amp / amx , we performed 
 growth inhibition assays employing mixtures of ent - amp / amx and varying 
 concentrations of unmodified ent ( figure 3h 
when these assays were performed under conditions of iron limitation , 
 the presence of exogenous ent attenuated the antibacterial activity 
 of ent - amp / amx .
a 1:1 molar ratio of ent - amx / ent afforded an od600 value comparable to that of the untreated control , whereas 
 higher equivalents of ent were required to block the antibacterial 
 action of ent - amp .
the origins of this difference are unclear and 
 may indicate that the hydroxyl group of amx has a negative effect 
 on the transport efficiency of the conjugate . in total , these ent 
 addition assays suggest that competition for ent - amp / amx and ent occurs 
 at the receptor(s ) and that the conjugates are delivered into the 
 bacteria via the same uptake machinery as ent .
l - serine is a biosynthetic 
 building block for ent , and a role 
 for chiral recognition in ent transport has been probed in prior studies . in one series of investigations ,
e. coli fepa was 
 found to bind ferric l - ent and ferric d - ent with 
 similar affinities ( kd = 21 and 17 nm , 
 respectively ; ascertained by measuring the binding of fe - loaded siderophores to e. coli bn1071 cells ) .
a lack of transport of ferric d - ent 
 into e. coli bn1071 was also reported in this work . 

a later study probed ent uptake in bacillus subtilis , and transport of both l- and d - ent analogues 
 was observed to occur with similar efficiency .
2457 t to hydrolyze l - ent and d - ent was 
 evaluated , and fes did not accept d - ent as a substrate . as 
 a result of the transport studies in b. subtilis and 
 enzymatic activity assays with s. flexneri fes , a 
 model in which both ent enantiomers are transported and chiral
taken together , these studies suggest that the ability 
 to transport d - ent may vary between species and even between 
 strains of a given species , and more studies are required to address 
 such possibilities .
based on the observation that e. coli fepa binds d - ent and that b. subtilis transports d - ent analogues , we synthesized the d - enantiomers of 
 the ent--lactam conjugates ( 13 and 14 , scheme 1 ) and evaluated the antibacterial 
 activity of these conjugates against e. coli k-12 , 
 25922 , cft073 , 35401 , and 43895 ( figures s9s13 ) .
d - ent - amp / amx exhibited reduced antibacterial activity 
 relative to ent - amp / amx for k-12 , 25922 , cft073 , and 43895 . under 
 conditions of iron limitation ,
complete growth inhibition was observed 
 with 1 m d - ent - amp / amx for e. coli k-12 , 25922 , and 43895 , compared to 100 nm for ent - amp / amx .
likewise , 
 a 10-fold reduction in antibacterial activity was observed for e. coli cft073 , where 100 nm d - ent was required 
 to inhibit growth completely .
in contrast , a negligible difference 
 in antibacterial activity of the l- and d - isomers 
 was observed for e. coli 35401 .
regardless of enantiomer , 
 all four ent - amp / amx conjugates provide enhanced antibacterial activity 
 against these e. coli strains relative to amp / amx . 

nevertheless , these data suggest that d - ent - amp / amx are less 
 readily transported into various e. coli strains 
 than the l - isomers .
although this modification provides no 
 appreciable benefit for this -lactam delivery system with periplasmic 
 targets , it is possible that ent - antibiotic conjugates based on d - ent may be desirable for delivering cargos to the cytosol , 
 precluding concomitant delivery of nutrient fe(iii ) . with support for ent - mediated delivery of ent - amp /
amx 
 to the e. coli periplasm , we sought to confirm the 
 essentiality of the -lactam warheads in antibacterial action . 

we therefore designed and prepared hydrolyzed ent - amp / amx analogues 15 and 16 ( scheme 2 ) where 
 the -lactam structure is destroyed .
hydrolysis of the amp / amx - alkynes 7 and 8 was achieved in the presence of 1% tfa 
 with heating at 37 c , and the decomposition products 9 and 10 were obtained as diastereomeric mixtures ( scheme 2 ) .
the formation of these species followed the reported 
 degradation pathways for ampicillin , where hydrolysis and subsequent 
 decarboxylation occur .
the diastereomeric 
 mixtures were employed to prepare the hydrolyzed conjugates ent - hydro - amp 15 and ent - hydro - amx 16 via a copper - catalyzed 
 click reaction .
( a , b ) antibacterial activity assays against e. coli k-12 ( a ) and cft073 ( b ) using ent - amp / amx and ent - hydro - amp / amx . 

( c ) antibacterial activity assays against e. coli atcc 35218 , which expresses a class a serine -lactamase , 
 using ent - amp / amx in the absence and presence of the -lactamase 
 inhibitors potassium clavulanate ( pc ) and sulbactam ( sb ) .
all assays 
 were performed in 50% mhb supplemented with 200 m dp ( mean 
  sem , n  3 ) .
we employed ent - hydro - amp / amx in antibacterial activity assays 
 against e. coli k-12 and observed negligible growth 
 inhibition ( figure 4a ) . when e. coli cft073 was treated with ent - hydro - amp / amx in the presence of dp
this result indicates that ent - hydro - amp / amx 
 are transported into the cytoplasm of e. coli cft073 , 
 where nutrient iron is released .
we also performed a series 
 of antibacterial activity assays with e. coli atcc 
 35218 , a strain that expresses a class a serine 
 -lactamase .
similar to unmodified amp / amx , ent - amp / amx were 
 inactive against e. coli atcc 35218 ( mic > 10 
 m ) 
 in the absence and presence of dp ( figures 4c and s14 ) .
slight growth inhibition was 
 observed at 10 m under conditions of iron limitation , which 
 may be attributed to iron chelation .
the addition of -lactamase 
 inhibitors restored the activities of ent - amp / amx and amp / amx , and 
 the conjugates exhibited greater antibacterial activity than the parent 
 antibiotics ( figures 4c and s14 ) . in total , the assays with ent - hydro - amp / amx and strains 
 expressing -lactamase demonstrate that an intact -lactam 
 is required for the antibacterial activity of ent - amp / amx .
moreover , 
 these studies indicate that the -lactams retain their original 
 function and inhibit pbps when conjugated to ent .
the remarkable sensitivity of e. coli cft073 to 
 ent - amp / amx ( figure 2c ) motivated us to investigate 
 the relative cell - killing kinetics 
 of ent - amp / amx and amp / amx to determine whether these conjugates kill e. coli cft073 more rapidly than the unmodified drugs . for 
 comparison between e. coli strains ,
ent - amp / amx 
 provide more rapid cell death than unmodified amp / amx ( figure 5 ) , and this behavior is most apparent for e. coli cft073 , where the od600 value was almost 
 reduced to the baseline value after 1 h incubation with 5 m 
 ent - amp / amx , corresponding to a 2-fold log reduction in cfu / ml . in 
 contrast , the change in od600 and cfu / ml for e. 
 coli cft073 treated with 50 m unmodified amp / amx is 
 negligible over this time period .
the time - kill kinetics for e. coli k-12 , conducted with 50 m of both unmodified 
 and modified -lactams , indicate a slight increase in kill kinetics 
 for ent - amp / amx relative to amp / amx , and that the kinetics of cell - killing 
 are slower for k-12 than cft073 ( figure 5 ) . 

these results support a model whereby ent modification facilitates 
 uptake of amp / amx relative to the unmodified drugs .
this effect is 
 more dramatic for e. coli cft073 than k-12 , which 
 is in accordance with the enhanced antibacterial activity observed 
 for cft073 relative to the other e. coli strains 
 considered in this work .
time - kill kinetic assays for treatment of e. coli k-12 ( top panel ) and cft073 ( bottom panel ) with 
 amp / amx and ent - amp / amx .
e. coli k-12 ( 10 cfu / ml ) was treated 
 with 50 m of amp / amx or 50 m ent - amp / amx .
e. 
 coli cft073 ( 10 cfu / ml ) was treated with 
 50 m of amp / amx or 5 m ent - amp / amx .
the assays were 
 conducted in 50% mhb medium containing 200 m dp at 37 c 
 ( mean  sem , n = 3 ) . to determine whether ent - amp / amx exhibit broad - spectrum or species - selective 
 activity , we performed antibacterial activity assays with two additional 
 gram - negative and two gram - positive species in both the absence and 
 presence of dp .
these species include klebsiella pneumoniae atcc 13883 , pseudomonas aeruginosa pao1 , s. aureus atcc 25923 , and bacillus cereus atcc 14579 .
k. pneumoniae is a gram - negative species 
 that biosynthesizes and utilizes ent for iron acquisition .
p. aeruginosa is a gram - negative bacterium that captures 
 ent as a xenosiderophore and expresses two ent receptors 
 pfea and pira.s .
aureus and b. cereus are both gram - positive bacterial species , and the ability to utilize 
 ferric ent as an iron source is reported for both species .
in contrast to gram - negative bacteria , where the pbps are located 
 in the periplasm , the targets of -lactam antibiotics are in 
 the extracellular peptidoglycan of gram - positive organisms .
k. pneumoniae atcc 13883 has a chromosomally encoded 
 class a -lactamase ( shv-1 ) and lacks sensitivity to amp / amx . 

we observed no effect of 100 m amp / amx on k. pneumoniae growth under our assay conditions ( figure s15 ) .
only 50% growth inhibition was observed when k. 
 pneumoniae was treated with high concentrations ( 10 m ) 
 of ent - amp / amx in the absence of dp , and the ent - amp conjugate provided 
 the greatest activity under conditions of iron limitation with 90% 
 growth inhibition at 10 m .
when -lactamase inhibitors 
 were included in the assays , k. pneumoniae exhibited 
 greater sensitivity to amp / amx ( mic = 100 m ) and ent - amp / amx 
 ( mic = 10 m ) ; however , we observed some growth inhibitory activity 
 of the -lactamase inhibitor sulbactam alone under these assay 
 conditions ( 100 m sb , figure s15 ) .
thus , the possibility of a synergistic effect from the inhibitors 
 and conjugates can not be ruled out completely .
the lack of activity 
 of ent - amp / amx against k. pneumoniae atcc 13883 is 
 reminiscent of results obtained during investigations of amp / amx - functionalized 
 tripodal triscatecholate ligands .
these 
 compounds were inactive against k. pneumoniae , and 
 the behavior was attributed to either an inability of the k. pneumoniae iron transport machinery to import the conjugates 
 or the development of resistance over the course of the assay .
an 
 alternative explanation is that -lactamase expression by k. pneumoniae resulted in inactivation of the -lactams .
the p. aeruginosa pao1 strain employed in this 
 work exhibited little sensitivity to both amp / amx and the conjugates 
 under the antibacterial assay conditions ( figure 
 s16 ) .
amp / amx exhibited no activity up to 100 m , whereas 
 ent - amp / amx provided growth inhibition at 10 m in both the 
 absence and presence of dp . whether these results indicate that ent - amp / amx 
 will be ineffective against multiple p. aeruginosa strains is unclear .
p. aeruginosa strains exhibit 
 different phenotypes , and highly variable and strain - dependent mic 
 values have been reported for triscatecholate-lactam 
 conjugates against p. aeruginosa .
we previously reported that p. aeruginosa pao1 imports ent - cargo conjugates , and 
 we speculate that the lack of activity observed for this strain stems 
 from its inherent insensitivity to amp / amx .
b. cereus atcc 14579 was also insensitive to amp / amx , 
 which only afforded growth inhibition at 100 m .
some growth 
 inhibition was observed for b. cereus treated with 
 10 m ent - amp / amx , which may result from iron sequestration 
 ( figure s17 ) .
s. aureus atcc 25923 is susceptible to amp / amx , with complete growth inhibition 
 observed at 1 m . in this case , a 10-fold reduction in antibacterial 
 activity was observed for ent - amp / amx relative to unmodified amp / amx 
 ( figure 6 ) . although the origins of this attenuation 
 are unclear , we speculate that ent - amp / amx may have trouble penetrating 
 the thick peptidoglycan of s. aureus .
an alternative 
 possibility is that recognition of ent - amp / amx by the s. aureus ent receptor diverts the -lactams from the pbps . in 
 total , the results from these assays indicate that ent - amp / amx 
 exhibit antibacterial activity enhancements that are species - selective , 
 providing increased potency against e. coli strains 
 and not for the other strains evaluated in this work . we therefore 
 reasoned that ent - amp / amx , at low concentrations , should selectively 
 kill e. coli in the presence of other less sensitive 
 species .
we treated co - cultures of e. coli cft073 
 and s. aureus with ent - amp / amx or amp / amx and analyzed 
 the species composition following a 19-h incubation using harty - uti 
 plates .
these agar plates are employed in medical microbiology laboratories 
 for the diagnosis of urinary tract infections and provide species 
 identification by the colony color . when grown on harty - uti plates ,
s. aureus are off - white and e. coli are 
 purple - pink . in figure 6 , we present representative 
 images of the colonies that resulted from treating co - cultures of e. coli and s. aureus with amp / amx or ent - amp / amx . 
 in the absence of antibiotic ,
the cultures provide a mixture of off - white 
 and purple - pink colonies , indicating that both e. coli and s. aureus grow when cultured together .
when 
 the co - cultures are treated with 1 m amp / amx , only purple - pink 
 colonies are present , which reveals that only e. coli survives .
in contrast , treatment of the co - cultures with 1 m 
 ent - amp / amx results in only off - white colonies from s. aureus .
these comparisons demonstrate that ent - amp / amx selectively kill e. coli in the presence of s. aureus and 
 that the siderophore modification reverses the inherent species 
 selectivity of the parent antibiotics .
achieving such species - selective 
 and single - pathogen antibiotic targeting is an important goal and 
 unmet need for pharmaceutical development that will allow for treating 
 disease with minimal perturbation to the commensal microbiota .
ent - amp / amx 
 selectively kill e. coli cft073 in 
 the presence of s. aureus atcc 25923 .
( a , b ) antimicrobial 
 activity assays against s. aureus atcc 25923 in the 
 absence ( a ) and presence ( b ) of 200 m dp .
( c , d ) bacterial growth 
 monitored by od600 for cultures of e. coli only , s. aureus only , and 1:1 e. coli / s .
aureus mixtures treated with amp / amx or ent - amp / amx 
 in the absence ( c ) and presence ( d ) of 200 m dp .
( e ) representative photographs of colonies 
 from mixed cultures of e. coli cft073 and s. aureus atcc 29523 treated with ent - amp / amx ( 1 m ) 
 or amp / amx ( 1 m ) in the presence of 200 m dp .
all assays 
 were conducted in 50% mhb medium ( t = 19 h , 30 c ) 
 ( mean  sem , n  3 for a  d ) .
we evaluated the cytotoxicity of ent - amp 
 against the human t84 colon 
 epithelial cell line .
cell survival was evaluated by mtt assay after 
 a 24 h treatment with apo or iron - bound ent - amp , amp , or ent .
the 
 iron - bound forms were assayed to determine whether iron chelation 
 in the growth medium is a factor .
no cytoxicity was observed for amp 
 or ent - amp , whereas apo ent itself decreased the survival of t84 cells 
 by approximately 30% at the highest concentration evaluated . when 
 pre - loaded with fe(iii ) ,
percent cell survival quantified by mtt assay after 
 a 24 h treatment with apo or iron - bound ent , ent - amp , and the parent 
 antibiotic amp in the absence and presence of 1 equiv of fe(iii ) ( mean 
  sem , n = 3 ) .
lipocalin-2 
 ( lcn2 , also known as siderocalin or ngal ) is a 22-kda protein produced 
 and released by neutrophils and epithelial cells .
it has a hydrophobic 
 binding pocket and coordinates ferric ent with sub - nanomolar affinity . by sequestering ferric ent
, this host - defense protein contributes 
 to the metal - withholding response and prevents bacterial acquisition 
 of this essential nutrient . to determine whether lcn2 also binds ent - amp / amx 
 and thereby blocks antibacterial activity ,
we performed antibacterial 
 activity assays with e. coli cft073 in m9 minimal 
 medium supplemented with lcn2 or bovine serum albumin ( bsa ) . under 
 these conditions , up to 1 m of lcn2 had no effect on the growth 
 of e. coli cft073 . addition of 1 m lcn2 to 
 the medium rescued the growth of e. coli cft073 treated 
 with 100 nm of ent - amp ( figure 8) whereas addition 
 of 1 m bsa had no effect on ent - amp cell killing .
these results 
 suggest that lcn2 binds ent - amp and blocks its recognition and uptake . 

to ascertain whether lcn2 binds ent - amp in the presence of exogenous 
 ent , we performed a series of experiments where e. coli cft073 were treated with fixed concentrations of ent - amp ( 100 nm ) 
 and lcn2 ( 1 m ) and the concentration of ent was varied ( 0 , 
 0.5 , and 1 m ) .
moreover , when 
 ent - amp was combined with a 1:1 molar ratio of ent and lcn2 at 10-fold 
 excess over the conjugate , no e. coli growth was 
 observed , which suggests that lcn2 preferentially binds ent .
prior 
 work demonstrated that lcn2 can not bind glucosylated ent , which was 
 attributed to a steric clash between the glucose moieties and the 
 ent binding site of the protein , and 
 decreased hydrophobicity of the siderophore may also be a factor . 

thus , our data suggest that the nature of linker attachment at c5 
 and the peg3 moiety of ent - amp / amx do no abrogate lcn2 
 binding as effectively as the glucose moieties exhibited by the salmochelins
. antibacterial 
 activity of ent - amp against e. coli cft073 in the 
 presence of lcn2 or bsa .
( a ) e. coli cft073 treated 
 with 100 nm ent - amp and varying concentrations of 
 lcn2 or bsa control .
( b ) e. coli cft073 treated with 
 ent - amp , varying concentration of ent , and varying concentrations 
 of lcn2 or bsa control .
the assays were performed in m9 minimal medium 
 ( 24 h , 37 c ) ( mean  sem , n  3 ) . 

ent - amp / amx are two siderophore-lactam 
 conjugates 
 based on the native enterobactin scaffold .
these molecules hijack 
 siderophore - based iron uptake pathways and provide potent antibacterial 
 activity against various e. coli strains , including 
 human pathogens .
our investigations of ent - amp / amx establish the following : 
 ( i ) ent - amp / amx provide up to 1000-fold enhanced antibacterial activity 
 against e. coli strains ; ( ii ) ent - amp / amx are transported 
 into e. coli by fepa and potentially other catecholate 
 siderophore receptors ( iron , iha ) employed by pathogenic ctf073 
 and uti98 ; ( iii ) ent - amp / amx are captured by pbps in the periplasm , 
 which results in pbp inhibition and cell death ; ( iv ) selective killing 
 of e. coli in the presence of less susceptible organisms 
 such as s. aureus is achieved because of the enterobactin 
 scaffold ; ( v ) ent - amp / amx exhibit negligible cytotoxicity to human 
 t84 intestinal epithelial cells ; and ( vi ) although lcn2 has the ability 
 to bind ent - amp / amx , this siderophore - scavenging protein prefers 
 to capture native ent . in total
, these studies demonstrate that modification 
 of antibiotic cargo with the native enterobactin platform provides 
 many desirable features for antibiotic delivery and efficacy .
the 
 large molecular weight of the conjugates resulting from the native 
 ent scaffold ( as opposed to a smaller mimic ) enhances rather than 
 diminishes uptake for gram - negative e. coli .
moreover , 
 we observed no evidence for the development of resistance to ent - amp / amx 
 over the course of the antibacterial activity assays performed during 
 these investigations .
our studies confirm that the enhanced 
 antibacterial activity observed 
 for ent - amp / amx requires both enterobactin recognition by outer membrane 
 receptors and an intact -lactam moiety .
the results are reminiscent of the dramatic 
 antibacterial activity enhancements observed for albomycin , a secondary 
 metabolite produced by actinomyces subtropicus .
albomycin 
 is comprised of the siderophore ferrichrome and a trna synthetase 
 inhibitor , and it exhibits antimicrobial activities that are 30,000-fold 
 greater than those of the unmodified trna synthetase inhibitor against e. coli and s. aureus . nonetheless , the ent - amp / amx cell - killing mechanism may 
 be more complex than only more efficient -lactam delivery across 
 the gram - negative outer membrane .
binding of ent - amp / amx to the pbps 
 presumably results in accumulation of ferric enterobactin in the e. coli periplasm for some period of time , which may have 
 deleterious consequences .
a recent study of an e. colitolc- mutant revealed that enterobactin accumulation 
 in the periplasm affords growth defects and abnormal cellular morphologies .
a fascinating observation that stems 
 from our current work is the 
 variable susceptibilities and responses of different e. coli strains to ent - amp / amx , which contrast the effects of unmodified 
 amp / amx .
such differences are manifest in the mic values to some degree 
 and time - kill kinetics ; however , the results presented in figure 2 indicate that mic values alone do not provide a 
 full description of how ent - amp / amx susceptibility differs between e. coli strains .
these results suggest underlying complexity 
 in microbial physiology related to iron - uptake pathways that can not 
 be fully explained by the presence or absence of a gene for a particular 
 receptor ( i.e. , fepa , iron ) .
the heightened sensitivity of uropathogenic e. coli cft073 is particularly noteworthy , and it will be 
 interesting to decipher the physiological origins of this effect as 
 well as the differential behavior of various e. coli pathogens toward ent - antibiotic conjugates . there is a clear 
 and unmet need for new antibacterial agents to 
 treat bacterial infections in humans , including antibiotics that target 
 specific bacterial sub - populations . preventing undesirable consequences of antibiotic treatment on the 
 commensal microbiota , which contributes to human health in beneficial 
 ways , is a challenge that needs to be addressed .
such targeted therapeutics will be valuable not only for treating 
 bacterial infections when the causative agent is known ( e.g. , urinary 
 tract infection and e. coli , cystic fibrosis lung 
 infection and p. aeruginosa ) but also for other pathologies 
 that involve microbial dysbiosis , such as irritable bowel disease .
our studies of ent - amp / amx provide one step 
 toward addressing species - specific antibiotic targeting as well as 
 overcoming gram - negative outer membrane permeability . from the standpoint 
 of the host environment , commensal e. coli employ 
 ent for acquiring iron in the host , and thus further elaboration of 
 this strategy to specifically target pathogenicity and evade host 
 responses ( e.g. , lcn2 ) is desirable .
 the design , synthesis , and characterization 
 of enterobactin  
 antibiotic 
 conjugates , hereafter ent - amp / amx , where the -lactam antibiotics 
 ampicillin ( amp ) and amoxicillin ( amx ) are linked to a monofunctionalized 
 enterobactin scaffold via a stable poly(ethylene glycol ) linker are 
 reported . under conditions of iron limitation , 
 these siderophore - modified 
 antibiotics provide enhanced antibacterial activity against escherichia coli strains , including uropathogenic e. coli cft073 and uti89 , enterohemorrhagic e. coli o157:h7 , and enterotoxigenic e. coli o78:h11 , compared to the parent -lactams . 
 studies with e. coli k-12 derivatives defective in ferric enterobactin 
 transport reveal that the enhanced antibacterial activity observed 
 for this strain requires the outer membrane ferric enterobactin transporter 
 fepa . a remarkable 1000-fold decrease in minimum inhibitory concentration 
 ( mic ) value is observed for uropathogenic e. coli cft073 relative to amp / amx , and time - kill kinetic studies demonstrate 
 that ent - amp / amx kill this strain more rapidly at 10-fold lower concentrations 
 than the parent antibiotics . 
 moreover , ent - amp and ent - amx selectively 
 kill e. coli cft073 co - cultured with other bacterial 
 species such as staphylococcus aureus , and ent - amp 
 exhibits low cytotoxicity against human t84 intestinal cells in both 
 the apo and iron - bound forms . 
 these studies demonstrate that the native 
 enterobactin platform provides a means to effectively deliver antibacterial 
 cargo across the outer membrane permeability barrier of gram - negative 
 pathogens utilizing enterobactin for iron acquisition . 
Introduction
Experimental Section
Results and Discussion
Summary and Perspectives