Enterobactin-mediated delivery of ?-lactam antibiotics enhances antibacterial activity against pathogenic escherichia coli

Article abstract of DOI:10.1021/ja503911p

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(ethy

Full text of DOI:10.1021/ja503911p

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Article
Enterobactin-Mediated Delivery of #-Lactam Antibiotics Enhances
Antibacterial Activity Against Pathogenic Escherichia coli
Tengfei Zheng, and Elizabeth M. Nolan
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 13 Jun 2014
Downloaded from http://pubs.acs.org on June 17, 2014
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Enterobactin-Mediated Delivery of β-Lactam Antibiotics Enhances Antibacterial Activity
Against Pathogenic Escherichia coli
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Tengfei Zheng and Elizabeth M. Nolan*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
*Corresponding author: lnolan@mit.edu
Phone: 617-452-2495
Fax: 617-324-0505
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Abstract
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The design, synthesis, and characterization of enterobactin-antibiotic conjugates,
hereafter Ent-Amp/Amx, where the β-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx)
are linked to a monofunctionalized enterobactin scaffold via a stable polyethyleneglycol 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 β-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.
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Introduction
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Bacterial infections as well as the emergence and spread of antibiotic resistance in
human pathogens are serious public health problems that plague hospital and community
settings across the globe.1 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 therapeutic strategies.2-8
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Metal ions are essential nutrients for all organisms. Almost all bacterial species have a
metabolic iron requirement and therefore employ various strategies to acquire this metal ion
when colonizing.9,10 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.10,11 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).12 Numerous studies support the
importance for siderophore-based iron acquisition during bacterial infections.13-19 Thus, the
potential of using siderophores,20-25 or targeting siderophore biosynthetic and transport
machineries,26-28 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-antibiotic conjugates for “Trojan horse” antibiotic delivery.29-58 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.6-8
Both native siderophores and synthetic siderophore mimics have been evaluated as
platforms for therapeutic development.20-25,59,60 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
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siderophore-antibiotic conjugates.61 The sideromycins are secondary metabolites comprised of
a siderophore moiety and a toxic cargo; the siderophore portion targets sideromycins to
bacterial strains expressing the siderophore receptor. Microcin E492m, a siderophore-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.62 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.
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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;63 (ii) modification of
antibiotics such that the antibacterial activity is attenuated or lost completely;42,44,51,58 (iii)
bridging the siderophores and antibiotics with problematic linkers, including linkers designed for
drug release that are too stable or are too labile and cause premature release;25,45-48 (iv) and
antibiotic resistance.34,63 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.63 Their results indicated that competition between the siderophore-conjugated
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monobactam MB-1 and native siderophores resulted in poor in vivo efficacy against
Pseudomonas aeruginosa, and provide support for designing and evaluating siderophore-
antibiotic conjugates based on native siderophore platforms. One recent and successful
example based on a native siderophore platform is a mycobactin-artemisinin conjugate that
provides enhanced antibacterial activity against Mycobacterium tuberculosis compared to
unmodified artemisinin.64
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Enterobactin (Ent, Figure 1) is a triscatecholate siderophore biosynthesized by enteric
bacteria and used for iron acquisition in the vertebrate host.65 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.66 Moreover, we established that the native Ent platform,
when monofunctionalized at the C5 position of one catecholate ring (Figure 1), affords delivery
of non-toxic small-molecule cargo across the outer membrane of Gram-negative organisms that
express Ent uptake machinery (e.g., FepABCDG of Escherichia coli).66 As described herein, this
proof-of-concept delivery 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.
O
OH
OH
O
O
OH
OH
N
H
O
O
NH
O
O
NH
O
O
O
O
O
O
O
O
O
O
R
O
O
O
OH HN
N
H
HO
OH HN
N
H
HO
HO
HO
O
O
OH
OH
1, Enterobactin (Ent)
Enterobactin-cargo conjugate
R = a cargo
Figure 1. Structures of enterobactin (1, Ent) and a generalized enterobactin-cargo conjugate.
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In this work, we present the syntheses and characterization of siderophore-antibiotic
conjugates based on the native Ent platform that harbor the clinically-relevant β-lactam
antibiotics ampicillin (Amp) and amoxicillin (Amp). 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-β-lactam conjugates exhibit significantly enhanced
antibacterial activity (up to 1000-fold) against pathogenic E. coli and provide more rapid cell
killing than the parent β-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 β-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.
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Experimental Section
Synthetic Reagents. 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. HPLC-grade
acetonitrile (MeCN) was purchased from EMD. L-Ent 1,67 the D-enantiomer of benzyl-protected
Ent-CO2H 2, and the benzyl-protected Ent-PEG3-N3 3 were synthesized according to previously
reported procedures.66 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. General synthetic methods are provided as Supporting
Information.
N1-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-N3-((3S,7S,11S)-7,11-bis(2,3-dihy-
droxybenzamido)-2,6,10-trioxo-1,5,9-trioxacyclododecan-3-yl)-4,5-dihydroxy-isophthal-
amide (4). Benzyl-protected Ent-azide 3 (80 mg, 55 µmol) and pentamethyl benzene (PMB, 147
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mg, 990 µmol) were dissolved in 5 mL of anhydrous CH2Cl2 to give a light yellow solution. This
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solution was cooled to -78 C in an acetone/dry ice bath under N2, and BCl3 (660 µL of 1 M
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solution in CH2Cl2, 660 µmol) was added slowly along the flask wall. After stirring for 1.5 h,
DIPEA (300 µL, 1.73 mmol) was added to the flask, followed by addition of 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 33-60% B over 20 min, 10
mL/min). The product eluted at 17 min and was lyophilized to yield compound 4 as white solid
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(13.9 mg, 28%). H NMR (DMSO-d6, 500 MHz), δ 3.35-3.57 (16H, m), 4.38-4.41 (3H, m), 4.63-
4.69 (3H, m), 4.89-4.96 (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), 8.33-8.35 (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). 13C NMR (CDCl3,
125 MHz), δ 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-1), 3389, 2954, 2928, 2868, 2111, 1754, 1645, 1589, 1535, 1460, 1384, 1329,
1266, 1176, 1132, 1074, 992, 846. HRMS (ESI): [M+Na]+ m/z calcd., 932.2506; found,
932.2520.
N1-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-4,5-bis(benzyloxy)-N3-((3R,7R,
11R)-7,11-bis(2,3-bis(benzyloxy)benzamido)-2,6,10-trioxo-1,5,9-trioxacyclo-dodecan-3-yl)
iso-phthalamide (5). 11-Azido-3,6,9-trioxaundecan-1-amine (36.4 µL, 186 µmol) and D-Bn6Ent-
COOH (2, 177 mg, 142 µmol) were dissolved in 5 mL of dry CH2Cl2. PyAOP (147 mg, 283 µmol)
and DIPEA (98.5 µL, 568 µmol) were added to give a light yellow solution. The reaction was
stirred for 4 h at rt and concentrated, and the crude product was purified by preparative TLC
(50% EtOAc/CH2Cl2) to afford 5 as a white foam (159 mg, 77%). TLC Rf = 0.6 (10%
1
MeOH/CH2Cl2). H NMR (DMSO-d6, 500 MHz), δ 3.33 (2H, J = 5.2 Hz), 3.62-3.69 (14H, m),
4.02-4.06 (3H, m), 4.15-4.18 (3H, m), 4.91-4.94 (3H, m), 5.04-5.21 (12H, m), 6.96 (1H, s), 7.11-
7.45 (36H, m), 7.65-7.67 (2H, m), 7.85-7.85 (1H, m), 7.97-7.97 (1H, m), 8.50-8.54 (3H, m). 13C
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NMR (CDCl3, 125 MHz), δ 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 (neat, cm-1), 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): [M+H]+ m/z calcd., 1476.5323; found, 1476.5318.
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N1-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-N3-((3R,7R,11R)-7,11-bis(2,3-dihy-
droxybenzamido)-2,6,10-trioxo-1,5,9-trioxacyclododecan-3-yl)-4,5-dihydroxyiso-phthal-
amide (6). Compound 6 was synthesized from 5 (153 mg, 105 µmol) following the same
procedure as for compound 4. The crude reaction was purified by preparative HPLC (33% B for
5 min and 33-60% 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%). 1H NMR (CDCl3, 500 MHz), δ 3.34-
3.56 (16H, m), 4.39-4.41 (3H, m), 4.61-4.66 (3H, m), 4.88-4.94 (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), 8.31-8.33
(1H, m), 9.12-9.13 (2H, m), 9.27-9.28 (1H, m), 9.50 (2H, bs), 9.84 (1H, bs), 11.6 (2H, bs), 11.9
(1H, bs). 13C NMR (CDCl3, 125 MHz), δ 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-1), 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.
(2S,5R,6R)-6-((R)-2-(Hex-5-ynamido)-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylic acid (7). 5-Hexynoic acid (113 µL, 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
acetyl 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
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(210 mg, 2.5 mmol) in 4:1 water/acetone (2.5 mL) and cooled on ice, to which the acetyl
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 x 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 x 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%). This solid was used in the following
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steps without further purification. TLC Rf = 0.1 (10% MeOH/CH2Cl2). H NMR (DMSO-d6, 500
MHz), δ 1.41 (3H, s), 1.55 (3H, s), 1.64-1.69 (2H, m), 2.13-2.16 (2H, m), 2.29-2.32 (2H, m),
2.77-2.78 (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), 7.25-7.43 (5H, m), 8.57 (1H, d, J = 8.0 Hz), 9.11 (1H, d, J = 8.0 Hz). 13C
NMR (DMSO-d6, 125 MHz), δ 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-1), 3297, 3058,
3023, 2970, 2937, 2863, 2626, 2526, 2120, 1780, 1688, 1518,1455, 1437, 1390, 1373, 1324,
1295, 1208, 1139, 1027, 1001, 843. HRMS (ESI): [M+H]+ m/z calcd., 444.1588; found,
444.1585.
(2S,5R,6R)-6-((R)-2-(Hex-5-ynamido)-2-(4-hydroxyphenyl)acetamido)-3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (8). Compound 8 was synthesized
as described for compound 7 except that amoxicillin (760 mg, 2.08 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. TLC Rf = 0.1 (10% MeOH/CH2Cl2). 1H NMR
(DMSO-d6, 500 MHz), δ 1.42 (3H, s), 1.56 (3H, s), 1.63-1.68 (2H, m), 2.12-2.15 (2H, m), 2.25-
2.29 (2H, m), 2.76-2.78 (1H, m), 4.19 (1H, s), 5.39 (1H, d, J = 4.0 Hz), 5.51-5.54 (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). 13C NMR (DMSO-d6, 125 MHz), δ 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-
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1), 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.
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8
9
(4S)-2-(((R)-2-(Hex-5-ynamido)-2-phenylacetamido)methyl)-5,5-dimethylthiazolidine
-4-carboxylic acid (9). 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 oC for
24 h and purified by preparative HPLC (20-50% B over 25 min, 10mL/min), which afforded a
white powder (12 mg, 25%). The white powder is a diastereomeric mixture of products and no
further separation was performed. 1H NMR (DMSO-d6, 500 MHz), δ (mixture of two
diastereomers) 1.22-1.23 (3H, pair of s), 1.54-1.58 (3H, pair of s), 1.62-1.68 (2H, m), 2.12-2.14
(2H, m), 2.28-2.30 (2H, m), 2.77 (1H, s), 3.18-3.24 (0.5H, m), 3.30-3.36 (0.5H, m), 3.44-3.49
(0.5H, m), 3.55-3.60 (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), 5.45-5.48 (1H, m), 7.27-7.40 (5H, m), 8.50-8.61 (2H, m). 13C
NMR (DMSO-d6, 125 MHz), δ (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-1), 3297, 3071, 3041, 2967, 2938, 2535, 2124, 1734, 1653, 1527, 1456, 1427,
1375, 1299, 1199, 1137, 1070, 1027, 836. HRMS (ESI): [M+Na]+ m/z calcd., 440.1614; found,
440.1626.
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(4S)-2-(((R)-2-(Hex-5-ynamido)-2-(4-hydroxyphenyl)acetamido)methyl)-5,5-dime-th-
ylthiazolidine-4-carboxylic acid (10). 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 (20-50% B over 25 min, 10mL/min), and obtained as white
1
powder (14.5 mg, 24%). H NMR (DMSO-d6, 500 MHz), δ (mixture of two diastereomers) 1.26
(3H, s), 1.55-1.59 (3H, pair of s), 1.61-1.67 (2H, m), 2.12-2.14 (2H, m), 2.24-2.27 (2H, m), 2.77
(1H, s), 3.20-3.26 (0.5H, m), 3.34-3.39 (0.5H, m), 3.42-3.47 (0.5H, m), 3.56-3.60 (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), 5.28-
5.31 (1H, m), 6.69 (2H, d, J = 8.5 Hz), 7.17 (2H, d, J = 8.5 Hz), 8.36-8.50 (3H, m). 13C NMR
(DMSO-d6, 125 MHz), δ (mixture of two diastereomers) 17.5, 24.3, 26.9, 27.5, 27.7, 28.8, 33.8,
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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-1), 3301, 3071, 3028, 2973, 2928, 2548, 2111, 1737, 1662, 1606, 1593,
1515, 1435, 1377, 1197, 1139, 837. HRMS (ESI): [M+Na]+ m/z calcd., 456.1564; found,
456.1569.
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Ent-Amp (11). Ampicillin-alkyne 7 (120 µL of an 80-mM solution in DMSO, 9.6 µmol)
and Ent-PEG3-N3 (4, 250 µL of a 13-mM solution in 1,4-dioxane, 3.25 µmol) were combined and
400 µL of DMSO was added. CuSO4 (100 µL of a 90-mM solution in water, 9.0 µmol) and
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 200 µL of a 50-mM solution in DMSO,
10 µmol) were combined and 100 µL of DMSO was added to give a blue-green solution, to
which NaAsc (400 µL of a 90-mM solution in water, 36.0 µmol) was added. This solution
became light yellow and was immediately added to the alkyne/azide solution. The reaction was
shaken on a bench-top rotator for 2 h at room temperature 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 β-lactam). The HPLC fractions containing 11
were collected manually and flash frozen in liquid N2 immediately after collection to prevent β-
1
lactam decomposition. The product was obtained as white powder (3.3 mg, 76%). H NMR
(DMSO-d6, 500 MHz), δ 1.40 (3H, s), 1.54 (3H, s), 1.78-1.81 (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), 4.38-4.44 (5H, m), 4.64-4.66
(3H, m), 4.91-4.92 (3H, m), 5.39 (1H, d, J = 3.5 Hz), 5.51-5.52 (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), 7.26-7.35 (5H, m), 7.42-7.45 (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.
Ent-Amx (12). Compound 12 was synthesized as describe for 11 except that compound
8 was used instead of compound 7. The product was obtained as white powder (2.9 mg, 66%).
1H NMR (DMSO-d6, 500 MHz), δ 1.40 (3H, s), 1.54 (3H, s), 1.78-1.80 (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), 4.39-4.43 (5H, m),
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4.63-4.65 (3H, m), 4.90 (3H, bs), 5.38 (1H, s), 5.52-5.56 (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), 8.33-8.39 (2H, m), 8.94 (1H, d, J = 8.0 Hz), 9.11-9.12
(2H, m), 9.29 (1H, bs), 9.38-9.43 (3H, m), 9.75 (1H, s), 11.6 (2H, bs), 11.9 (1H, bs). HRMS
(ESI): [M+Na]+ m/z calcd., 1395.3970; found, 1395.3982.
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D-Ent-Amp (13). As described for 11 except that compound 6 was used instead of
compound 4. The product was obtained as white powder (1.6 mg, 36%). HRMS (ESI): [M+Na]+
m/z calcd., 1379.4021; found, 1379.4022.
D-Ent-Amx (14). As described for 12 except that compound 6 was used instead of
compound 4. The product was obtained as white powder (2.9 mg, 66%). HRMS (ESI): [M+Na]+
m/z calcd., 1395.3970; found, 1395.3995.
Ent-Hydro-Amp (15). As described for 11 except that compound 9 was used instead of
compound 7. The product was obtained as white powder (1.8 mg, 21%). HRMS (ESI): [M+H]+
m/z calcd., 1331.4409; found, 1331.4389.
Ent-Hydro-Amx (16). Compound 16 was synthesized as described for 12 except that
compound 10 was used instead of compound 8. The product was obtained as white powder (1.5
mg, 17%). HRMS (ESI): [M+Na]+ m/z calcd., 1369.4177; found, 1369.4191.
General Procedures for Antimicrobial Activity Assays. General microbiology
materials and methods, including details of Ent-Amp/Amx stock solution preparation and
storage, are detail 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
o
appropriate freezer stock. The overnight cultures were incubated at 37 C for 16-18 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 µM 2,2’-dipyridyl (DP) and
o
incubated at 37 C with shaking at 150 rpm until OD600 reached 0.6. Each culture was
subsequently diluted to in 50% reduced MHB medium (10.5 g/L) with or without 200 µM DP to
achieve an OD600 value of 0.001. A 90-µL aliquot of the diluted culture was combined with a 10-
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µL aliquot of a 10x 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 (endpoint 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 are reported and the error bars are the standard error of the mean obtained from the
independent replicates.
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Antimicrobial Assays in the Presence of β-Lactamase Inhibitors. 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 β-lactam/inhibitor mixtures were: sulbactam/Amp or Amp-Ent, 1.5:1; potassium
clavulanate/Amx or Amx-Ent, 0.9:1. These ratios were taken from the recipe of commercial drug
combinations.68 Sulbactam and potassium clavulanate were stored as DMSO stock solutions at
-20 oC.
Antimicrobial Assays in the Presence of Exogenous Ent. These assays were
performed with E. coli K-12 and CFT073 following the general procedure except that varying
concentrations (1 – 100 µM) of synthetic L-Ent were mixed with Ent-Amp/Amx.
Antimicrobial Activity Assays in the Presence of Lipocalin 2. This assay is based on
a published protocol69 and was conducted with E. coli CFT073. Overnight cultures of E. coli
CFT073 were grown in M9 minimal medium. Each overnight culture was serially diluted into M9
minimal medium to provide 103-104 CFU/mL. Lipocalin 2 was diluted in PBS to a concentration
o
of 10 µM upon arrival, aliquoted, and stored at -20 C until use. A 90-µL aliquot of the diluted
culture was added to 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 µL with sterile PBS. The 96-well
plate was incubated at 37 oC for 24 h in a tabletop incubator set at 150 rpm and bacterial growth
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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 standard error of the mean.
Time-Kill Kinetic Assays. 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 µM DP,
and this culture was incubated at 37 oC 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 x 10 min, rt)
and the resulting pellet was resuspended 50% MHB and centrifuged two times. The resulting
pellet was resuspended in 50% MHB with or without DP and the OD600 was adjusted to 0.3. A
90-µL aliquot of the resulting culture was mixed with a 10-µL aliquot of a 10x 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, 3 h
by using a plate reader. In a parallel experiment, a 10-µL aliquot of the culture was taken at t =
0, 1, 2, 3 h and serially diluted by using sterile phosphate-buffered saline (PBS) and plated on
LB-Agar plates for colony counting (CFU/mL). Each well condition was repeated at least three
times independently on different days. The resulting mean OD600 or CFU/mL is reported and the
error bars are the standard error of the mean.
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Mixed-Species Assays. A 5-mL overnight culture of each bacterial strain was grown in
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LB, diluted 1:100 into 5 mL of fresh LB media containing 200 µM 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 106 CFU/mL in 50% MHB with or without 200 µM
DP. For experiments requiring a mixture of two species, a 1:1 mixture was prepared (106
CFU/mL for each strain) in 50% MHB with or without 200 µM 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-µL aliquot of each culture was combined
with a 10-µL aliquot of a 10-µM solution of the antibiotic or Ent-antibiotic conjugate in a 96-well
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plate, which was covered and 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 standard error of the mean. Images of representative
CHROM-UTI plates are presented.
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Results and Discussion
Design and Syntheses of Enterobactin-Antibiotic Conjugates Harboring β-Lactams. 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 β-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, 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, napthalene, phenylmethyl
benzene) to the cytosol readily whereas largo cargos (e.g. vancomycin) are not transported to
the cytosol.66 Moreover, in prior studies, various β-lactams including Amp and Amx have been
linked to simple catechols29-32,34-36,40 and more complex catechol-containing siderophores or
siderophore mimics,43,44,46,48,53,56,70 which provides the opportunity to compare the outcomes
obtained for different siderophore-inspired design strategies.
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NH2
O
R
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1. SOCl2
reflux
NH
O
R
O
HN
O
+
7, R = H
8, R = OH
O
HO
HN
H
2. NaHCO3, rt
O
N
H
N
S
S
O
CO2
H
O
OH
OH
N
N
N
CO2
H
3
H
N
R = H, Ampicillin (Amp)
R = OH, Amoxicillin (Amx)
O
NH
R
O
O
NH
CuSO4, TBTA
NaAsc
O
HN
O
O
O
O
O
O
O
O
N
H
O
OBn
OBn
NH
O
O
OH
OH
O
H
N
DMSO/H2O, rt
O
H
N
OH
HN
S
N
H
HO
HO
COOH
O
O
O
O
NH
OH
O
O
O
BCl3, PMB
11, R = H, Ent–Amp
12, R =OH, Ent–Amx
O
O
O
O
O
O
O
N3
O
N3
O
CH2Cl2, -78oC
O
O
13, R = H,
D
-Ent–Amp
D-Ent–Amp
BnO HN
N
H
BnO
OH HN
N
H
HO
14, R = OH,
BnO
O
HO
O
O
OBn
OH
4 or its enantiomer 6
3 or its enantiomer 5
Scheme 1. Syntheses of the L- and D-forms of Ent-Amp/Amx.
In Scheme 1, we present the syntheses of Ent-Amp 11 and Ent-Amx 12, which feature
installation of alkyne-modified β-lactam warheads onto Ent-azide 4 via copper-catalyzed click
chemistry in the final step. The catechol moieties of benzyl-protected Ent-azide 3 were
o
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;67,71 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 β-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 copper-catalyzed click
chemistry with Ent-azide 4 and the alkyne-modified β-lactams 7/8, prepared by thionyl chloride
coupling of 5-hexynoic acid to the amino group of Amp/Amx, failed because of copper-mediated
β-lactam decomposition.72 This problem was overcome by including the metal-ion chelator
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tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)73 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 β-lactam moieties. This synthetic route was employed to prepare the D-enantiomers of
Ent-Amp/Amx 13 and 14 (Scheme 1).
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This synthesis affords intermediates and reaction conditions of broad utility. Deprotected
Ent-azide 4 enables alkyne-functionalized molecules to be covalently linked to Ent via click
chemistry, 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. This synthesis also
provides opportunity to elaborate β-lactams for a variety of purposes. Indeed, very few
examples employing copper(I)-catalyzed click chemistry with fused β-lactams are reported in
the literature,74,75 and it is likely that this paucity stems from the fact that β-lactams are
incompatible with standard conditions for copper-catalyzed click chemistry.72 The conditions
defined in this work employing TBTA allow for copper-catalyzed triazole formation and preserve
the β-lactam warhead.
Ent-Amp/Amx Coordinate Fe(III). 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-
Amp causes the solution color to immediately change from colorless to purple-red and a broad
absorption feature in the 400-700 nm range occurs (Figure S1), confirming that both Ent-
Amp/Amx readily chelate Fe(III).
Ent-Amp/Amx Exhibit Enhanced Antibacterial Activity Against Various E. coli
Strains Including Human Pathogens. To ascertain whether Ent-Amp/Amx provide
antibacterial activity against E. coli, including pathogenic strains,76 we preformed antimicrobial
activity assays using six strains (Table S1, Figures 2, S2-S7). E. coli ATCC 25922 is a
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laboratory susceptibility test strain originally obtained as a clinical isolate. E. coli H9049 is a
non-pathogenic clinical isolate.77 E. coli UTI8978 and CFT07379 are both pathogens of the
human urinary tract (UPEC).80 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.76 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.81 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.82,83 Studies with Salmonella indicate that IroN has the ability to transport Ent as well as
its glucosylated forms.84 Of the strains considered in this work, E. coli CFT073 and UTI89
harbor the iroA gene cluster. E. coli H9049 does not produce salmochelins85 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),86 which suggests that its genome does not
encode the iroA cluster. Whether E. coli 35401 produces salmochelins is unclear from the
available literature. 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.87
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A
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C
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+DP
UTI89
+DP
CFT073
+DP
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10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
D
E
F
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
H9049
+DP
35401
+DP
43895
+DP
0.40
0.30
0.20
0.10
0.00
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
Amx
Amp
Ent-Amx
Ent-Amp
Figure 2. Antibacterial activity of Ent-Amp/Amx against various E. coli strains that include
human pathogens. (A) Laboratory test strain E. coli ATCC 25922. (B) Uropathogenic E. coli
UTI89. (C) Uropathogenic E. coli CFT073. (D) Non-pathogenic clinical isolate E. coli H9049. (E)
Pathogenic ETEC E. coli ATCC 35401. (F) Pathogenic EHEC E. coli ATCC 43895. All assays
were performed in 50% MHB media supplemented with 200 µM DP to provide iron-limiting
conditions (mean ± SEM, n ≥ 3). The data for assays performed in the absence of DP are
presented in Figures S2-S7.
We performed antibacterial activity assays using a 10-fold dilution series to compare the
abilities Ent-Amp/Amx and unmodified Amp/Amx to kill E. coli (Figure 2, S2-S7). These assays
were conducted in 50% MHB and in the absence or presence of 200 µM DP. The latter
condition provides iron limitation and results in expression of the Ent uptake machinery
FepABCDG. Amp/Amx exhibit minimum inhibitory concentration (MIC) values of 10 µM against
these E. coli strains and regardless of the presence of DP in the growth media (Figure 2, S2-
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S7). 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 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 S2-S7). A noteworthy characteristic of CFT073 is its multiple
mechanisms for iron acquisition, and we hypothesize that the presence of multiple receptors
that recognize Ent (FepA, IroN, Iha) contributes to this enhanced sensitivity. One or more of
these transporters (e.g., IroN, Iha) may afford Ent-Amp/Amx passage across the outer
membrane, hence more efficient uptake. CFT073 and UTI89 both express IroN, which may
indicate that Iha is responsible for the enhanced susceptibility of CFT073; however, we cannot
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.
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Ent-Amp/Amx Exhibit Enhanced Antimicrobial Activity Against E. coli K-12. 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 µM. 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 µM where Ent-Amp/Amx inhibit E. coli K-12 growth to
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varying degrees and Amp/Amx do not affect bacterial growth. Under conditions of iron limitation,
a 100-fold reduction in MIC value (10 µM 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, 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 β-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.
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FepA is Essential for Ent-Amp/Amx Antimicrobial Activity Against E. coli K-12. 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 knock-out E. coli K-12 strains obtained from
the Keio Collection,88 fepA-, fepC-, and fes- (Figures 3E-G, S8). We selected these mutants to
ascertain how components of the enterobactin transport and processing machinery contribute to
Ent-Amp/Amx antibacterial activity. E. coli fepA- 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. coli fes- lacks the cytoplasmic esterase
Fes responsible for hydrolysis of the Ent macrolactone for iron release. Based on our studies
with wild-type K-12 and other E. coli strains, we hypothesized that the activity of Ent-Amp/Amx
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D
C
A
B
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.16
wild-type
+DP
wild-type
wild-type
-DP
wild-type
0.40
0.30
0.20
0.10
0.00
0.16
0.12
0.08
0.04
0.00
+DP
+DP
0.12
0.08
0.04
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0.00
0
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-10 10-9 10-8 10-7 10-6 10-5
Concentration (M)
0
0
Amx
Amp
Ent-Amx
Ent-Amp
Ent-Amx +Fe(III)
Ent-Amp +Fe(III)
Ent
Ent + Amx
Ent + Amp
Ent-Amx
Ent-Amp
E
G
F
0.25
0.20
0.15
0.10
0.05
0.00
0.08
0.06
0.04
0.02
0.00
fepA-
+DP
fes-
fepC-
+DP
0.20
0.15
0.10
0.05
0.00
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
0
Amx
Amp
Ent-Amx
Ent-Amp
H
I
0.40
0.40
wild-type
-DP
wild-type
+DP
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
**
d
e
d
e
1:1
1:5
1:20
1:1
1:5
1:20
t
t
a
a
re
re
Conjugate : Ent
Conjugate : Ent
t
t
n
n
U
U
Conjugate
Conjugate
Ent-Amx
Ent-Amp
Figure 3. Antibacterial activity of Ent-Amp/Amx against wild-type and mutant E. coli K-12. (A
and 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/Amp and Ent-Amp/Amx. (H-I)
Growth of E. coli K-12 in the presence of 1 µM Ent-Amp/Amx (“conjugate”) and mixtures of Ent-
Amp/Amx (1 µM) 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 media with or
without 200 µM DP (see panels) (mean ± SEM, n ≥ 3). The data for additional assays performed
in the absence of DP is presented in Figure S8.
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would be attenuated for the fepA- mutant. Moreover, we questioned whether loss of FepC or
Fes would modulate the antimicrobial activity.
8
9
Overnight cultures of fepA- and fepC- reached similar OD600 values (~0.15) as wild-type
K-12 when grown in 50% MHB supplemented with 200 µM DP. In contrast, the fes- strain
exhibits 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 µM 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 µM (Table S2), which can be compared to 10 µM of a high-affinity
extracellular iron chelator. Indeed, we previously observed similar growth inhibition of fepA- with
10 µM of an Ent-vancomycin conjugate (extracellular iron chelation) and also 10 µM D-Ent (iron
chelator that cannot be used for iron acquisition) under these growth conditions.66 These data
confirm that FepA is essential for the potent antibacterial activity of Ent-Amp/Amx against E. coli
K-12.
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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 and 3G), comparable to what was
observed for the wild-type strain. The targets of β-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 down-stream 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
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PBPs and 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.
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Ent-Amp/Amx and L-Ent Compete for FepA Recognition. 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-I). When performed
under conditions of iron limitation, the presence of exogenous Ent attenuated the antibacterial
activity of Ent-Amp/Amx. A 1:1 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.
Approximately 75% recovery was observed when ≥5 equiv. of Ent was included in the assay for
both Ent-Amp and Ent-Amx. 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.
Antibacterial Activity of D-Ent-Amp/Amx. L-Serine is a biosynthetic building block for
Ent and a role for chiral recognition in Ent transport has been probed in prior studies.89.90 In one
series of investigations, E. coli FepA was found to bind ferric L-Ent and ferric D-Ent with similar
affinities (Kd = 21 nM and 17 nM, respectively; ascertained by measuring the binding of 59Fe-
loaded siderophores to E. coli BN1071 cells).89 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 analogs was observed to occur with similar efficiency.90
Moreover, the ability of Fes from Shigella flexneri 2a str. 2457T 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 recognition occurs at the level of the esterase was
proposed.90 Taken together, these studies suggest that the ability to transport D-Ent may vary
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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 analogs, we synthesized the D-enantiomers of the Ent-β-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 S9-S13). 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 µM 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, 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 β-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, avoiding concomitant delivery of nutrient Fe(III).
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The Ent-Amp/Amx β-lactam Warhead is Essential for Antimicrobial Activity. With
support for Ent-mediated delivery of Ent-Amp/Amx to the E. coli periplasm, we sought to confirm
the essentiality of the β-lactam warheads in antibacterial action. We therefore designed and
prepared hydrolyzed Ent-Amp/Amx analogs 15 and 16 (Scheme 2) where the β-lactam structure
is destroyed, which we reasoned would attenuate the antimicrobial activity observed for Ent-
Amp/Amx. Hydrolysis of the Amp/Amx-alkynes 7 and 8 was achieved in the presence of 1%
o
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.91
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The diastereomeric mixtures were employed to prepare the hydrolyzed conjugates Ent-Hydro-
6
7
Amp 15 and Ent-Hydro-Amx 16 via copper-catalyzed click chemistry.
O
O
OH
OH
8
9
N
N
N
3
H
N
O
O
S
O
NH
NH
R
10
11
12
13
14
15
16
17
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NH
O
O
NH
R
R
1% TFA
O
Ent-Azide 4
O
O
HN
HN
HN
O
O
O
O
O
H2O / MeCN, 37 o
C
CuSO4, TBTA, NaAsc
DMSO/H2O, rt
O
S
H
O
OH
HN
N
N
H
HO
NH
NH
S
HO
O
CO2H
COOH
COOH
OH
7, R = H
8, R = OH
9, R = H
10, R = OH
15, R = H, Ent–Hydro–Amp
16, R =OH, Ent–Hydro–Amx
Scheme 2. Syntheses of Ent-Hydro-Amp/Amx.
A
B
C
0.20
0.15
0.10
0.05
0.00
0.40
0.30
0.20
0.10
0.00
K-12
+DP
CFT073
+DP
35218
+DP
0.40
0.30
0.20
0.10
0.00
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-10 10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
10-9 10-8 10-7 10-6 10-5 10-4
Concentration (M)
0
Ent-Amx
Ent-Amp
Ent-Hydro-Amx
Ent-Hydro-Amp
Ent-Amx
Ent-Amp
Ent-Amx + PC
Ent-Amp + SB
Figure 4. The β-lactam is required for Ent-Amp/Amx antimicrobial activity. (A and 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 β-lactamase, using Ent-Amp/Amx in the absence and presence of
the β-lactamase inhibitors potassium clavulanate (PC) and sulbactam (SB). All assays were
performed in 50% MHB supplemented with 200 µM DP (mean ± SEM, n ≥ 3). Additional data
are presented in Figure S14.
We employed Ent-Hydro-Amp/Amx in antibacterial activity assays against E. coli K-12
and, as expected, observed negligible growth inhibition (Figure 4A). When E. coli CFT073 was
treated with Ent-Hydro-Amp/Amx in the presence of DP, growth recovery occurred at
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micromolar concentrations (Figure 4B). This result indicates that Ent-Hydro-Amp/Amx are
transported into the cytoplasm of E. coli CFT073 where nutrient iron is released.
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We also performed a series of antibacterial activity assays with E. coli ATCC 35218, a
strain that expresses a class A serine β-lactamase. Similar to unmodified Amp/Amx, Ent-
Amp/Amx were inactive against E. coli ATCC 35218 (MIC > 10 µM) in the absence and
presence of DP (Figures 4C and S14). Slight growth inhibition was observed at 10 µM under
conditions of iron limitation, which may be attributed to iron chelation. The addition of β-
lactamase inhibitors restored the activities Ent-Amp/Amx and Amp/Amx, and the conjugates
exhibited greater antibacterial activity than the parent antibiotics (Figure 4C). In total, the assays
with Ent-Hydro-Amp/Amx and strains expressing β-lactamase demonstrate that an intact β-
lactam is required for the antibacterial activity of Ent-Amp/Amx. Moreover, these studies indicate
that the β-lactams retain their original function and inhibit PBPs when conjugated to Ent.
Time-Kill Kinetics Reveal Rapid Killing of E. coli CFT073 by Ent-Amp/Amx. 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, we also evaluated the time-kill kinetics for E. coli K-12. 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 µM Ent-Amp/Amx, corresponding to a two-fold log reduction in
CFU/mL. In contrast, the change in OD600 and CFU/mL for E. coli CFT073 treated with 50 µM
unmodified Amp/Amx exhibits negligible change over this time period. The time-kill kinetics for
E. coli K-12, conducted with 50 µM of both unmodified and modified β-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
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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.
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K-12
0.16
0.12
0.08
0.04
0.00
0.16
0.12
0.08
0.04
0.00
Amx (50 µM)
Amp (50 µM)
Ent-Amx (50 µM)
Ent-Amp (50 µM)
Untreated
CFT073
Amx (50 µM)
Amp (50 µM)
Ent-Amx (5 µM)
Ent-Amp (5 µM)
Untreated
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Time (h)
Figure 5. 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 (~108 CFU/mL) were treated with 50 µM
of Amp/Amx or 50 µM Ent-Amp/Amx. E. coli CFT073 (~108 CFU/mL) were treated with 50 µM of
Amp/Amx or 5 µM Ent-Amp/Amx. The assays were conducted in 50% MHB medium containing
200 µM DP at 37 oC (mean ± SEM, n = 3).
Ent-Amp/Amx Provides Species-Selective Antimicrobial Activity. 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 bacteria that captures Ent as a xenosiderophore
and expresses two Ent receptors PfeA and PirA.92-94 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
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species.95,96 In contrast to Gram-negative bacteria, where the PBPs are located in the
periplasm, the targets of β-lactam antibiotics are in the extracellular peptidoglycan of Gram-
positive organisms.
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K. pneumoniae ATCC 13883 has a chromosomally-encoded class A β-lactamase (SHV-
1) and lacks sensitivity to Amp/Amx. We observed no effect of 100 µM Amp/Amx K.
pneumoniae growth under our assay conditions (Figure S15). Only ~50% growth inhibition was
observed when K. pneumoniae was treated with high concentrations (10 µM) 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 µM. When β-lactamase inhibitors
where included in the assays, K. pneumoniae exhibited greater sensitivity to Amp/Amx (MIC of
100 µM) and Ent-Amp/Amx (MIC of 10 µM); however, we observed some growth inhibitory
activity of the β-lactamase inhibitor sulbactam alone under these assay conditions (100 µM SB,
Figure S15). Thus, the possibility of a synergistic effect from the inhibitors and conjugates
cannot 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.53 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 β-lactamase expression by K. pneumoniae resulted in inactivation
of the β-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 µM whereas Ent-Amp/Amx provided growth inhibition at 10 µM 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. Varying P. aeruginosa strains
exhibit different phenotypes, and highly variable activity of triscatecholate-β-lactam conjugates
against multiple P. aeruginosa strains has been observed.53 We previously reported that P.
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