Biomimetic enterobactin analogue mediates iron-uptake and cargo transport intoE. coliandP. aeruginosa

Article abstract of DOI:10.1039/d1sc02084f

The design, synthesis and biological evaluation of the artificial enterobactin analogueEnt_KLand several fluorophore-conjugates thereof are described.Ent_KLprovides an attachment point for cargos such as fluorophores or antimicrobial p

Full text of DOI:10.1039/d1sc02084f

Showcasing research from the KlahnLab, Institute of Organic
Chemistry, Technische Universität Braunschweig,
Lower-Saxony, Germany.
As featured in:
Biomimetic enterobactin analogue mediates iron-uptake and
cargo transport into E. coli and P. aeruginosa
The design, synthesis and biological evaluation of the artificial
enterobactin analogue EntKL and several fluorophore-conjugates
thereof are described. EntKL provides an attachment point for
cargos such as fluorophores or antimicrobial payloads. Showing
similar iron binding behaviour compared to the natural product,
EntKL exhibited potent concentration-dependent growth
promoting effects in siderophore biosynthesis deficient mutants
of E. coli and P. aeruginosa and was demonstrated to be able
to deliver fluorescent molecular cargos via the bacterial iron
transport machinery into the bacteria.
See Philipp Klahn et al.,
Chem. Sci., 2021, 12, 10179.
The artwork was created by Ella Maru Studios and the
copyright is held by Dr. Philipp Klahn.
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Biomimetic enterobactin analogue mediates iron-
uptake and cargo transport into E. coli and P.
Cite this: Chem. Sci., 2021, 12, 10179
aeruginosa†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Robert Zscherp,‡a Janetta Coetzee,‡bc Johannes Vornweg,
Jorg Grunenberg,a
¨
a
Jennifer Herrmann,bc Rolf Mullerbc and Philipp Klahn
*
a
¨
The design, synthesis and biological evaluation of the artificial enterobactin analogue EntKL and several
fluorophore-conjugates thereof are described. EntKL provides an attachment point for cargos such as
fluorophores or antimicrobial payloads. Corresponding conjugates are recognized by outer membrane
siderophore receptors of Gram-negative pathogens and retain the natural hydrolyzability of the tris-
lactone backbone. Initial density-functional theory (DFT) calculations of the free energies of solvation
3ꢀ
(DG(sol)) and relaxed Fe–O force constants of the corresponding [Fe-EntKL
]
complexes indicated
a similar iron binding constant compared to natural enterobactin (Ent). The synthesis of EntKL was
achieved via an iterative assembly based on a 3-hydroxylysine building block over 14 steps with an
overall yield of 3%. A series of growth recovery assays under iron-limiting conditions with Escherichia
coli and Pseudomonas aeruginosa mutant strains that are defective in natural siderophore synthesis
revealed a potent concentration-dependent growth promoting effect of EntKL similar to natural Ent.
Additionally, four cargo-conjugates differing in molecular size were able to restore growth of E. coli
indicating an uptake into the cytosol. P. aeruginosa displayed a stronger uptake promiscuity as six
different cargo-conjugates were found to restore growth under iron-limiting conditions. Imaging studies
utilizing BODIPYFL-conjugates, demonstrated the ability of EntKL to overcome the Gram-negative outer
membrane permeability barrier and thus deliver molecular cargos via the bacterial iron transport
machinery of E. coli and P. aeruginosa.
Received 14th April 2021
Accepted 16th June 2021
DOI: 10.1039/d1sc02084f
rsc.li/chemical-science
negative bacteria. In addition, the pathogens recently priori-
tized by the WHO with a critical, high or medium need for the
Introduction
development of novel antimicrobial drugs are primarily Gram-
negative bacteria, including b-lactam-resistant Escherichia coli
and Pseudomonas aeruginosa or uoroquinolone-resistant
Salmonella and Shigella species.8 However, the development of
novel antimicrobial drugs against Gram-negative bacteria is
challenging, not only due to the presence of drug exporters as
also found in Gram-positive bacteria, but because many anti-
bacterial compounds fail to overcome the Gram-negative outer
membrane. As a consequence, many potential drugs displaying
antibacterial activity against Gram-positive bacteria remain
inactive against Gram-negative bacteria, although their biolog-
ical targets are generally present, as they fail to translocate over
the cell envelope barrier.9
The development of bacterial resistance towards antimicrobial
drugs is an intrinsic part of bacterial evolution and this, in turn,
necessitates the continuous development of novel drugs able to
kill these life-threatening multi-drug resistant human patho-
gens.1–3 Facing the current spread of bacterial resistance against
clinically used antibiotics, the development of novel antimi-
crobial drugs and innovative concepts is of vast importance to
counteract this serious threat for public health.1,4,5
Four of the six ESKAPE pathogens,6,7 represent bacterial
species considered as signicant threat for public health due to
a very high occurrence of multi-drug resistance, are Gram-
a
¨
Institute of Organic Chemistry, Technische Universitat Braunschweig, Hagenring 30,
An innovative approach to enable translocation of antimi-
crobial drugs or reporter molecules over the Gram-negative cell
envelope barrier and to develop novel antimicrobial drugs
against these pathogens is the conjugation and hybridization
with siderophores.4,10–12 Siderophores are small molecule iron
chelators,13,14 produced and secreted by bacteria, fungi and
plants to ensure their supply with iron, an essential growth
factor for all living organisms.15 Beyond iron uptake,
D-38106 Braunschweig, Germany. E-mail: p.klahn@tu-bs.de
bDepartment for Microbial Natural Products, Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS), Helmholtz Center for Infection Research and Department
¨
of Pharmacy at Universitat des Saarlandes, Campus Building
E 8.1, D-66123
¨
Saarbrucken, Germany
cGerman Center for Infection Research (DZIF), Site Hannover-Braunschweig, Germany
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1sc02084f
‡ Authors contributed equally.
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siderophores have multiple other biological functions in
bacteria,16–18 e.g. they are important virulence factors contrib-
uting to pathogenesis.17,19 Aer coordination of Fe(III), the
resulting ferri-siderophores are recognized by specic side-
rophore receptors, transmembrane proteins embedded in the
outer membrane, and are actively transported into the cytosol of
bacteria.4,20–23 Siderophore drug conjugates can enter bacterial
cells via the same pathway through siderophore receptor
mediated uptake.1,4,24–32
The general concept of this Trojan Horse approach is derived
from natural antetypes. The sideromycins,33 such as albomy-
cins34–36 or the microcins E492 (ref. 37–40) and H47 (ref. 41) are
utilized by different bacteria to defend their ecological niche
against competing bacteria. These natural siderophore drug
conjugates reveal a remarkable increase in antibacterial activity
compared to the parental free antibiotics. Inspired by this
natural concept researchers have developed articial side-
rophore drug conjugates based on synthetically modied side-
rophore analogues,42–55 leading to active drug accumulation and
tremendously increased antibacterial activity compared to the
parental drugs.42,43,48 Furthermore, siderophore drug conjugates
can expand the activity of Gram-positive-only drugs towards
Gram-negative pathogens.50,51,56,57 Additionally, this strategy
allows for the design of narrow-spectrum antibiotics,10,49,57
selectively targeting specic virulent pathogens, thereby
reducing the selection pressure on the bacterial resistome.
Importantly, the siderophore-b-lactam hybrid cederocol
developed by Shionogi Pharmaceuticals was recently approved
by the FDA for the treatment of pneumonia caused by Gram-
negative bacteria.58
Scheme 1 (A) Structures of enterobactin (Ent) and ferri-enterobactin
([Fe-Ent]3ꢀ); (B) retrosynthetic analysis and design concept of EntKL
.
catechol units might interfere with [Fe-Ent]3ꢀ receptor
recognition.
In this context, the tris-catechol siderophore enterobactin
(Ent, Scheme 1A)14 displaying an extraordinarily high Fe(III)
binding constant (Kd ¼ 10ꢀ49 M)59,60 as well as its glycosylated
derivatives, the salmochelins61 evading lipocalin-2 inactiva-
tion,62,63 are of high interest.
Ent and salmochelins are biosynthesized by Gram-negative
bacteria of the family Enterobacteriaceae, such as E. coli, Kleb-
siella pneumonia, Salmonella typhimurium, Yersinia enterocolitica
and Shigella exneri, to ensure their iron supply.64 However, Ent
has also been found to be produced by Gram-positive Strepto-
myces species65 and it serves as a xenosiderophore for the
Therefore, we envisaged the synthesis of the novel, biomi-
metic enterobactin analogue EntKL (Scheme 1B). Our synthesis
is based on the incorporation of the 3-hydroxy lysine derivative 1
into Ent backbone, aiming to generate an attachment point for
antimicrobial payloads in the backbone out of the recognition
site of the siderophore and simultaneously retain the natural
hydrolyzability of the tris-lactone backbone.52,87
Result and discussion
opportunistic human pathogen P. aeruginosa, where internali- Retrosynthetically, the nal siderophore could be derived from
zation is mediated by the outer membrane siderophore receptor a modied asymmetric tris-lactone backbone by decoration
PfeA.66–69 Ent and salmochelins also play a crucial role during with catechol units via amide coupling (Scheme 1B). As the tin-
infection18,70 and they have been demonstrated to sequester iron template mediated trans-esterication approach utilized earlier,
from human transferrin.17,19 Previously reported articial Ent by Shanzer,88,89 later by Gutierrez90 and Raymond91 for the
analogues used for the development of siderophore drug synthesis of the enterobactin tris-serine lactone backbone,
conjugates or siderophore-based imaging tools71,72 were based leads exclusively to the formation of the thermodynamic,
on modications at one of the catechol units42,43,46,49,52,73–75 or the symmetric product, it is not applicable for the synthesis of the
natural tris-serine lactone backbone was substituted by articial envisaged asymmetric tris-lactone backbone. Therefore, we
moieties to generate an attachment point for payloads in the decided to follow an approach of iterative assembly of a linear
backbone of the siderophore.48,76–78 Although a co-crystal struc- trimer containing the 3-hydroxy lysine derivative 1 with subse-
ture of [Fe-Ent]3ꢀ with its E. coli siderophore receptors of quent macro lactonization as used in the early synthesis of Ent
FepA79,80 and IroN80–82 is not available, recognition of [Fe-Ent]3ꢀ by Corey,92 Rastetter93 and Rogers94 in order to generate the
at other proteins such as FeuA,83–85 VctP,86 PfeA69 or lipocalin-2 desired asymmetric backbone of EntKL
.
(ref. 83) suggests that the attachment of larger payloads at the
In order to estimate if the envisaged substituent at the
asymmetric tris-lactone backbone would negatively impact the
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solution thermodynamics and/or the kinetic stability of the while being thermodynamically stable, as found earlier for [Fe-
Fe(III) complex [Fe-EntKL
3ꢀ, we computed the free energies of Ent]3ꢀ by Raymond and co-workers.101,102
]
solvation DG(sol) as well as the relevant (relaxed) Fe–O force
Thus, expecting no negative impact of an axial backbone
substituent on the Fe(III) complex stability, we developed
3ꢀ
constants for [Fe-Ent]3ꢀ and two stereoisomers of [Fe-EntKL
]
bearing an axial or equatorial substituent (Table 1) following a robust, scalable synthetic route to the required 3-hydroxy
the protocol of Baramov et al.95 (see ESI†).
lysine derivative 1 starting from commercially available 4-ami-
In a rst step, we tried to reproduce the experimentally well- nobutanol (2) as outlined in Scheme 2. A sequence of Boc
known free energy of complexation (DG(sol): ꢀ66.8 kcal molꢀ1 protection, Swern oxidation of the resulting N-Boc-4-
(ref. 59 and 60)) for [Fe-Ent]3ꢀ
.
aminobutanol (3) to aminal 4 and nal Wittig olenation in
Our calculated value of ꢀ64.5 kcal molꢀ1 for DG(sol) [Fe- presence of methyl (triphenyl-phosphoranylidene) acetate (5) at
Ent]3ꢀ is very close to the literature ref. 59 and 60 positively 100 ꢁC led to formation of the E-congured a,b-unsaturated
validating our applied in silico procedure. Interestingly, the methyl ester 6 in excellent diastereoselectivity of E : Z ¼ 100 : 1
articial ferri-enterobactin analogues seem to be even better and 89% yield over 3 steps with only a single chromatographic
iron binders than the unsubstituted [Fe-Ent]3ꢀ by purication step being required. An asymmetric sharpless
several kcal molꢀ1. Furthermore, the value for [Fe-EntKL(eq)]3ꢀ dihydroxylation using AD-mix-a nally gave diol 7 in 83% yield.
was calculated to be energetically more favored as compared to The formation of the corresponding cyclic sulte in the pres-
[Fe-EntKL(ax)]3ꢀ by a value of 3.5 kcal molꢀ1
.
ence of thionyl chloride and triethyl amine at 0 C and subse-
ꢁ
Nevertheless, the absolute values need to be considered with quent a-inversion in the presence of sodium azide afforded a-
caution and we hypothesize that both articial enterobactin azido methyl ester 8 in 64% yield. At this step the absolute
analogues, EntKL(eq) and EntKL(ax), lead to complexes which
are at least of similar thermodynamic stability compared to [Fe-
Ent]3ꢀ and that there is no signicant energetical advantage of
one analogue over the other. While siderophore Fe(III)
complexes are thermodynamically very stable, they are kineti-
cally labile. This is in contrast to, for example, their chromic
counterparts, which are both thermodynamically and kineti-
cally stable. In order to nd out if this kinetic lability holds true
also for our articial ferri-enterobactin analogues, we calculated
the relaxed Fe–O force constants, a method which was recently
successfully applied in several other weakly bound systems.
According to our computational results, all three complexes
seem to be kinetically labile with relaxed Fe–O force constants
well below 1 N cmꢀ1 (see Table 1), indicating very weak bonds
with less or no covalency.96–100 (Note, that the averaged Cr–O
value for [CrEnt]3ꢀ is 1.25 N cm; unpublished results J. G.)
Furthermore, our calculated Fe–O values are very similar. In
summary, all three complexes seem to be kinetically labile,
Scheme 2 Synthesis of 3-hydroxy lysine derivative 1.
Table 1 Calculated DG(sol) and averaged, relaxed force constants of the Fe–O single bonds of [Fe-EntKL
]
3ꢀ, [Fe-EntKL(ax)]3ꢀ and [Fe-
EntKL(eq)]3ꢀ
[Fe-Ent]3ꢀ
[Fe-EntKL(ax)]3ꢀ
[Fe-EntKL(eq)]3ꢀ
DG(sol)a,b [kcal molꢀ1
]
ꢀ64.5
ꢀ269.9
0.95
ꢀ67.2
ꢀ281.6
0.97
ꢀ70.7
ꢀ296.0
0.98
DG(sol)a,b [kJ molꢀ1
]
Fe–O bond force constant (av)a [N cmꢀ1
]
a
Computed for DMSO as solvent. b Separate consideration of all hydronium ions.
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conguration at the C3 position introduced by the sharpless 18 was obtained using Gerlach's modication107 of the Corey–
dihydroxylation was determined to be S-congured with an Nicolaou macro lactonization,108 rst formation of the inter-
enantiomeric excess of 94%ee using Mosher's method.103
mediate thioester using Corey's disulfane and subsequent
ꢁ
Finally, a Staudinger reduction and subsequent Cbz protec- silver-catalyzed cyclization at 23 C in benzene (Entry 3, Table
tion generated the methyl ester 9 in 87% yield, which was 2).
saponied in the presence of trimethyltin hydroxide in 1,2-
However, as the yield of this reaction was hardly reproduc-
dichloro ethane at 65 C using Nicolaou's protocol104 yielding ible, we nally applied 2,20-dipyridyldisulde to form the
the desired 3-hydroxy lysine derivative 1 in 94%. intermediate thioester and furnished macro lactonization in
ꢁ
With 1 in hands, we achieved the assembly of the linear the presence of silver acetate giving 32% yield of the desired tris-
trimer 17 as outlined in Scheme 3. First, the 2-methylan- lactone 18 reliably (Entry 4, Table 2).
thraquinyl ester 11 was formed with 2-bromomethylan-
The nal quantitative, hydrogenolytic Cbz deprotection
thraquinone 10 (MaqBr) in the presence of DBU in 55% yield.93 required 20 atm of hydrogen gas in a mixture of methanol, ethyl
A two times consecutive sequence of Steglich esterication with acetate and TFA. Subsequent acylation under Schotten–Bau-
Cbz-Ser(TBS)-OH 12 and subsequent desilylation in the pres- mann conditions with 2,3-diacetoxybenzoic acid chloride 19 or
ence of a mixture of aqueous, concentrated HF solution and 2,3-dimethoxybenzoic acid chloride 20, respectively, led to the
TBAF led to the Maq-protected linear trimer 16 obtained in 64% formation of (AcO)EntKL and (MeO)EntKL. EntKL could be ob-
yield over 4 steps. Ester 16 was then photolytically cleaved at tained in low yield from (AcO)EntKL by mild saponication in
360 nm in the presence of N-methyl morpholine (NMM) in diluted methanolic ammonia.
a mixture of iso-propanol and chloroform giving the unpro-
However, as it has been reported that acetylated siderophore-
tected linear trimer 17 as precursor for the backbone cyclization prodrugs can be activated in situ and are favorable to prevent
in 65% yield.93
the inactivation of catecholates by enzymatic methyla-
As expected for the formation of a 12-membered ring with tion,48,76,109,110 we employed (AcO)EntKL as siderophore-prodrug
several axial substituents, the cyclization of 17 to the tris-lactone and (MeO)EntKL as starting point for the synthesis of corre-
18 was challenging.
sponding negative control probes, unable to bind iron or
The activation of the carboxylic acid via several reagents mediate uptake in our approach. In order to determine if the
predominantly led to elimination under formation of the cor- synthesized enterobactin derivatives are internalized by E. coli
responding dihydro amino acid (Table 2).
and P. aeruginosa during iron limitation, growth recovery assays
Aer extensive screening of different methods for the macro were done with mutant strains that lack the ability to bio-
lactonization, a rst success with a low yield of 13% was ach- synthesize siderophores, and therefore require external side-
ieved using Shiina's reagent105 (Entry 1, Table 2). Any other rophores to be able to grow under iron-limiting conditions.
screened coupling reagent such as different carbodiimides, Importantly, iron-limiting conditions are typically found in vivo
PyBOP, T3P (propylphosphonic anhydride) or Yamaguchi's at the site of infections being part of the host immune response
reagent106 could not furnish the macro lactonization, favouring to prevent bacterial growth.58,111,112 E. coli K-12 DentA grew to
elimination. The activation of the alcohol through intra- OD600 z 0.35 in 50% MHB II medium (37 ꢁC, t ¼ 24 h), and this
molecular Mitsunobu reaction gave similarly a low yield of 14% value decreased to <0.05 when 200 mM 2,20-bipyridine (DP) was
(Entry 2, Table 2). Finally, the highest yield of 50% for tris-lacton added to the media in order to simulate iron-limiting
Scheme 3 Assembly of a linear trimer, cyclization and final catechol decoration.
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Table 2 Selected conditions for the cyclization of the linear trimer 17
Entry
Conditions
Result
1
2
3
4
MNBA (5.0 eq.), DMAP (8.0 eq.), (CH2Cl2), 0–23 ꢁC, 20 h
13%a
PPh3 (1.5 eq.), DIAD (1.3 eq.), (THF), 0–23 ꢁC, 24 h
14%a
(1) Corey's disulfane92 (1.4 eq.), PPh3 (1.4 eq.), (CH2Cl2), 23 ꢁC, 10 min; (2) AgBF4 (5.0 eq.), (PhH), 25 ꢁC, 16 h
5–50%a,b
(1) 2,20-Dipyridyldisulde (1.4 eq.), PPh3 (1.4 eq.) in (CH2Cl2), 30 ꢁC, 90 min; (2) AgOAc (2.0 eq.), (PhH), 30 ꢁC, 3 h
32%a
a
b
Elimination. Bad reproducibility.
conditions. Low-micromolar concentrations of Ent restored
At higher concentrations of Ent of 10 mM and 15 mM the
growth, and the E. coli cultures reached OD600 z 0.1, 0.15 and growth promoting response was even exceeding the growth in
0.3 in the presence of 1.0 mM, 10 mM and 15 mM Ent, respectively absence of DP, with P. aeruginosa K648 Dpvd/pch growing to
(Fig. 1A).
OD600 z 0.5 and 0.6, respectively. Again, (AcO)EntKL was also
Intriguingly, the addition of (AcO)EntKL led to restore the able to restore the growth of P. aeruginosa K648 Dpvd/pch under
growth of E. coli K-12 DentA in the presence of DP in a concen- iron-limiting conditions in a concentration-dependent manner
tration dependent-manner and with a similar efficiency as the (Fig. 1D and E). While the growth promoting effect of (AcO)
natural siderophore Ent (Fig. 1A and B). A signicant increase in EntKL at a concentration of 1.0 mM was slightly smaller
growth was observed starting at 1 mM concentration, while at 10 compared to Ent, the growth promoting was clearly surpassing
mM and 15 mM the cultures grew to OD600 z 0.15 and 0.3, the effect of Ent at 10 mM. In contrast to the uptake of Ent in E.
respectively, indicating the ability of (AcO)EntKL to be inter- coli, Ent never reaches the cytosol of P. aeruginosa as iron release
nalized into the cytosol of E. coli. Similarly, P. aeruginosa K648 takes place in the periplasm through cleavage of the side-
Dpvd/pch grew to OD600 z 0.4 (37 C, t ¼ 24 h) and this value rophore backbone by the periplasmic esterase PfeE.113 This
ꢁ
decreased to <0.25 in the presence of 600 mM DP. Supplemen- indicates that the compounds are accepted as substrates by
tation of the iron-reduced growth medium with 1.0 mM of Ent PfeE. As expected, the permethylated probe (MeO)EntKL, was
resulted in the restoration of P. aeruginosa growth to OD600
0.35 (Fig. 1D).
z
not able to restore growth of neither E. coli K-12 DentA nor P.
aeruginosa K648 Dpvd/pch (Fig. 1C and F).
Fig. 1 E. coli K-12 DentA and P. aeruginosa K648 Dpvd/pch growth recovery assays employing Ent in comparison to (AcO)EntKL and (MeO)EntKL
(50% MHB II, ꢂ200 or 600 mM DP (2,20-bipyridine), t ¼ 24 h, 37 ꢁC). Gray bars: OD600 of bacteria cultured in the absence of DP. Black bars: OD600
of bacteria cultured in the presence of 200 mM (E. coli) or 600 mM (P. aeruginosa) DP. (A) Ent promotes growth recovery of E. coli. (B) (AcO)EntKL
affords growth recovery of E. coli. (C) (MeO)EntKL shows no growth recovery of E. coli. (D) Ent promotes growth recovery of P. aeruginosa. (E)
(AcO)EntKL affords growth recovery of P. aeruginosa. (F) (MeO)EntKL shows no growth recovery of P. aeruginosa. Each bar indicates the average
of three independent replicates (two wells per replicate) and the error bars are the standard deviation of the mean. As P. aeruginosa shows
a growth of <0.25 OD in the absence of an external siderophore, Y-axis is shown starting at an OD600 of 0.2. Statistical significance is indicated as
a p-value < 0.05and was determined by an unpaired t-test using GraphPad Prism 9 (version 9.1.1). ***(P-value: <0.001), **(P-value: <0.005) and
*(P-value: <0.05).
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Next, we synthesized a series of cargo-conjugates of (AcO) conjugates (AcO)EntKL-PEG4-BODIPY and (AcO)EntKL-PEG4-
EntKL and (MeO)EntKL attaching uorophores and dyes that BODIPYFL as well as their respective negative control probes
differ in their size in order to investigate whether (AcO)EntKL is (MeO)EntKL-PEG4-BODIPY and (MeO)EntKL-PEG4-BODIPYFL in
able to deliver different cargo molecules to E. coli and P. aeru- moderate to good yields (Scheme 4). Finally, we synthesized
ginosa. Therefore, we cleaved the Boc group at the amino handle (AcO)EntKL-PEG3-MG and (MeO)EntKL-PEG3-MG via a sequence
in the presence of TFA in dichloromethane and reacted the of initial Boc cleavage, installation of a PEG3-NHBoc chain via
resulting free amine in the presence of N-hydroxy succinimide NHS ester coupling, nal Boc cleavage and reaction of the free
(NHS) esters of the corresponding uorophores and dyes amine with MG-NHS in moderate yields.
(SulfoCy5-NHS, BODIPYFL-NHS and MG-NHS) obtaining the
When incubating E. coli K-12 DentA under iron-limiting
conjugates (AcO)EntKL-BODIPYFL, (AcO)EntKL-MG and (AcO) conditions with (AcO)EntKL-PEG4-BODIPY, (AcO)EntKL-PEG4-
EntKL-SulfoCy5 as well as their respective negative control BODIPYFL, (AcO)EntKL-BODIPYFL and (AcO)EntKL-SulfoCy5 led
probes (MeO)EntKL-BODIPYFL
, (MeO)EntKL-MG and (MeO) to a concentration-dependent growth recovery (Fig. 2A–D)
EntKL-SulfoCy5 in moderate yields as outlined in Scheme 4. clearly indicating the uptake of these cargo-conjugates to the
Similarly, initial Boc cleavage in the presence of TFA and cytosol of E. coli. The two conjugates (AcO)EntKL-MG and (AcO)
subsequent installation of a PEG4-N3 chain via NHS ester EntKL-PEG3-MG bearing a malachite green derivative as cargo
coupling gave access to the corresponding (AcO)EntKL-PEG4-N3 molecule did not lead to a growth recovery in E. coli (Fig. 2E and
and (MeO)EntKL-PEG4-N3. Final copper(I)-mediated azide– F). Furthermore, high concentrations of 10 mM and 15 mM
alkyne click reaction with alkyne functionalized BODIPYs seemed to reduce overall growth in E. coli K-12 DentA (Fig. 1E
(BODIPY-alkyne and BODIPYFL-alkyne) furnished the and F) as well as in the E. coli wild-type strain (see ESI: Fig. S7
Scheme 4 Synthesis of different EntKL-cargo conjugates bearing different fluorophores and chromophores as payload.
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Fig. 2 E. coli K-12 DentA (A–F) and P. aeruginosa K648 Dpvd/pch (G–L) growth recovery assays employing (AcO)EntKL-PEG4-BODIPY, (AcO)
EntKL-BODIPYFL, (AcO)EntKL-PEG4-BODIPYFL, (AcO)EntKL-SulfoCy5, (AcO)EntKL-MG and (AcO)EntKL-PEG3-MG (50% MHB II, ꢂ 200 or 600 mM
DP (2,20-bipyridine), t ¼ 24 h, 30 ꢁC). Gray bars: OD600 of bacteria cultured in the absence of DP. Black bars: OD600 of bacteria cultured in the
presence of 200 mM (E. coli) or 600 mM (P. aeruginosa) DP. (A) (AcO)EntKL-PEG4-BODIPY promotes growth recovery of E. coli. (B) (AcO)EntKL
-
SulfoCy5 promotes growth recovery of E. coli. (C) (AcO)EntKL-BODIPYFL promotes growth recovery of E. coli. (D) (AcO)EntKL-PEG4-BODIPYFL
promotes growth recovery of E. coli. (E) (AcO)EntKL-MG is not able to restore growth of E. coli. (F) (AcO)EntKL-PEG3-MG is not able to restore
growth of E. coli. As P. aeruginosa shows a growth of <0.25 OD in the absence of an external siderophore, Y-axis is shown starting at an OD600 of
0.2. (G) (AcO)EntKL-PEG4-BODIPY promotes growth recovery of P. aeruginosa. (H) (AcO)EntKL-SulfoCy5 promotes growth recovery of P.
aeruginosa. (I) (AcO)EntKL-BODIPYFL promotes growth recovery of P. aeruginosa. (J) (AcO)EntKL-PEG4-BODIPYFL promotes growth recovery of
P. aeruginosa. (K) (AcO)EntKL-MG promotes growth recovery of P. aeruginosa. (L) (AcO)EntKL-PEG3-MG promotes growth recovery of P. aer-
uginosa. Each bar indicates the average of three independent replicates (two wells per replicate) and the error bars are the standard deviation of
the mean. Fig. S3–S8 (see ESI)† contain the assay results for the corresponding negative control probes (MeO)EntKL-PEG4-BODIPY, (MeO)
EntKL-BODIPYFL, (MeO)EntKL-PEG4-BODIPYFL, (MeO)EntKL-SulfoCy5, (MeO)EntKL-MG and (MeO)EntKL-PEG3-MG, none of which were able to
restore growth in neither E. coli nor or P. aeruginosa. Statistical significance is indicated as a p-value < 0.05 and was determined by an unpaired t-
test using GraphPad Prism 9 (version 9.1.1). ***(P-value: <0.001), **(P-value: <0.005) and *(P-value: <0.05).
˚
and S8†). This might be due to the fact that these compounds approximately 13.7 A (extracted from an energy minimized
are not taken up by the bacteria and therefore, they further structure by Chem3D® Ultra 15.1.0.144), size seems not to be
reduce the available iron in the surrounding medium. the exclusive parameter for uptake. In comparison, (AcO)EntKL
-
Although, the malachite green derivative attached within (AcO) SulfoCy5, bearing the rigid SulfoCy5 uorophore with a length
˚
EntKL-MG and (AcO)EntKL-PEG3-MG has a large diameter of diameter of approximately 19.9 A (extracted from an energy
© 2021 The Author(s). Published by the Royal Society of Chemistry
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minimized structure by Chem3D® Ultra 15.1.0.144) of its
indocyanine-backbone, led to a growth recovery under same
conditions. Looking at the crystal structure of the enterobactin
specic siderophore receptor FepA,79 the transmembrane pore
opening upon recognition of Ent seems to have an elliptical
˚
inner diameter between 20-30 A, large enough to harbor any of
the reported conjugated discussed here. Therefore, additional
parameters beyond size seem to determine the uptake of
a bound cargo at the outer membrane receptor.
However, size exclusion might still play a role at the ABC-type
transporter mediating uptake of hydrolyzed fragments across
the inner membrane.
When incubating the conjugates with P. aeruginosa K648
Dpvd/pch under iron-limiting media conditions all cargo
conjugates led to clear concentration-dependent growth
recovery, including (AcO)EntKL-MG and (AcO)EntKL-PEG3-MG,
indicating the uptake of these cargo-conjugates to the peri-
plasm of P. aeruginosa (Fig. 2G–L). These results are in line with
the ndings of Nolan and co-workers42 reporting that P. aeru-
ginosa exhibits greater promiscuity for the uptake compared to
E. coli. Looking at the crystal structure of PfeA,69 the outer
membrane siderophore receptor of P. aeruginosa, a similar
˚
elliptical inner diameter between 25–35 A can be assumed.
Consistently, none of the corresponding permethylated nega-
tive control probes led to a growth recovery neither in E. coli nor
P. aeruginosa (see ESI, Fig. S3–S8†). The overall growth recovery
response to the added compounds was signicantly higher for
P. aeruginosa K648 Dpvd/pch compared to E. coli K-12 DentA,
under non-iron-limiting conditions (Fig. 3B). The comparison
of the growth recovery over all tested compounds in E. coli
revealed a similar effciency of all conjugates compared to the
natural siderophore Ent (Fig. 3A).
A similar picture was observed for the growth recovery in P.
aeruginosa. However, for some of the compounds such as (AcO)
EntKL and (AcO)EntKL-PEG4-BODIPY an even improved perfor-
mance compared to Ent was observed (Fig. 3B). Furthermore,
we were able to demonstrate that growth of P. aeruginosa
mutants could be restored with (AcO)EntKL in the presence of
comparably high concentrations (0.01 mM, 0.1 mM, 1.0 mM and
10 mM) of the human iron scavenger protein apo-transferrin (Kd
¼ 10ꢀ22 M)114 (see ESI, Fig. S9†).
Fig. 3 Comparison of E. coli K-12 DentA (A) and P. aeruginosa K648
Dpvd/pch (B) growth recovery assays employing 10 mM concentration
of (AcO)EntKL-PEG4-BODIPY, (AcO)EntKL-BODIPYFL
, (AcO)EntKL-
PEG4-BODIPYFL (AcO)EntKL-SulfoCy5, (AcO)EntKL-MG and (AcO)
,
EntKL-PEG3-MG under iron-limiting and non-limiting conditions (50%
MHB II, ꢂ200 or 600 mM DP (2,20-bipyridine), t ¼ 24 h, 37 ꢁC). Gray
bars: OD600 of bacteria cultured in the absence of DP. Black bars:
OD600 of bacteria cultured in the presence of 200 mM (E. coli) or 600
mM (P. aeruginosa) DP. Each bar indicates the average of three inde-
pendent replicates (two wells per replicate) and the error bars are the
standard deviation of the mean. As P. aeruginosa shows a growth of
<0.25 OD in the absence of an external siderophore, Y-axis is shown
starting at an OD600 of 0.2. Statistical significance is indicated as a p-
value < 0.05and was determined by an unpaired t-test using GraphPad
Prism 9 (version 9.1.1). ***(P-value: <0.001), **(P-value: <0.005) and
*(P-value: <0.05).
the bacteria, providing additional evidence for the uptake into
the bacteria.115
Further, an additional growth recovery assay was conducted
with (AcO)EntKL in the presence of high concentrations (1.0 mM,
10.0 mM, 50 mM and 100 mM) bovine serum albumin (see ESI,
Fig. S9†). It is worth to mention, that growth recovery was
achieved with (AcO)EntKL. Albumin is responsible for the
transport of lipophilic compounds and it is therefore able to
bind Ent115 These results indicate the potential ability of our
designed enterobactin derivative (AcO)EntKL to compete with
human iron-binding and lipophilic transport serum proteins.
To our delight, all assessed bacteria were uorescently
labelled when treated with either (AcO)EntKL-PEG4-BODIPYFL or
(AcO)EntKL-BODIPYFL at concentrations of 10 mM (Fig. 4A, B, D
and E, see ESI, Fig. S11, S13, S16 and S18†), further conrming
the uptake into the wild-type and mutant strains of E. coli and P.
aeruginosa. Weaker uorescence was observed at 1.0 mM
concentration of the uorophore conjugates on all tested
strains (see ESI, Fig. S11, S13, S16, S18 and Table S3†). In
general, the uorescence uptake into both mutant strains was
higher compared to the wild types, and E. coli showed a signif-
icantly higher uptake than P. aeruginosa (see ESI, Table S3†).
Furthermore, we observed slight differences in the labeling
performance of (AcO)EntKL-BODIPYFL and (AcO)EntKL-PEG4-
Next, we incubated (AcO)EntKL-BODIPYFL
, (MeO)EntKL-
BODIPYFL, (AcO)EntKL-PEG4-BODIPYFL and (MeO)EntKL-PEG4-
BODIPYFL at different concentrations with E. coli K-12 DentA, P.
aeruginosa K648 Dpvd/pch and their corresponding wild-type
strains under iron-limiting conditions in order to proof
whether uptake of the probes leads to uorescence labelling of
BODIPYFL for the different bacteria. While (AcO)EntKL
-
BODIPYFL led to more prominent labelling of E. coli BW25113
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ESI, Fig. S12, S14, S15, S17, S19 and S20†). Fluorescence labeling
of E. coli K-12 DentA and P. aeruginosa K468 Dpvd/pch was also
observed when treating the bacteria with mixtures of (AcO)
EntKL-BODIPYFL : Ent/1 : 10, 1 : 1, 10 : 1 or 10 : 10 (Fig. S21 and
S22†).
Interestingly, the uorescence labelling for E. coli and P.
aeruginosa mutant strains seems to be increased in the presence
of Ent, as lower concentrations of 1.0 mM of the uorophore
conjugates led to strong uorescence signals of similar intensity
(Fig. 4C and F, see also ESI, Table S3†) compared to the 10-fold
higher concentration in absence of Ent (Fig. 4B and E, see also
ESI, Table S3†). We assume that the overall tness of the
bacteria is increased in the presence of additional siderophore,
leading to a more efficient uptake of the uorophore conju-
gates. These results underline the potential of EntKL-based
reporter conjugates to serve as molecular tools for infection
detection.
Finally, (AcO)EntKL, (MeO)EntKL and all derived cargo-
conjugates were investigated for their antibacterial activity
against the E. coli and P. aeruginosa strains and the compounds'
cytotoxic activity against human HepG2 cells was assessed (see
ESI, Table S4†). All tested compounds lack antibacterial and
cytotoxic activity, which is important looking at (AcO)EntKL in
the context of being potentially used as safe carrier molecule for
the future development of antimicrobial siderophore drug
conjugates that help to prevent fast resistance development. In
addition, all compounds lack any cytotoxic activity against
human HepG2 cells, enabling their possible application in
mammalian cells. Notably, although the close derivative of
Miller's articial enterobactin analogue47 (AcO)EntM displayed
similar growth recovery efficacy in siderophore biosynthesis
decient mutants of E. coli and P. aeruginosa compared to (AcO)
EntKL (see ESI, Fig. S10†), we found signicant cytotoxic activity
against human HepG2 cells with an IC50 value of 12.17 mM in
our assay (see ESI, Table S4†).
Conclusions
In summary, we designed and synthesized the novel enter-
obactin derivative (AcO)EntKL
, which retains the natural
Fig. 4 Fluorescence microscopy of E. coli BW25113, E. coli K-12
DentA, P. aeruginosa PA01 and P. aeruginosa K468 Dpch/pvd culti-
vated under iron-limiting conditions (50% MHB II, +200 or 600 mM DP
hydrolyzability of the tris-lactone scaffold, while providing an
amino handle for easy attachment of cargos in the backbone. In
silico studies at the DFT level of theory predicted a high ther-
modynamic stability combined with kinetic lability for the
iron(III) complexes similarly as demonstrated earlier for the
(2,20-bipyridine), t ¼ 24 h, 37 ꢁC) and treated with 10 mM of (AcO)EntKL
-
BODIPYFL (A and B), 10 mM of (AcO)EntKL-PEG4-BODIPYFL (D and E) or
a mixture (AcO)EntKL-BODIPYFL (1 mM) : Ent (10 mM) (C and F). All
images were captured using a 40ꢃ/1.30 objective (overall magnifica-
tion of 400ꢃ). BF: bright field, F: fluorescence GFP filter set (excitation:
480 nm, 20 nm bandwidth; emission: 527 nm, 15 nm bandwidth). For
corrected total cell fluorescence (CTCF) of each experiment see Table
S3 in the ESI.†
natural ferri-siderophore [Fe-Ent]3ꢀ
.
Growth recovery experiments with siderophore biosynthesis
decient mutants of E. coli and P. aeruginosa under iron-
limiting conditions revealed an uptake of (AcO)EntKL and
other EntKL-based derivatives into the tested bacteria, while, in
contrast, permethylated probes based on (MeO)EntKL unable to
bind iron, did not lead to any growth recovery.
Furthermore, imaging experiments under iron-limiting
conditions utilizing E. coli and P. aeruginosa decient mutants
and wild-type strains showed labelling of the bacteria, further
underlining the uptake of cargo conjugates.
and E. coli K-12 DentA, (AcO)EntKL-PEG4-BODIPYFL preferred of
P. aeruginosa PA01 and P. aeruginosa K468 Dpvd/pch (see ESI,
Table S3†). Consistently, no labelling was observed when cells
were treated with either (MeO)EntKL-BODIPYFL, (MeO)EntKL
-
PEG4-BODIPYFL or non-conjugated BODIPYFL-alkyne alone (see
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As uorophore uptake into both, E. coli K-12 DentA and P.
aeruginosa K648 Dpvd/pch, was increased in the presence of
supplemented Ent, we assume that overexpression of Ent would
not contribute to any resistance against EntKL-based drug
conjugates.
Since growth recovery was also observed in the presence of
high concentrations of human apo-transferrin or albumin and
no antimicrobial activity or cytotoxicity was observed, (AcO)
EntKL holds potential to serve as good starting point for the
assembly of antimicrobial siderophore drug conjugates to
tackle infections caused by Gram-negative bacterial pathogens
in humans.
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