Facile and Versatile Chemoenzymatic Synthesis of Enterobactin Analogues and Applications in Bacterial Detection

Article abstract of DOI:10.1002/anie.201603921

Siderophores, such as enterobactin (Ent), are small molecules that can be selectively imported into bacteria along with iron by cognate transporters. Siderophore conjugates are thus a promising strategy for delivering functional reagents into bacteria. In this work, we present an easy-to-perform, one-pot chemoenzymatic synthesis of functionalized monoglucosylated enterobactin (MGE). When functionalized MGE is conjugated to a rhodamine fluorophore, which affords RhB-Glc-Ent, it can selectively label Gram-negative bacteria that utilize Ent, including some E. coli strains and P. aeruginosa. V. cholerae, a bacterium that utilizes linearized Ent, can also be weakly targeted. Moreover, the targeting is effective under iron-limiting but not iron-rich conditions. Our results suggest that the RhB-Glc-Ent probe is sensitive not only to the bacterial strain but also to the iron condition in the environment.

Full text of DOI:10.1002/anie.201603921

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International Edition: DOI: 10.1002/anie.201603921
German Edition: DOI: 10.1002/ange.201603921
Siderophore Conjugates
Facile and Versatile Chemoenzymatic Synthesis of Enterobactin
Analogues and Applications in Bacterial Detection
Albert A. Lee, Yi-Chen S. Chen, Elisa Ekalestari, Sheng-Yang Ho, Nai-Shu Hsu, Tang-Feng Kuo,
and Tsung-Shing Andrew Wang*
Abstract: Siderophores, such as enterobactin (Ent), are small
molecules that can be selectively imported into bacteria along
with iron by cognate transporters. Siderophore conjugates are
thus a promising strategy for delivering functional reagents into
bacteria. In this work, we present an easy-to-perform, one-pot
chemoenzymatic synthesis of functionalized monoglucosy-
lated enterobactin (MGE). When functionalized MGE is
conjugated to a rhodamine fluorophore, which affords RhB-
Glc-Ent, it can selectively label Gram-negative bacteria that
utilize Ent, including some E. coli strains and P. aeruginosa. V.
cholerae, a bacterium that utilizes linearized Ent, can also be
weakly targeted. Moreover, the targeting is effective under iron-
limiting but not iron-rich conditions. Our results suggest that
the RhB-Glc-Ent probe is sensitive not only to the bacterial
strain but also to the iron condition in the environment.
Enterobactin (Ent) is a catecholate siderophore secreted
by Gram-negative enteric bacteria. It is composed of an l-
[
9]
serine trilactone linked to three 2,3-dihydroxybenzoic acid
[2b,10]
(DHBA) moieties by amide bonds (Figure 1a).
The six
hydroxyl groups on DHBA can form a hexadentate ligand for
[
11]
binding iron(III).
The Ent–iron(III) complex will be
[12]
internalized by an outer membrane receptor, FepA, and
transported to the cytosol by FepBCDG.
[
4,13]
Interestingly,
Ent can be glucosylated by IroB at the C5 position of DHBA,
producing monoglucosylated Ent (MGE) and diglucosylated
[14]
Ent (DGE), also known as Salmochelin S4 (Figure 1a). The
[
15]
glucosylation does not compromise the iron binding. The
glucose (Glc) moiety provides additional water solubility
[
14a]
compared to Ent.
In K. pneumoniae, microcin E492 (Mcc
E492, Figure 1b) uses its peptide moiety as an active toxin
and Ent as a targeting probe against FepA-expressing
[
16]
S
iderophores are secondary metabolites, which are secreted
by microorganisms, with high chelation affinity for iron(III).
strains. Strikingly, the large peptide cargo can be carried
through the outer membrane by the Ent-FepA uptake
[1]
[16a,17]
These molecules acquire iron(III) from extracellular space
system.
This implies that the MccE-492-like modifica-
under iron-limiting conditions, and are critical to microbe
survival. Siderophores differ between microbe species and
tions do not compromise the internalization of Ent. We
therefore hypothesized that using the same modification
strategy on Ent, we should be able to transport other
conjugated cargos into the bacteria.
[2]
[
1c,3]
exhibit highly variable chemical structures.
bound siderophores are recognized and transported into the
The iron-
[
4]
cells by dedicated receptors. Only the microbes expressing
Guided by MccE492, we aimed to devise a strategy for
synthesizing MGE analogues with different functional groups
on the C6 position of Glc for conjugation. The current method
for obtaining MGE requires a nine-step synthesis of the
glucosylated DHBA, and there is no evidence regarding
[5]
the cognate receptor can take up the siderophore. Owing to
the selective and essential nature of siderophore-based iron
acquisition, siderophores can potentially serve as a probe to
target microbes.
[
18]
Natural siderophore mimics, or sideromycins, selectively
whether the Glc-functionalized MGE can be made. More-
over, the synthesis of nonglucosylated monofunctionalized
[
6]
deliver toxic cargos into bacteria by conjugating toxins.
[7b]
These mimics demonstrate the feasibility of the sidero-
phore-based targeting strategy and have inspired the devel-
opment of many synthetic conjugates, including antibiotic
Ent can be non-trivial.
The complexity stems from the
requirement of synthesizing the modified DHBA and the
stochastic nature of linking two types of DHBA onto the
trilactone. Herein, to circumvent the arduous synthesis, we
developed a facile and versatile chemoenzymatic synthesis of
Ent analogues with conjugatable groups, and then we
demonstrated the feasibility of using Ent conjugates to
target bacteria with selectivity.
[
7]
conjugates for Trojan-horse-type delivery, and surface-
conjugated siderophores for bacterial adsorption and detec-
[
8]
tion. All of these applications require a handle on the
siderophore for site-specific modification.
[
*] A. A. Lee, Y.-C. S. Chen, S.-Y. Ho, N.-S. Hsu, T.-F. Kuo,
Dr. T.-S. A. Wang
Department of Chemistry, National Taiwan University
No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 (Taiwan) (R.O.C.)
E-mail: tswang@ntu.edu.tw
We attempted to use the IroB enzyme to introduce Glc
analogues to achieve functionalized microcin-like MGE
(
(
Figure 1c). IroB is a glycosyltransferase that uses UDP-Glc
[14b,19]
2a) to glucosylate Ent.
To introduce Glc analogues,
UDP-Glc analogues (2a–d) are needed as sugar donors.
Although practical methods have been reported, it is not
trivial to prepare UDP-sugars in large quantities. It has been
shown that certain UDP-sugars can be made from 2-chloro-4-
nitrophenyl glycosides (CNP-sugars) by reversed glycosyla-
E. Ekalestari
Department of Chemistry & Biochemistry
University of California, Los Angeles
Los Angeles, CA 90095 (USA)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201603921.
[
20]
tion using OleD mutants.
We synthesized several CNP-
[
20]
sugars (1a–d) for testing (Figure 1c). To our surprise, wild-
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Figure 1. Structures and synthesis of Ent analogues. a) IroB enzymatically modifies Ent at the C5 position of DHBA, producing MGE, DGE, or
TGE. b) A truncated chemical structure of microcin E492. c) Left: Schematic of combined OleD and IroB enzymatic reactions starting from CNP-
sugars to produce functionalized MGEs. Right: Designation of the compounds used in the reactions.
type OleD, cloned from S. antibioticus (ATCC11891), can
readily catalyze the formation of 2a–d from corresponding
the CNP-Glc analogues (1a–d) in good yields, as judged by
the yellow color of the released nitrophenate. HPLC and ESI-
MS analysis of the reaction mixtures clearly showed the
disappearance of UDP and emergence of UDP-Glc ana-
logues, confirming the validity of the reaction (Supporting
Information, Figure S1).
a one-pot double-enzymatic conversion started with incubat-
ing 1a–d, UDP, and OleD. Subsequently, without purifying
the UDP-sugars, Ent and IroB were added directly to the
mixtures. LC/MS analysis confirmed the formation of glyco-
sylated Ent for all the CNP-Glc analogues (Figure 2 and the
Supporting Information, Figure S2). In our test condition
(Ent:CNP-sugar is 1:6), 1a produces DGE ((Glc) Ent) and
2
triglucosylated Ent (TGE or (Glc) -Ent), consistent with
3
[
14b,21]
We then sought to combine the OleD reaction with the
IroB glucosylation in one pot to afford functionalized MGE,
bypassing the need to purify the UDP-sugars (Figure 1c).
Prior efforts have shown the feasibility of coupling a reverse
glycosylation, which generates UDP-Glc in situ, to a second
previous reports.
1b produces mainly (6azGlc) Ent and
2
some (6azGlc) Ent, while 1c and 1d produce mainly
3
(6BrGlc) Ent (3c) and (6HS-Glc) Ent (3d), respectively
1
1
(Supporting Information, Figure S2). The number of gluco-
sylations can be tailored by adjusting the ratio of Ent and
CNP-sugars, providing a convenient method to obtain tri, di,
[
20]
glycosylation. After optimization, our working protocol for
Figure 2. LC/MS analysis of the combined OleD-IroB enzymatic reaction of CNP-6azGlc and Ent. Chromatograms and mass spectra of major Ent
species are shown. The ratio of Ent and CNP-sugars used is shown in red at the top-left corner. a) For the test ratio 1:6, the major products are
(
6azGlc) Ent and (6azGlc) Ent. b) For the optimal ratio 1:3, the major product is (6azGlc) Ent.
3
2
1
2
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or monoglucosylated products (Figure 2). Using HPLC, we
Fluorescently labeled Ent that targets bacteria can be
are able to purify the products (Supporting Information,
Figure S3). Currently, only small quantities (a few milligrams)
can be obtained by a single reaction. Although the scale was
sufficient for preliminary tests, practical larger-scale produc-
tion would demand further improvements in reaction con-
ditions and separation. Toward this end, we have demon-
strated a facile double enzymatic reaction to obtain a family
useful for detecting bacteria. To confirm the capacity of Ent
conjugates to target bacteria, we treated E. coli CFT073 with
RhB-Glc-Ent and Dan-Glc-Ent, and examined the treated
bacteria by fluorescent microscopy. CFT073 was incubated in
M63 minimal medium to provide an iron-limiting environ-
ment. Whereas the RhB-Glc-Ent and Dan-Glc-Ent treated
groups were mostly fluorescently labeled, the 26- and dansyl
disulfide 27-treated control groups showed no fluorescent
bacteria (Figure 3c and Supporting Information, Figure S6).
Some bacteria were only weakly labeled by our probes, which
could result from the heterogeneity of protein expression in
of functionalized MGEs, such as (6azGlc) Ent (3b), 3c, and
1
3
d (Figure 1c). These versatile functional groups can provide
synthetic flexibility in further applications.
We next evaluated the conjugation reactions with the
functionalized MGEs (3b–d). Azido-functionalized 3b was
reacted with rhodamine B-alkyne (RhB-alkyne, 26) using
a convenient copper-catalyzed azide–alkyne cycloaddition
[
22]
the population. Other possibilities for weak or no labeling
cannot be ruled out at this time and further characterization is
required. Nevertheless, heterogeneous labeling was fre-
quently observed with other probes, including siderophore-
(
CuAAC) reaction and purified by HPLC to afford RhB-Glc-
[
23]
Ent (Figure 3a). Thiol-functionalized 3d can be linked to
a dansyl group by a disulfide bond exchange using 2-
mercaptopyridine activated reactant 29 (Dansyl-PDS) to
afford Dan-Glc-Ent (Figure 3b and the Supporting Informa-
tion, Figure S5 and Scheme S10). However, bromo-function-
alized 3c did not react with dansylthiol (28) in any condition
tested, and not even with 2-mercaptoethanol, owing probably
to the hindered environment around C6-Br, on which the
substitution takes place.
based probes. We further confirmed the labeling by flow
cytometry. When 10 mm RhB-Glc-Ent was added, a significant
3-fold increase in fluorescence intensity was observed after
1 h, and it continued to increase to 6-fold over 7 h. In contrast,
when 10 mm 26 was treated as a control, the increase was less
than 2-fold even over an extended time period (Supporting
Information, Figures S7 and S8). Our combined results clearly
indicate that the Ent portion contributed to the higher
fluorescence increase, owing to the Ent uptake machinery.
We expanded our experiment to other strains to examine
the selectivity, including E. coli CFT073, K12, O157:H7; P.
aeruginosa; V. cholerae; S. aureus; and B. subtilis (Figure 4).
The E. coli strains and P. aeruginosa were previously reported
[
7b,24]
to use Ent,
whereas V. cholerae uses linearized Ent but
[25]
not cyclic Ent. The Gram-positive S. aureus and B. subtilis
use other siderophores and were not reported to take up
[26]
glucosylated Ent. In the minimal medium, the two Gram-
positive bacteria did not take up RhB-Glc-Ent. All three E.
coli strains and P. aeruginosa showed an over 5-fold fluores-
cence increase when treated with RhB-Glc-Ent (Figure 4a
and the Supporting Information, Figure S9). However,
although V. cholerae was reported not to take up cyclic Ent,
RhB-Glc-Ent treatment resulted in a weaker increase. One
possibility is the low usage of cyclic Ent by V. cholerae, as
[
27]
observed in the literature. To further explore whether the
probe targeting is iron dependent, we cultured all bacteria in
an iron-rich medium. The bacteria tested showed no signifi-
cant fluorescence increase (Figure 4b and the Supporting
Information, Figure S10). The combined results demon-
strated that the uptake of RhB-Glc-Ent is dependent on
culture conditions in E. coli, P. aeruginosa, and V. cholerae. To
rule out the possibility of other factors in LB rich medium, we
cultured CFT073 in M63 minimal medium supplemented with
1
00 mm FeCl (Figure 4b and the Supporting Information,
3
Figure S11). Compared to the M63 minimal medium, CFT073
showed no significant fluorescence increase in the iron-rich
environment of M63 + FeCl . This again supports the
3
conclusion that RhB-Glc-Ent targeting is indeed sensitive to
iron conditions.
In conclusion, we have provided a convenient chemo-
enzymatic synthesis of functionalized MGE that would
facilitate the application of siderophore conjugates. Although
many siderophore conjugates have been developed, the
Figure 3. Synthesis of a) RhB-Glc-Ent through CuAAC and b) Dan-Glc-
Ent through disulfide bond exchange. c) Microscopy images of E. coli
CFT073 treated with RhB-Glc-Ent (top) and Dan-Glc-Ent (bottom).
Left: bright field (BF), middle: fluorescence channel, right: merge.
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conjugates to only iron-limiting conditions, it does provide
selectivity towards bacteria in iron-limiting environments. In
mammalian hosts, free iron is extremely scarce and bacteria
[
1a]
would rely on a siderophore pathway to obtain iron. This
makes the bacteria in hosts a great target for Ent conjugates,
while rendering bacteria in non-host, iron-rich environments
free of targeting.
The current limitation of our method is the reaction scale.
An improvement in making Ent conjugates is required for
practical use. Another limitation is the dependency of our
targeting probes on the activity of the iron-acquisition
pathway, which could lead to heterogeneity in targeting
efficiency. While this makes it less potent as a universal
method, relying on siderophore pathway enables the method
to perform unique applications, such as iron-limited specific
labeling during host infection and cargo delivery into bacteria
inside hosts. We believe that these applications should not be
over-shadowed by the limitations.
Acknowledgements
We thank the Ministry of Science and Technology, Taiwan,
(MOST 104-2119-M-002-021- and NSC 103-2113-M-002-001-
MY2) and Center for Emerging Material and Advanced
Devices, National Taiwan University (NTU) (NTU-ERP-
1
05R8956-4) for financial support. We also thank Dr. Ching-
Hao Teng at National Cheng Kung University for providing
E. coli CFT073, and Dr. Ya-Wen Liu at School of Medicine,
NTU for providing the fluorescent microscope. Finally, we
thank the Proteomics MS Core Facility and Chemical Biology
Core Facility at Department of Chemistry, NTU, for instru-
mental support.
Figure 4. Bacterial labeling with 10 mm RhB-Glc-Ent. Bars represent
average of the medium of fluorescence intensity of bacteria measured
by flow cytometry in triplicate. (+): treated with RhB-Glc-Ent, (À):
bacteria alone. a) Bacteria cultured in minimal medium. b) Bacteria in
LB. Inset: E. coli CFT073 cultured in M63 minimal medium supple-
mented with 100 mm FeCl . (statistical analysis: *** as p<0.001, n.s.
as not significant, n=3 for all tests).
3
Keywords: bacteria detection · chemoenzymatic synthesis ·
click chemistry · enterobactin · siderophores
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Received: April 22, 2016
Revised: July 7, 2016
Published online: && &&, &&&&
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Communications
Siderophore Conjugates
A. A. Lee, Y.-C. S. Chen, E. Ekalestari,
S.-Y. Ho, N.-S. Hsu, T.-F. Kuo,
T.-S. A. Wang*
&&&& — &&&&
Facile and Versatile Chemoenzymatic
Synthesis of Enterobactin Analogues and
Applications in Bacterial Detection
Siderophore-based bacteria labeling: A
one-pot chemoenzymatic synthesis
approach that is capable of functionaliz-
ing enterobactin with different reactive
groups is developed. The functionalized
enterobactin can be further conjugated
with fluorophores to perform specific
detection of bacteria. This strategy can
serve as a convenient way to deliver
cargos into bacteria with selectivity.
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
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