Metabolic inhibitors of bacterial glycan biosynthesis

Article abstract of DOI:10.1039/c9sc05955e

The bacterial cell wall is a quintessential drug target due to its critical role in colonization of the host, pathogen survival, and immune evasion. The dense cell wall glycocalyx contains distinctive monosaccharides that are absent from human cells, and

Full text of DOI:10.1039/c9sc05955e

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Pradhan, A. Paul, I. R. Olin, O. T. Tuck, K. D. Moulton, S. S. Kulkarni and D. H. Dube, Chem. Sci., 2020,
DOI: 10.1039/C9SC05955E.
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DOI: 10.1039/C9SC05955E
ARTICLE
Metabolic inhibitors of bacterial glycan biosynthesis
a
b
Daniel A. Williams,† Kabita Pradhan,† Ankita Paul,b Ilana R. Olin,a Owen T. Tuck,a Karen D.
Moulton,a Suvarn S. Kulkarni,*b and Danielle H. Dube*a
Received 00th January 20xx,
Accepted 00th January 20xx
The bacterial cell wall is a quintessential drug target due to its critical role in colonization of the host, pathogen survival, and
immune evasion. The dense cell wall glycocalyx contains distinctive monosaccharides that are absent from human cells, and
proper assembly of monosaccharides into higher-order glycans is critical for bacterial fitness and pathogenesis. However,
the systematic study and inhibition of bacterial glycosylation enzymes remains challenging. Bacteria produce glycans
containing rare deoxy amino sugars refractory to traditional glycan analysis,1, 2 complicating the study of bacterial glycans
and the creation of glycosylation inhibitors. To ease the study of bacterial glycan function in the absence of detailed
structural or enzyme information, we crafted metabolic inhibitors based on rare bacterial monosaccharide scaffolds.
Metabolic inhibitors were assessed for their ability to interfere with glycan biosynthesis and fitness in pathogenic and
symbiotic bacterial species. Three metabolic inhibitors led to dramatic structural and functional defects in Helicobacter
pylori. Strikingly, these inhibitors acted in a bacteria-selective manner. These metabolic inhibitors will provide a platform for
systematic study of bacterial glycosylation enzymes not currently possible with existing tools. Moreover, their selectivity will
provide a pathway for the development of novel, narrow-spectrum antibiotics to treat infectious disease. Our inhibition
approach is general and will expedite the identification of bacterial glycan biosynthesis inhibitors in a range of systems,
expanding the glycochemistry toolkit.
DOI: 10.1039/x0xx00000x
3
colonization and pathogenesis.1,
Indeed, an analysis of
Introduction
bacterial glycan structures reveals the presence of rare,
exclusively bacterial deoxy amino monosaccharides that are
absent from eukaryotes.8 For example, pseudaminic acid,9
The main therapies targeting bacterial glycans have had
remarkable success in the clinic. Indeed, our antibiotic arsenal
contains small molecule inhibitors of enzymes involved in
bacterial glycoconjugate biosynthesis.3 Among the most
prominent of these are the blockbusters penicillin,4
vancomycin,5 and bacitracin,6 all of which interfere with
peptidoglycan biosynthesis. Although these antibiotics have
saved countless lives, the emergence of World Health
Organization-listed antibiotic resistant “priority pathogens,”
including Helicobacter pylori and Campylobacter jejuni, has
bacillosamine,10
2,4-diacetamido-2,4,6-trideoxygalactose
(DATDG),11 N-acetylfucosamine (FucNAc),12 and legionaminic
acid9 are a sampling of the unusual building blocks used by
medically significant priority pathogens (Fig. 1A). Pathogenic
bacteria that cannot synthesize glycoproteins have attenuated
virulence. For example, Pseudomonas aeruginosa and
Haemophilis influenzae glycosylation mutants have reduced
14
adhesion to host cells,13,
Staphylococcus aureus and H.
influenzae glycosylation mutants are defective at forming
biofilms,15 and C. jejuni, H. pylori, and P. aeruginosa mutants
with interruptions in flagellin glycosylation are immotile and
cannot colonize the host.9, 16, 17 Small molecules that inhibit
glycosylation enzymes and recapitulate the effects of genetics
experiments have the potential to expand our antibiotic arsenal
with agents that are minimally disruptive to beneficial
microflora. Pathogen-specific glycan interference could
circumvent problems caused by broad-spectrum agents, which
alter the gut microbiome and precipitate deleterious health
consequences.18-20
prompted the search for alternative treatments.7
A
preponderance of biochemical and genetics evidence suggests
that glycoprotein biosynthesis enzymes within priority
pathogens are compelling potential targets of therapeutic
intervention.1, 3
Bacterial glycoproteins, and the glycosyltransferases that
synthesize them, have emerged as intriguing targets because
they are produced only by select bacteria, they contain
distinctive structures that are markedly different from their
eukaryotic counterparts, and they often play important roles in
Numerous strategies including transition state analogue
design, structure-based design, fragment-based and library
screening approaches have successfully yielded inhibitors of
eukaryotic glycan biosynthesis enzymes.21 Inspired by these
precedents, Walker, Kahne, and coworkers used structure-
based design to produce a novel inhibitor of peptidoglycan
a. Department of Chemistry & Biochemistry, Bowdoin College, 6600 College Station,
Brunswick, ME, 04011, USA. E-mail: ddube@bowdoin.edu
b. Department of Chemistry, Indian Institute of Technology Bombay, Powai,
Mumbai, 400076, India. E-mail: suvarn@chem.iitb.ac.in
† These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: Fig. S1-S5 and full
experimental details. See DOI: 10.1039/x0xx00000x
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containing sugars are taken up by cells, then metabolically
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DOI: 10.1039/C9SC05955E
incorporated into cellular glycans. Azide-containing glycans are
subsequently tagged with bioorthogonal reaction partners (e.g.
alkynes, phosphines) to enable detection, identification, and
26, 29
visualization in live cells and animals (Fig. 1B).25,
This
approach has been expanded to bacterial systems3, 30, 31 and led
to, for example, the discovery of a general protein glycosylation
system in Helicobacter pylori using the azidosugar peracetylated
N-azidoacetylglucosamine (Ac4GlcNAz) as
substrate.32, 33
a
metabolic
In addition to yielding
a readout of cellular glycan
biosynthesis, MOE provides information about which
monosaccharides are metabolically processed by particular
bacterial species. Azide-containing analogues of the rare
bacterial
sugars
bacillosamine,
2,4-diacetamido,2,4,6-
trideoxygalactose (DATDG), N-acetylfucosamine (FucNAc),34
altrose35, trehalose,36, 37 pseudaminic acid,38 N-acetylmuramic
acid (MurNAc)39, 40, and 3-deoxy-
D-manno-octulosonic acid
(Kdo)41 have been developed and deployed in metabolic
labeling experiments. Experiments have revealed differential
incorporation of rare bacterial monosaccharides into glycans on
bacterial cells, presumably due to species-specific differences in
carbohydrate biosynthetic pathways. These observations
provide the basis for the development of inhibitors based on
rare bacterial monosaccharide scaffolds.
Here, we report a strategy for the production of bacterial
glycan biosynthesis inhibitors in the absence of detailed
pathway or glycan information. We designed and synthesized
two classes of metabolic inhibitors based on rare bacterial
monosaccharide scaffolds, then used a cellular MOE-based
assay to assess the ability of these substrate-based analogue
inhibitors to terminate or divert glycan biosynthesis (Fig. 1C).
The “hits” were then screened for their ability to induce fitness
defects in a variety of bacterial pathogens. Importantly,
features involved in the ability of bacteria to initiate and
maintain infections within a host were scored. These assays
yielded metabolic inhibitors that interfere with glycan
biosynthesis in the gastric pathogen H. pylori and precipitate
functional defects in growth, motility, and biofilm formation.
Intriguingly, these metabolic inhibitors have differential effects
on glycan biosynthesis in other bacteria explored in these
studies. The selectivity of these inhibitors has the potential to
set the stage for the development of novel, narrow-spectrum
antibiotics to treat infectious disease. This approach is a general
one that promises to enable systematic study of bacterial
glycosylation enzymes and expedite the identification of
bacterial glycan biosynthesis inhibitors in a range of systems,
further expanding the glycochemistry toolkit.
Fig. 1 Bacteria synthesize complex glycans bearing distinctive monosaccharides
that can be detected via metabolic oligosaccharide engineering (MOE); disrupted
glycan biosynthesis yields a diminished MOE-based signal. A) Bacteria coat their
cells with glycans comprised of rare, deoxy amino monosaccharides that are
crucial for fitness and absent from human cells. B) Schematic overview of MOE,
which facilitates detection and visualization of cellular glycan biosynthesis. Cells
are first metabolically labeled with an unnatural azide-containing sugar, then
azide-labeled glycans are covalently elaborated via Staudinger ligation with a
phosphine-based probe to yield detectable glycans on cells or in cell lysates. C)
MOE was used as a cell-based readout of interrupted glycan biosynthesis. The
effect of metabolic inhibitors on glycan biosynthesis was detected by diminished
metabolic incorporation of unnatural, azide-containing sugars into cellular
structures.
glycosyltransferase that displayed potent antibacterial
activity,22 Imperiali and coworkers employed fragment-based
and library screening approaches to develop nM inhibitors of
UDP-bacillosamine biosynthesis,23 while Logan and coworkers
invoked a library-based screen of pseudaminic acid biosynthesis
enzymes to discover two potent inhibitors of flagellin
glycosylation.24 While these strategies are directly transferable
to enzymes involved in bacterial glycan synthesis, they typically
require in-depth knowledge of the targets, including the
structures of substrates and products as well as robust in vitro
assays to monitor enzyme-catalyzed product formation. A lack
of precise glycomics information has impeded the development
of suitable inhibitors of glycoprotein biosynthesis in many
priority pathogens.
A method that sheds light on bacterial glycoprotein
biosynthesis in intact cells could circumvent the need for full
pathway and glycan characterization to screen inhibitors.
Metabolic oligosaccharide engineering (MOE), which was
pioneered by Bertozzi, Reutter, and coworkers, is well suited to
this task as it tracks metabolic incorporation of unnatural
monosaccharides into cellular glycans via endogenous
carbohydrate biosynthetic pathways.25-28 In essence, azide-
RESULTS AND DISCUSSION
Design of substrate-based metabolic inhibitors
Motivated by the successful development of small molecule
inhibitors of glycosyltransferases in mammalian systems,42-45
two classes of metabolic inhibitors were explored. The first class
of inhibitors was inspired by Esko’s seminal work with metabolic
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substrate decoys such as benzyl-
ARTICLE
a
-GalNAc (Bn-GalNAc).45-48
According to the original design, the presence of these
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DOI: 10.1039/C9SC05955E
Metabolic substrate decoys are small molecule analogues of fluoro sugars in glycans should terminate subsequent
endogenous glycoproteins that are recognized as acceptor elaboration by glycosyltransferases as they lack sites (C3-OHs)
substrates by glycosyltransferases, ultimately diverting glycan for additional extension. Recent work in mammalian systems
synthesis onto decoy substrates and leading to the synthesis of with fluoro analogues of sialic acid, fucose, GlcNAc and GalNAc
truncated glycans on proteins. Successful decoys in mammalian has demonstrated that these compounds indeed act as global
systems led us to design benzyl glycosides of Bac, DATDG, and metabolic inhibitors, though by a different mechanism than
FucNAc as substrate decoys of bacterial glycosyltransferases originally intended.44, 52, 53 In mammalian systems, fluoro sugars
(Fig. 2A; compounds 1, 2 and 3). We focused on derivatives of are activated as nucleotide sugars in situ but not effectively
the rare bacterial monosaccharides Bac, DATDG, and FucNAc utilized by glycosyltransferases, leading to a buildup of the
due to expedient syntheses of these scaffolds and their activated unnatural nucleotide sugars in cells and metabolic
utilization in priority pathogens.34
feedback inhibition that prevents de novo synthesis of natural
The second class of inhibitors was inspired by classic and activated substrates.44, 53 Regardless of the precise molecular
modern examples of chain-terminating substrates, including mechanism, fluoro sugars based on rare bacterial
Sanger’s pioneering use of di-deoxy chain terminators of DNA monosaccharide scaffolds are likely to serve as global metabolic
synthesis and more recent extensions of this concept with inhibitors of bacterial glycan biosynthesis.
deoxy- and fluoro-sugars as terminators of glycan
Following these rational design criteria, benzyl glycoside and
biosynthesis.43, 44, 49-51 Based on these successful precedents, we 3-F analogues of Bac, DATDG, and FucNAc were our target
designed 3-fluoro analogues of Bac, DATDG, and FucNAc to act metabolic inhibitors of bacterial glycosylation (Fig. 2). As a key
as global metabolic inhibitors of bacterial glycoprotein design element of putative inhibitors, the hydrophilic hydroxyl
biosynthesis (Fig. 2B; compounds 4, 5, and 6). Fluoro-sugars are groups were temporarily masked with hydrophobic acetyl
expected to be converted into the corresponding nucleotide- groups47 to maximize efficiency of uptake by bacteria.32 We
activated monosaccharide within cells, then impede glycan previously validated this transient protection strategy in H.
elaboration.
pylori by demonstrating via mass spectrometry that H. pylori
treated with Ac4GlcNAz metabolically incorporated the free
sugar GlcNAz into glycoproteins,33 in line with reports that this
bacteria has endogenous esterase activity.54, 55 In addition to
these novel compounds, we included commercially available
substrate decoys and chain terminators based on the common
monosaccharide scaffolds N-acetylglucosamine (GlcNAc) and N-
acetylgalactosamine (GalNAc) (Fig. 2C; compounds 7-10) in our
studies due to their ability to elicit glycosylation defects in
mammalian systems and the ubiquity of these monosaccharides
in bacterial glycans.
Synthesis of bacterial monosaccharide analogue inhibitors
We synthesized a panel of substrate decoy OBn analogues 1-3
by adaptation of our published protocols for synthesis of Bac,
DATDG, and FucNAc, but with slight improvements (using
inexpensive KNO2 in place of expensive TBANO2).56-58 The
synthesis of benzyl glycosides involved one-pot nucleophilic
displacements of 2,4-bis-trifluoromethanesulfonates (triflates,
OTf) to access the corresponding thioglycoside donors, followed
by glycosylation with benzyl alcohol under standard conditions.
Synthesis of chain terminator 3-fluoro analogues 4-6 demanded
a novel approach. Fluorination of a sugar hydroxyl involves
inversion at the chosen carbon. Therefore one has to gain
access to its epimeric precursor, in our case, the C3 epimer 6-
Fig. 2 Potential metabolic inhibitors based on rare bacterial monosaccharide scaffolds
(A, B) or common monosaccharide scaffolds (B) used in this study. Substrate-based
metabolic inhibitors were designed based on the rare bacterial monosaccharide
scaffolds bacillosamine, DATDG, and FucNAc. A) Benzyl glycosides 1, 2 and 3 were
created to compete with and divert glycan biosynthesis onto decoy substrates. B) Fluoro
analogues 4, 5 and 6 were designed to interrupt glycan biosynthesis. C) Established
mammalian glycosylation inhibitors 7-10, based on the common monosaccharide
scaffolds GlcNAc and GalNAc, were also explored as potential inhibitors of bacterial
glycosylation.
deoxy-
synthesis of 3-F analogues involved regioselective and
orthogonal protection of -rhamnosyl triol and sequential
D-allosamine thioglycoside derivative. Accordingly, the
D
nucleophilic displacements of the C2-OTf and C3-OTf by azide
and nitrite anions as nucleophiles, respectively, to access the
key D-allo 3-OH intermediate. Subsequently, inversion at the C3
position with the fluorinating agent diaminoethyl sulfur
59-61
trifluoride (DAST)52,
afforded the C3-F quinovose as a
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common synthon. Finally, inversions at C4 by various double displacement with 10 equiv of NaN3 in DMF at room
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57
nucleophiles, followed by functional group transformations, temperature afforded di-azido derivativeD1O5I: 10in.108389%/Cy9iSeCl0d5o95v5eEr
furnished compounds 4-6.
To synthesize benzyl glycoside analogues 1-3, the common with 1 equiv of BnOH by using NIS, TfOH as promoter at 0 °C in
precursor -rhamnosyl 2,4-diol 11 was readily prepared starting CH2Cl2:Et2O (1:1 ratio) to obtain benzyl glycoside 16 in 93% yield
from commercially available isomers. Subsequent reduction of azide
-mannose in six steps.57 Synthesis as a 1:1 mixture of α:
two steps. The thioglycoside donor 15 was then glycosylated
D
D
b
of Bac-diNAc-OBn (Bn-Bac) 1 is shown in Scheme 1. Triflation of groups in 16 with Zn, AcOH in THF, followed by treatment with
diol 11 with Tf2O and pyridine in dry CH2Cl2 gave the Ac2O and catalytic DMAP in THF, furnished benzyl-2,4-
corresponding 2,4-bis-triflate intermediate, which upon a brief diacetamido-2,4,6-trideoxy
work-up was subjected to a one pot, double nucleophilic in 86% yield over two steps.
displacement reaction. The crude 2,4-bis-triflate derivative of
D-galactose 2 (DATDG-diNAc-OBn)
compound 11 was transformed to the corresponding C2 azide
1. Tf2O (3 equiv)
Py (6 equiv)
CH2Cl2, 0 °C, 1 h
N3
displaced product by treatment with 0.85 equiv of TBAN3 in
CH3CN at -30 ºC for 18 h. Concomitant treatment with 10 equiv
of KNO2 in DMF at room temperature displaced the remaining
C4-OTf to afford compound 12 in 70% yield over three steps
after a single column chromatographic purification.
OH
O
O
HO
AcO
SPh
SPh
AcO
2. NaN3 (10 equiv)
DMF, rt, 5 h
88% over 2 steps
N3
15
11
NIS (2 equiv)
TfOH (0.6 equiv)
N3
1. Zn (12 equiv), AcOH
THF, rt, 9 h
O
BnOH, 3Å MS, 0 °C
1. Tf2O (3 equiv)
Py (6 equiv)
CH2Cl2, 0 °C, 1 h
2. TBAN3 (0.85 equiv)
CH3CN, -30 °C, 18 h
AcO
OBn
CH2Cl2:Et2O (1:1), 1 h
N3
16
2. Ac2O (4 equiv)
DMAP (0.01 equiv)
THF, 0 °C to rt, 5 h
86% over 2 steps
93% (α:β =1:1)
HO
OH
O
O
HO
AcO
SPh
SPh
AcO
AcHN
3. KNO2 (10 equiv)
DMF, rt, 5 h
70% over 3 steps
N3
12
11
O
AcO
OBn
AcHN
2
NIS (2 equiv)
TfOH (0.6 equiv)
BnOH, MS 3Å , 0 °C
1. Tf2O (1.5 equiv)
Py (3 equiv)
CH2Cl2, 0 °C, 1 h
O
N3
AcO
Scheme 2 Synthesis of DATDG-diNAc-OBn (Bn-DAT) 2.
SPh
2. NaN3 (10 equiv)
DMF, rt, 5 h
78% over 2 steps
N3
CH2Cl2:Et2O (1:1)
1 h, 90% (α only)
13
To synthesize FucNAc-OBn (Bn-FucNAc) 3, we started from
compound 12 (Scheme 3). The free hydroxyl group in compound
12 was acetylated with AcCl and pyridine in CH2Cl2 to give 3,4-
diacetylated compound 17 in 92% yield. Donor 17 was then
activated with NIS, TfOH in CH2Cl2:Et2O (1:1 ratio) and coupled
with BnOH at 0 °C to provide exclusively α-glycosylated
compound 18 in 87% yield. Subsequently, the azide was
converted to acetamide using Zn, AcOH in THF followed by
1. Zn (12 equiv), AcOH
THF, rt, 9 h
O
O
N3
AcO
AcHN
AcO
N3
2. Ac2O (4 equiv)
DMAP (0.01 equiv)
THF, 0 °C to rt, 5 h
82% over 2 steps
AcHN
1
OBn
OBn
14
Scheme 1 Synthesis of Bac-diNAc-OBn (Bn-Bac) 1.
AcCl (3 equiv)
Py (3 equiv)
CH2Cl2, 0 °C to rt
HO
AcO
AcO
AcO
O
O
The free C4-hydroxyl group in compound 12 was treated
with Tf2O and pyridine in dry CH2Cl2 at 0 °C followed by inversion
using 10 equiv of NaN3 in DMF at room temperature to afford
SPh
SPh
3 h, 92%
N3
12
N3
17
2,4-diazido-2,4,6-trideoxy D-glucose (Bac) derivative 13 in 78%
NIS (2 equiv)
TfOH (0.6 equiv)
BnOH, 3Å MS, 0 °C
over two steps. Donor 13 was further glycosylated with 1 equiv
of benzyl alcohol (BnOH) by using NIS, TfOH as promoter at 0 °C
in CH2Cl2:Et2O (1:1 ratio) to deliver exclusively α-linked OBn
glycoside 14 in 90% yield. Azide groups of OBn glycoside 14
were reduced with Zn, AcOH in THF, which was followed by
acetamide formation with Ac2O and catalytic amount of DMAP
AcO
1. Zn (12 equiv), AcOH
THF, rt, 9 h
O
AcO
N3
2. Ac2O (4 equiv)
DMAP (0.01 equiv)
THF, 0 °C to rt, 5 h
82% over 2 steps
CH2Cl2:Et2O (1:1), 1 h
OBn
87% (α only)
18
AcO
in THF to furnish benzyl-2,4-diacetamido-2,4,6-trideoxy
D-
O
glucose 1 (Bac-diNAc-OBn) in 82% yield over two steps.
AcO
Synthesis of the rare sugar analogue DATDG-diNAc-OBn 2 is
outlined in Scheme 2. The known 2,4-diol substrate 11 was
reacted with Tf2O and pyridine in dry CH2Cl2 at 0 °C to provide
the corresponding 2,4-bis-triflate compound, which upon
AcHN
OBn
3
Scheme 3 Synthesis of FucNAc-OBn (Bn-FucNAc) 3.
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ARTICLE
Scheme 4 Synthesis of common 3-fluoro-D-quinovosamine 26.
treatment with Ac2O, and catalytic amount of DMAP in THF to different nucleophiles (NaN3 and KNO2). The synthesis of 3F-
obtain benzyl-2-acetamido-2,6-dideoxy
OBn) 3 over two steps in 82% yield.
D
-galactose (FucNAc- Bac-diNAc (F-Bac) 4 is outlined in Scheme 5. The free C4-OH of
compound 26 was subjected to O-triflation using triflic
The synthesis of fluoro analogues 4-6 involved two stages – anhydride and pyridine, followed by inversion of O-triflate with
1) the synthesis of common precursor 3-fluoro-quinovosamine 15 equiv of KNO2 to afford compound 27 in 60% yield over two
derivative 26, and 2) its C4 functionalization with inversion using steps. Then, triflation of 27 and subsequent nucleophilic
azide or nitrite anions, followed by functional group inter displacement by 10 equiv of NaN3 furnished 3-fluoro
conversions. The known D
-rhamnose triol 1957 was deemed to bacillosamine derivative 28 with 80% yield. The C2 and C4 azide
be the appropriate starting material for the synthesis of the groups in compound 28 were reduced by Zn and AcOH to give
common 3-fluoro building block 26 (Scheme 4). First, the C2 and
1. Tf2O (1.5 equiv)
C3 hydroxyl groups in 19 were simultaneously capped by
acetonide protecting groups. Next, the remaining C4-hydroxyl
group was naphthylmethylated with NapBr and NaH to obtain
compound 20 in 82% yield over two steps. Acetonide
deprotection upon acid hydrolysis followed by a highly
regioselective acetylation at O3 using 5 mol% Me2SnCl2
afforded compound 21 in very good yield over two steps. The
C2-OH was converted to the corresponding O-triflate, which
was subsequently displaced by azide to give compound 22 in
89% yield. Deacetylation of 22 followed by its O-triflation and
concomitant treatment with 15 equiv of KNO2 transformed 2-
HO
F
Py (3 equiv)
CH2Cl2, 0 °C, 0.5 h
O
O
HO
SPh
SPh
F
2. KNO2 (15 equiv)
DMF, rt, 4 h
60% over 2 steps
N3
N3
26
27
1. Zn (12 equiv)
AcOH, THF
RT, 8 h
1. Tf2O (1.5 equiv)
Py (3 equiv)
CH2Cl2, 0 °C, 0.5 h
O
N3
F
SPh
2. NaN3 (10 equiv)
DMF, rt, 4 h
80% over 2 steps
N3
28
2. Ac2O (4 equiv)
DMAP (0.1 equiv)
THF, 0 °C to rt, 4 h
75% over 2 steps
azido-2,6-dideoxy glucose to 2-azido-2,6-dideoxy D-allose 24 in
53% yield over two steps. Fluoride was installed at the C3
position using 3 equiv of DAST at -20 °C to room temperature to
1. NBS (3 equiv)
THF:H2O (4:1)
rt, 15 min
O
O
AcHN
AcHN
afford 2-azido-3-fluoro-2,3,6-trideoxy
D-glucose 25 in 71% yield.
SPh
F
F
2. Ac2O (5 equiv)
Et3N (5 equiv)
DMAP (0.1 equiv)
CH2Cl2, rt, 5 h
AcHN
Finally, removal of Nap group using 1.8 equiv of DDQ in CH2Cl2
NHAc
OAc
and H2O fashioned the common
D
-quinovosamine 3-F building
29
4
(α only)
block 26 in 93% yield.
75% over 2 steps
To access the fluoride containing rare sugar analogues 4-6,
the C4 center of compound 26 was triflated and inverted using Scheme 5 Synthesis of 3F-Bac-diNAc (F-Bac) 4.
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AcCl (1.5 equiv)
Py (1.5 equiv),
CH2Cl2, rt, 1 h
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HO
O
AcO
1. Tf2O (1.5 equiv)
Py (3 equiv)
CH2Cl2, 0 °C, 0.5 h
N3
O
SPh
SPh
N3
F
F
O
O
HO
F
SPh
SPh
F
N3
27
73%
N3
2. NaN3 (10 equiv)
DMF, rt, 4 h
N3
32
26
30
70% over 2 steps
1. NBS (3 equiv)
THF:H2O (4:1)
rt, 15 min
AcO
1. Zn (12 equiv), AcOH
THF, rt, 8 h
1. NBS (3 equiv)
THF:H2O (4:1)
rt, 15 min
O
AcHN
O
SPh
NHAc
F
1. Zn (12 equiv) AcOH
THF, rt, 8 h
2. Ac2O (5 equiv)
Et3N (5 equiv)
DMAP (0.1 equiv)
CH2Cl2, rt, 5 h
2. Ac2O (4 equiv)
DMAP (0.1 equiv)
THF, 0 °C to rt, 4 h
73% over 2 steps
SPh
NHAc
31
F
33
2. Ac2O (5 equiv)
Et3N (5 equiv)
DMAP (0.1 equiv)
CH2Cl2, rt, 5 h
2. Ac2O (4 equiv)
DMAP (0.1 equiv)
THF, 0 °C to rt, 4 h
72% over 2 steps
75% over 2 steps
70% over 2 steps
AcO
AcHN
O
F
O
F
AcHN
OAc
6
(α only)
OAc
AcHN
5
(α/β = 5:1)
Scheme 7 Synthesis of 3F-FucNAc (F-FucNAc) 6.
Scheme 6 Synthesis of 3F-DATDG-diNAc (F-DAT) 5.
to detect the 125 glycoproteins synthesized by H. pylori’s
general protein glycosylation system32, 33 (Fig. 1B). Though the
identify of these glycosylated proteins has been revealed by
mass spectrometry analyses,33 the structure of the higher-order
glycan on these glycoproteins is not yet characterized. Thus, this
system is a test bed for structural perturbation in the absence
of full information. In essence, we assessed the ability of each
of these analogues to inhibit azide-labeled glycoprotein
biosynthesis (Fig. 1C) in H. pylori. Following our published
methods,29 metabolic labeling experiments were performed
with Ac4GlcNAz as a positive control, with the azide-free sugar
peracetylated N-acetylglucosamine (Ac4GlcNAc) as a negative
control, or with Ac4GlcNAz in the presence of increasing
concentrations (0.5-2 mM) of putative inhibitors 1-10 as
experimental samples. After four days of metabolic labeling,
proteins were harvested from lysed cells and reacted via
Staudinger ligation with a phosphine probe comprising a FLAG
peptide63 (Phos-FLAG). As expected, analysis of positive control
samples from H. pylori treated with Ac4GlcNAz yielded the full
complement of azide-labeled glycoproteins detectable by
Western blot analysis with anti-FLAG antibody, whereas control
samples from cells treated with the azide-free compound
Ac4GlcNAc yielded negligible azide-dependent signal (Fig. 3).
These samples benchmark the most and least signal,
respectively, that we would expect to observe in any given
sample. We performed an additional control in which H. pylori
cells were treated with Ac4GlcNAz in the presence of increasing
concentrations (0.5-2 mM) of the control sugar Ac4GlcNAc,
which lacks fluoro and OBn modifications and therefore was not
expected to act as a glycosylation inhibitor. This control was
included due to the possibility that 2 mM of any non-azide
containing sugar might be a sufficiently high concentration to
outcompete the enzymes (e.g. deacetylases, carbohydrate
biosynthesis enzymes, glycosyltransferases) that process the
azidosugar and install it into glycoproteins. Samples treated
with up to 2 mM of Ac4GlcNAc had no appreciable effect on H.
pylori’s ability to synthesize azide-labeled glycoproteins, as
the corresponding diamine compound, which was then treated
with Ac2O and catalytic amount of DMAP in THF to afford 3-
fluoro compound 29 in 75% yield over two steps. Thioglycoside
29 was hydrolyzed to hemiacetal upon treatment with NBS, THF
and H2O followed by acetylation using Ac2O, Et3N and catalytic
amount of DMAP in CH2Cl2 to obtain 3F-Bac-diNAc 4 in 75% yield
(α only) over two steps.
Scheme 6 outlines our straightforward synthesis of F-DAT
compound 5. Starting from compound 26, the C4 hydroxyl
group was subjected to O-triflation and concomitant
nucleophilic displacement using NaN3, DMF to afford diazido
derivative 30 in 70% yield over two steps. The C2 and C4 azide
groups were then reduced to the corresponding diamine, which
was then acetylated to obtain 2,4 di-NHAc derivative 31 in 72%
yield. Finally, thioglycoside 31 was activated and hydrolyzed to
hemiacetal followed by treatment with Ac2O, Et3N and catalytic
amount of DMAP in CH2Cl2 to furnish 3F-DATDG-diNAc (F-DAT)
analogue 5 in 70% yield (α:β = 5:1) over two steps.
To synthesize 3F-FucNAc 6 (Scheme 7), the C4 hydroxyl
group of compound 27 was protected with AcCl and pyridine to
render compound 32 in 73% yield. On treatment with Zn and
AcOH, the azide group in 32 was converted to amine, then
acetylated to give compound 33 in 73% yield. The anomeric
thioglycoside was converted to corresponding hemiacetal
which was acetylated using Ac2O, Et3N and catalytic amount of
DMAP in CH2Cl2 to deliver 3F-FucNAc (F-FucNAc) 6 in 75% yield
(α only) over two steps.
Glycoprotein biosynthesis is impeded in H. pylori
To ascertain whether the newly developed 3-F and benzyl
glycoside analogues of rare bacterial monosaccharides (1-6, Fig.
2) and the established mammalian metabolic inhibitors (7-10)
act as metabolic inhibitors of bacterial glycosylation, we took
advantage of the established utility of Ac4GlcNAz,62 an azide-
containing analogue of the common monosaccharide GlcNAc,
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glycosylation in H. pylori. Similarly, three rare bacterial
View Article Online
DOI: 10.1039/C9SC05955E
monosaccharide analogues, BnDAT (2), F-Bac (4), and F-FucNAc
(6), had little to no apparent impact on H. pylori’s ability to
synthesize the full profile of azide-labeled glycoproteins (Fig. 3).
By contrast, the rare bacterial monosaccharide analogues
BnBac (1), BnFucNAc (3), and F-DAT (5) elicited a concentration-
dependent diminishment in the azide-dependent glycoprotein
profile (Fig. 3). Coomassie staining of electrophoresed samples
confirmed that all samples contain proteins (Fig. S1†), indicating
that the lack of detectable azides in some samples was not due
to low protein levels. Taken together, these results suggest that
three of the novel compounds are metabolic inhibitors that
impede H. pylori’s ability to synthesize glycoproteins.
We next sought to confirm via an orthogonal method that
H. pylori’s glycosylation is disrupted upon treatment with select
metabolic inhibitors. Toward this end, we assessed the binding
of the carbohydrate-binding lectin concanavalin A (ConA) to
untreated wildtype H. pylori versus H. pylori treated with 2 mM
of compounds 1-6. Flow cytometry analysis of intact cells
revealed that ConA bound to wildtype H. pylori (Fig. 3C). Pre-
incubation of ConA with its known ligand mannose mitigated
binding to wildtype H. pylori, consistent with the carbohydrate-
dependent nature of this binding interaction (Fig. 3C). H. pylori
treated with BnDAT (2) exhibited ConA binding comparable to
untreated wildtype H. pylori (Fig. 3), suggesting no appreciable
alteration in cell wall glycans. By contrast, treatment of H. pylori
with the rare bacterial monosaccharide analogues BnBac (1),
BnFucNAc (3), F-Bac (4), F-DAT (5), and F-FucNAc (6) elicited an
increase in ConA binding relative to untreated H. pylori (Fig. 3C;
Fig. S1†). Enhanced ConA binding is consistent with metabolic
glycan inhibitors altering cell wall glycan architecture, perhaps
by unmasking underlying glycan epitopes.
Fig. 3 Western blot and flow cytometry analyses indicate select monosaccharide
analogues act as metabolic inhibitors of H. pylori’s glycan biosynthesis. A) Benzyl
glycosides (1, 2, 3, 7, 8) and B) fluoro analogues (4, 5, 6, 9, 10) were screened for their
ability to interfere with biosynthesis of the full profile of azide-labeled glycoproteins in
H. pylori treated with Ac4GlcNAz. H. pylori were grown for four days in media
supplemented with 0.5 mM Ac4GlcNAz (Az), Ac4GlcNAc (Ac), or with 0.5 mM Ac4GlcNAz
(Az) alongside increasing concentrations (0.5, 1.0, or 2.0 mM; indicated by the increasing
shaded triangle, with the shortest point representing 0.5 mM and the tallest part
representing 2 mM except for the BnDAT (2) samples, which are 0.5 and 1.0 mM) of
compounds 1-10 or Ac4GlcNAc (GlcNAc), then harvested by lysis. The presence of azides
in cellular glycoproteins was detected by reacting lysates with 250 µM Phos-FLAG for 12
h at 37 °C and then analyzing samples via Western blot with anti-FLAG antibody. C)
Benzyl glycosides (1, 2, 3) and fluoro analogues (4, 5, 6) were screened for their ability
to alter lectin binding to H. pylori. H. pylori were grown for four days in media
supplemented with 2.0 mM of compounds 1-6 or left as untreated wildtype (WT), then
probed with ConA conjugated to Alexa Fluor 488 and analysed by flow cytometry.
Alternatively, ConA was pre-treated with 400 mM mannose (carbo block) prior to
probing untreated H. pylori. The data shown are representative of replicates (n ³ 2).
The combination of Western blot and flow cytometry data
suggest that BnBac (1), BnFucNAc (3), and F-DAT (5) alter cell
wall glycan architecture by inhibiting glycoprotein biosynthesis.
By contrast, F-Bac (4) and F-FucNAc (6) did not appreciably alter
glycoprotein biosynthesis but did alter global cell wall glycan
architecture; these data suggest that these analogs may impact
biosynthesis of
a different glycoconjugate in H. pylori.
Collectively, these data support the hypothesis that metabolic
inhibitors alter H. pylori’s cell wall glycan architecture.
Metabolic inhibitors elicit functional defects in H. pylori
With compelling evidence that this panel of metabolic inhibitors
interferes with glycan biosynthesis in H. pylori, we next explored
whether glycosylation inhibitors impact fitness. We queried
effects on critical functions that are absolutely required for H.
pylori to colonize the host and sustain an infection. In particular,
we scored the effect of analogues on growth by monitoring
OD600, motility by quantifying halo formation on soft agar,64 and
biofilm formation by measuring crystal violet staining.65
Untreated wildtype H. pylori grew to stationary phase (Fig.
4A), were viable (Fig. S3†), were motile (Fig. 4B), and formed a
dense biofilm that stained with crystal violet (Fig. 4C). H. pylori
treated with 1 mM BnGlcNAc (7), F-Bac (4), and F-FucNAc (6)
exhibited growth, motility, and biofilm formation comparable
indicated by the robust azide-dependent glycoprotein profile
present in these samples (Fig. 3A). This result allayed concerns
that a diminishment in azide-dependent signal results from
competition alone.
With the MOE-based assay validated, we turned to our
experimental samples. Of the previously reported common
monosaccharide analogues, BnGalNAc (8) and F-GalNAc (10)
had no appreciable effect on the azide-labeled glycoprotein
fingerprint detected, whereas BnGlcNAc (7) and F-GlcNAc (9)
appeared to elicit only a very minor effect on detected signal
intensity (Fig. 3). Therefore, previously reported metabolic
inhibitors do not appear to dramatically impact protein
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Chemical Science
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DOI: 10.1039/C9SC05955E
Fig. 5 Metabolic inhibitors have a negligible effect on Campylobacter jejuni’s glycan
biosynthesis or fitness. C. jejuni were treated with the indicated metabolic inhibitors (1-
8) and scored for (A) synthesis of azide-labeled glycoproteins upon metabolic labeling
with 1 mM Ac4GalNAz (Gal), (B) motility on soft agar, and (C) biofilm formation using a
crystal violet assay. A) C. jejuni were grown for four days in media supplemented with 1
mM Ac4GalNAz (Gal), Ac4GlcNAc (Ac), or with 1 mM Ac4GalNAz alongside increasing
concentrations (0.5, 1.0, or 2.0 mM; indicated by the increasing shaded triangle, with the
shortest point representing 0.5 mM and the tallest part representing 2 mM except for
the BnGalNAc (8) samples, which are 0.5 and 1.0 mM; BnBac (1) samples, which are 1.0
and 2.0 mM; and BnDAT (2) samples, which are 0.5 and 2.0 mM) of compounds 1-8, then
harvested by lysis. The presence of azides in cellular glycoproteins was detected by
reacting lysates with 250 µM Phos-FLAG for 12 h at 37 °C and then analyzing samples via
Western blot with anti-FLAG antibody. B, C) C. jejuni were treated with 2 mM of the
indicated metabolic inhibitors (1, 2, 4, 5, 6, 7) and scored for (B) motility on soft agar
after 4 days, and (C) biofilm formation after 4 days using a crystal violet assay. Untreated
wildtype C. jejuni (WT) served as a reference point in motility and biofilm formation
assays. The data shown are representative of replicates (n ³ 2).
Fig. 4 Fitness assays reveal defects in growth, motility and biofilm formation for H. pylori
treated with metabolic inhibitors. H. pylori were treated with 1 mM of benzyl glycosides
(1, 3, 7) or fluoro analogues (4, 5, 6, 9, 10) and scored for (A) growth by monitoring optical
density at 600 nm (OD600), (B) motility on soft agar (shown at day 8), and (C) biofilm
formation using a crystal violet assay. Untreated wildtype H. pylori (WT) and an H. pylori
protein glycosylation mutant containing an insertional inactivation in a predicted
glycosyltransferase gene (DGT, HPG27_580::CmR) were also scored in these assays. The
data shown are representative of replicates (n ³ 2).
to untreated H. pylori. By contrast, H. pylori treated with BnBac
(1), F-DAT (5) and BnFucNAc (3) displayed slowed growth,
decreased viability, diminished motility, and impaired biofilm
formation relative to untreated H. pylori (Fig. 4; Fig. S3†). These
fitness defects were also observed with an H. pylori protein
glycosylation mutant containing an insertional inactivation of a
putative H. pylori glycosyltransferase gene
(DGT,
HPG27_580::CmR)(Fig. 4); inactivating this gene impedes H.
pylori’s general protein glycosylation system (Fig. S2†) by an as-
yet uncharacterized mechanism, yet has no effect on
lipopolysaccharide biosynthesis.66 Small molecule-induced
functional defects appeared even more striking than those
induced via genetic interruption. Fitness appeared to be most
impaired in H. pylori treated with BnBac (1), F-DAT (5) and
BnFucNAc (3) (Fig. 3), and quantification of motility and biofilm
production confirmed this qualitative finding (Fig. S3†).
Profound defects in motility were sustained even up to fifteen
days after allowing cells to swarm (data not shown).
Intriguingly, the three compounds that impaired all fitness
attributes also produced the most pronounced glycoprotein
biosynthesis defects. Somewhat surprisingly, F-GlcNAc (9) and
F-GalNAc (10) led to disrupted motility (Fig. 4B) despite not
appreciably altering H. pylori’s general glycosylation system
(Fig. 3B). This effect could be due, potentially, to inhibition of
flagellin glycosylation, which impacts only motility.1 Small-
molecule induced functional defects in attributes that are
absolutely critical for establishment and maintenance of
infection within a host suggest the potential functional
importance of H. pylori’s protein glycosylation machinery and
ensuing suitability as a drug target.
Metabolic inhibitors act in a bacteria-selective manner
Encouraged by the observation that metabolic inhibitors based
on rare bacterial monosaccharide scaffolds interfere with H.
pylori’s glycan biosynthesis and induce functional defects, we
next evaluated the effect of these metabolic inhibitors on an
additional Gram-negative pathogen that colonizes the human
gut, Campylobacter jejuni. C. jejuni is a close relative of H. pylori
that was selected because it synthesizes well-characterized
glycoproteins and incorporates azide-containing sugars into its
glycans via MOE.34, 67 Analogous to the experiments performed
with H. pylori, we assessed the ability of our novel
monosaccharide analogues to inhibit azide-labeled glycoprotein
biosynthesis in C. jejuni. For these experiments, peracetylated
N-azidoacetylgalactosamine (Ac4GalNAz),68 an azide-containing
analogue of N-acetylgalactosamine, was used to metabolically
label cellular glycans. Robust azide-labeled glycan biosynthesis
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turned to this azidosugar as a metabolic reporter of intact
View Article Online
polysaccharide A biosynthesis. B. fragiliDsOsI:u1p0p.1l0e3m9/eCn9tSeCd05w95i5thE
Ac4GalNAz displayed robust azide-dependent glycan labeling.
Additional supplementation with 0.5-2 mM of select
metabolic inhibitors (1-6, 8) caused no appreciable
diminishment of the prominent azide-dependent signal
observed ~72-95 kDa (Fig. 6A). A subtle diminishment of the
high molecular weight signal, >95 kDa, was noted in a
concentration-dependent manner with several of the analogues
(Fig. 6A). In all cases Coomassie staining of electrophoresed
samples confirmed equivalent protein levels (Fig. S5A†),
indicating that subtle changes in detectable azides in some
samples was not due to low protein content. Since Western blot
data were difficult to interpret, we turned to a complementary
approach to quantify changes in cell surface glycans.
To explore the possibility that these analogues may impact
synthesis of fully-elaborated glycans, we probed cell surface
azide expression using a flow cytometry-based assay.69 Cells
were metabolically labeled with Ac4GalNAz or the azide-free
control sugar Ac4GlcNAc, and surface accessible azides were
probed on live cells using strain-promoted azide-alkyne
cycloaddition with fluorescent AF488-DBCO.34 B. fragilis treated
with Ac4GalNAz exhibited
a high fluorescence intensity,
Fig. 6 Metabolic inhibitors have a subtle effect on Bacteroides fragilis’ glycan
biosynthesis. B. fragilis were treated with 0.5-2 mM metabolic inhibitors (1-6, 8
corresponding to abundant expression of azides and consistent
with previous reports34, 69 (Fig. 6B; Fig. S5B†). Treatment with
Ac4GalNAz in tandem with 1 mM BnFucNAc (3), and to a lesser-
extent BnDAT (2), led to a substantial diminishment in the
fluorescence intensity that was comparable to the Ac4GlcNAc
control (Fig. 6B) and occurred in a concentration-dependent
manner (Fig. S5D†). In contrast, relatively subtle shifts in
fluorescence intensity inconsistent with concentration-
dependent inhibition were observed with the other analogues
tested (Fig. 6B, Fig. S5C†). These data indicate that
modifications in B. fragilis glycan biosynthesis can be induced
by treatment with the metabolic inhibitors BnDAT (2) or
BnFucNAc (3). Taken together, the extent of glycan disruption
appears to be dependent on both the bacterial strain explored
and the choice of metabolic inhibitor. Thus, tailoring of glycans
in a bacteria-selective manner should be possible.
)
and 0.1 mM Ac4GalNAz for three days, with 0.1 mM Ac4GalNAz (Gal or GalNAz) in
the absence of inhibitor, or with the azide-free control sugar Ac4GlcNAc (Ac), then
probed for the presence of azide-labeled glycans within lysates or on cells. (A)
Lysates from treated cells were reacted with 250
detected with anti-FLAG antibody via Western blot analysis. (B) Treated cells were
reacted with 20 M AF488-DBCO and fluorescence intensity of each cell
µM Phos-FLAG and subsequently
µ
population was measured by flow cytometry analysis.
was observed in C. jejuni treated with Ac4GalNAz, whereas
control samples from cells treated with the azide-free
compound Ac4GlcNAc benchmark background antibody binding
that occurs in an azide-independent manner (Fig. 5A). None of
the metabolic inhibitors elicited an appreciable concentration-
dependent diminishment of the azide-labeled glycan profile
(Fig. 5A) nor an alteration in protein expression (Fig. S4A†).
Moreover, an analysis of C. jejuni’s growth (Fig. S4B†), motility
(Fig. 5B) and biofilm formation (Fig. 5C; Fig. S4C†) yielded
comparable fitness for untreated C. jejuni versus cells treated
with 2 mM BnBac (1), BnDAT (2), F-Bac (4), F-DAT (5) F-FucNAc
(6), or BnGlcNAc (7). These data indicate that metabolic
inhibitors act in a cell-selective manner; BnBac (1) and F-DAT (5)
elicit glycosylation and phenotypic effects in H. pylori yet
appears to have a negligible effect on C. jejuni. Therefore,
bacteria-selective effects are possible with metabolic glycan
inhibitors. This result suggests the potential for narrow-
spectrum approaches to sensitize particular bacterial
populations for destruction.
Discussion
Bacterial glycans are intriguing molecular targets as they
contain rare monosaccharides that are expressed on a small
number of bacterial pathogens, are absent from human cells,
and are frequently linked to pathogenesis.1, 3, 70 Thus, expanding
the repertoire of tools to experimentally modulate their
biosynthesis should be a high priority. Such efforts will aid in
defining the functions of these molecules and have the
potential to guide the development of urgently needed
antibiotics. Although systematic efforts to screen for inhibitors
of glycan biosynthesis have been undertaken in mammalian
systems71-74, there is a relative paucity of bacterial glycosylation
inhibitors. The progress that has been made in this area has
relied on in-depth characterization of glycosyltransferases,75-78
information that is often a rate-limiting step in inhibitor
development. As an alternative to direct screening of
With compelling evidence of bacteria-selective effects, we
next explored whether analogues impact glycan biosynthesis in
the Gram-negative gut symbiont Bacteroides fragilis. This
information is critical to assess the potential undesired impacts
of glycan modulators on innocent bystanders. Based on the
previous demonstration by Kasper and coworkers that B. fragilis
robustly incorporates Ac4GalNAz into polysaccharide A,69 we
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glycosyltransferases, Vocadlo,42 Paulson44 and others21, 45 have
Chemical Science
Small molecule-induced functional defectsVieiwnArtgicrleoOwnltinhe,
demonstrated the potential of unnatural monosaccharides or motility, and biofilm formation – threeDaOtIt:r1i0b.1u0t3e9s/Cw9ShCic0h595a5rEe
glycosides bearing key structural features (e.g. O-benzyl absolutely critical for establishment and maintenance of
glycosides, fluorinated analogues) that act as global metabolic infection within a host – suggest the potential functional
inhibitors of glycosylation within mammalian cells. Inspired by importance of H. pylori’s general protein glycosylation
these precedents, we produced a series of benzyl glycosides and machinery and ensuing suitability as a drug target. It is well
fluoro-containing analogues of rare bacterial monosaccharides established that flagellin glycosylation is a virulence factor in H.
to metabolically inhibit bacterial glycan biosynthesis.
pylori, as targeted genetic disruption of the dedicated flagellin
Here we explored metabolic oligosaccharide engineering as protein glycosylation system impedes flagella formation,
an approach to guide the design and discovery of substrate- motility, and colonization of the host.9 In contrast, the link
based metabolic inhibitors of bacterial glycosylation. Our data between H. pylori’s general protein glycosylation system and
indicate that established mammalian glycosylation inhibitors virulence is not known, as the general protein glycosylation
have minimal effect on glycan biosynthesis and fitness in H. system is not well characterized. Metabolic inhibitor-induced
pylori, C. jejuni, and B. fragilis, yet metabolic inhibitors based on functional defects, coupled to those induced via genetic
rare bacterial monosaccharide scaffolds elicit species-selective interruption of H. pylori’s general protein glycosylation system
defects in glycan biosynthesis and function. For example, BnBac
(DGT, Fig. 4), provide the first evidence that H. pylori’s general
(1), BnFucNAc (3), and F-DAT (5) dramatically reduced H. pylori’s protein glycosylation system may be a virulence factor.
ability to synthesize glycoproteins and led to diminished Metabolic inhibitor-induced functional defects appeared even
growth, motility, and biofilm formation. In contrast, these more striking than those induced via genetic interruption. This
analogues had no effect on glycan biosynthesis or fitness in C. observation could stem from the differential glycan
jejuni, while BnFucNAc (3) and an entirely different analogue, biosynthesis disruption caused by metabolic inhibitors relative
BnDAT (2), led to subtle changes in B. fragilis glycosylation. to genetic glycosyltransferase deletion. Metabolic inhibitors
These data indicate that the choice of metabolic inhibitor can might interrupt glycan biosynthesis at an earlier point in
be tailored to elicit strain-selective effects in bacteria of biosynthesis. Thus, metabolic inhibitors have the potential to
interest. Thus, these compounds are useful probes and have the yield insightful information, complementary to genetics
potential to form the basis of new glycosylation-based experiments, about the suitability of glycan biosynthesis as a
strategies to interfere with bacterial pathogenicity.
virulence factor and potential drug target.
The differential effect of BnBac (1), BnDAT (2), BnFucNAc
With metabolic inhibitors as probe compounds, it is now
(3), and F-DAT (5) in bacterial strains could reflect underlying possible to query how changes in glycan structure precipitate
variation in glycosylation machinery present within each altered function. Molecular-level information concerning how
species. H. pylori, C. jejuni, and B. fragilis use some of the same glycan structure changes in the presence of metabolic inhibitors
monosaccharide building blocks, yet the structures of the will be critical to gain incisive information on the structure-
glycans produced by these bacteria and the glycosyltransferases function relationship. Recent reports by Paulson and
that stitch these higher order glycans together diverge.79-82 For coworkers44 and Wang et al.,45 for example, indicate successful
example, although H. pylori and C. jejuni have analogous strategies for characterizing the effects of metabolic inhibitors
dedicated flagellin glycosylation systems, in which pseudaminic on cellular glycosylation in mammalian systems that should
acid is installed onto serine and threonine residues on flagellin translate to bacterial systems. Moreover, molecular-level
proteins,1, 3 their general glycosylation systems differ.79, 82 These evidence will reveal the mechanism by which these analogues
differences have manifested in species-selective utilization of inhibit bacterial glycosylation. Benzyl glycosides likely act as
azidosugars in metabolic labeling experiments.34 Metabolic substrate decoys that divert glycan biosynthesis onto mock
inhibitors may exploit inherent differences in glycan substrates; buildup of glycans on benzyl glycosides within cells
biosynthesis, such as glycosyltransferase substrate specificity would support this hypothesis. By contrast, fluoro analogues
and relative permissiveness for unnatural substrates, to induce are presumably activated in situ as unnatural nucleotide sugars
species-selective glycosylation defects. Metabolic inhibitors that then directly (via glycan installation) or indirectly (via
that impact glycosylation could be leveraged to yield insight into feedback inhibition) terminate glycan biosynthesis; an analysis
glycan composition and assembly. This information could, in of nucleotide sugar pools and of fluoro-sugar fate within treated
turn, provide information about how glycan structure correlates cells would yield insight into these possible mechanisms.
to function.
Glycan-disrupting analogues have the potential to disarm
Some surprising effects were observed in light of what is bacteria, rendering them more vulnerable to host immune
documented in the literature. For example, C. jejuni defenses, sensitized to existing antibiotics, and susceptible to
glycoproteins have bacillosamine at the core,78 yet BnBac had competition with other microflora.83, 84 Based on precedents in
no effect on this bacteria’s protein glycosylation. Likewise, B. mammalian systems, metabolic inhibitors modulate
fragilis contains GalNAc in polysaccharide A,80 yet BnGalNAc glycosylation on cells in vitro and in animal models.50 Therefore,
had no appreciable effect on PSA biosynthesis. These the compounds developed here could be deployed in animal
observations reveal unknowns worthy of further exploration, infection models to perturb glycans in complex microbial
including relative substrate promiscuity of glycosyltransferases communities. Ultimately, this strategy has the potential to lead
and carbohydrate uptake mechanisms.
to the development of a narrow-spectrum glycosylation-based
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antibacterial strategy that could treat priority pathogen cytometry or lysed for subsequent Western bloVtieawnAartlicylseiOs,nlianes
DOI: 10.1039/C9SC05955E
infections while minimizing the deleterious effects on beneficial described below.
bacteria.
Western Blot. Lysates from metabolically labeled cells were
standardized to a protein concentration of ~2 mg mL-1 and
reacted 1:1 with 500
detection of azide-labeled glycans. Reacted lysates were loaded
onto 12% Tris–HCl SDS-PAGE gel, separated by
µ
M Phos-FLAG63 overnight at 37 °C for
Conclusions
a
Rare bacterial sugars and associated biosynthetic machinery
have untapped potential as targets of selective interference.
Despite their appeal, no general strategy has been developed to
block the biosynthesis of elaborated bacterial glycans. This work
represents the first demonstration, to our knowledge, of using
MOE and unnatural monosaccharides to globally inhibit the
bacterial glycome. While this work focused on a small panel of
metabolic inhibitors, the general approach presented here will
pave the way for the development of a broad array of metabolic
inhibitors to study and alter bacterial glycans in diverse species.
Indeed, metabolic inhibitors based on the plethora of
exclusively bacterial monosaccharides have the potential to
form the basis of tailor-made probe compounds that provide
insight into the functional importance of bacterial glycans and
serve as agents that selectively disarm bacteria within a mixed
population. Broadly, this work introduces new approaches to
study and harness glycans of priority pathogens to create
urgently needed antibiotics.
electrophoresis, and transferred to nitrocellulose paper. Anti-
FLAG-HRP was employed to visualize FLAG-tagged proteins via
chemiluminescence.
Lectin Binding. H. pylori treated with 2 mM inhibitor or with no
inhibitor (wildtype) for 4 days were probed with Alexa Fluor
488-conjuaged Concanavalin A (ConA), then analysed by flow
cytometry on a BD Accuri C6 (BD Biosciences) instrument. As a
negative control, ConA was pre-incubated with 400 mM
mannose (carbo-block) prior to binding to untreated H. pylori.
Flow cytometry of B. fragilis. Metabolically labeled intact B.
fragilis were reacted with Alexa Fluor Dye 488 DBCO (AF488-
DBCO, 20 µM) for 5 hours in the dark for strain-promoted azide-
alkyne cycloaddition detection of azides (Click Chemistry Tools;
ex: 488/em: 519). Cells were analysed by flow cytometry on a
BD Accuri C6 (BD Biosciences) instrument, with 30,000 live cells
gated for each replicate experiment.
Growth curves. Growth was measured over the course of 3-5
days, as indicated. Bacteria treated were inoculated at a starting
OD600 of 0.1-0.3 into culture tubes containing 3 mL of rich media
along with 1 mM (H. pylori) or 2 mM (C. jejuni) of the indicated
inhibitor (1-10). Cultures were kept at 37 °C and 14% CO2 with
gentle shaking. The OD600 of each culture was measured using
spectrophotometry at the indicated timepoints. We note that
the relative change in OD600 depended upon whether cultures
were started in the exponential phase of the growth curve or
closer to saturation.
Experimental
Materials and Chemical Synthesis. Organic chemicals and anti-
FLAG antibodies were purchased from Sigma-Aldrich unless
otherwise noted. 4F-GlcNAc (9) and 4F-GalNAc (10) were
acquired from Sussex Research. H. pylori strain G2785 was a gift
of Manuel Amieva (Stanford University) and protein
glycosylation mutant
(DGT), H. pylori G27 insertionally
Motility assays. The ability of the bacteria to swarm in the
presence of compounds 1-8 was monitored over the course of
4-8 days. Bacteria were standardized to an OD600 of 1.0, then
incubated in rich media containing 1 mM (H. pylori) or 2 mM (C.
jejuni) of the indicated inhibitor (1-8) or no inhibitor overnight
at 37 °C and 14% CO2 with gentle shaking. An aliquot of the
overnight culture was concentrated by centrifugation and
resuspension in media, and 10 μL of the concentrated
suspension was plated into soft agar plates supplemented with
4% agar and 10% fetal bovine serum. Plates were incubated at
37°C and 14% CO2, and colony diameter was measured daily.
Biofilm formation assays. Biofilm formation for bacteria
cultured in the presence versus absence of 1 mM (H. pylori) or
2 mM (C. jejuni) compounds 1-8 was measured after 4 days
following to a literature protocol.88 Briefly, bacteria were
standardized to an OD600 of 1.0 in rich media containing 2 mM
compounds 1-8 or no inhibitor, plated in triplicate in the side
wells of a 96 well plate, and incubated at 37 °C and 14% CO2.
Following 4 days of growth, media was removed from the wells
and the remaining biofilm was stained with 0.15% crystal violet.
Sideview images of the triplicate wells were taken after staining.
inactivated in HPG27_580 with a chloramphenicol resistance
cassette,86 was a gift from Nina Salama (Fred Hutchinson Cancer
Research Center). Other bacterial cells (C. jejuni ATCC 33560; B.
fragilis ATCC 23745) were purchased from ATCC and grown
according to the supplier’s instructions. Ac4GlcNAc, Ac4GlcNAz,
Ac4GalNAz and Phos-FLAG were synthesized as previously
described.26, 87 Compounds 1-6 were synthesized using standard
organic chemistry procedures and characterized by standard
1
techniques including H and 13C NMR spectroscopy and mass
spectrometry. Compounds 1-6 were purified using flash silica
gel chromatography.
Metabolic Labeling. Bacterial cells were grown in rich liquid
media supplemented with 0.5 mM Ac4GlcNAz, Ac4GalNAz, or
the azide-free control Ac4GlcNAc for 2–4 days, depending on
the doubling rate of the organism. H. pylori was metabolically
labeled for 4 days under microaerophilic conditions (14% CO2,
37 °C), C. jejuni was metabolically labeled for 3 days under
microaerophilic conditions, and B. fragilis was metabolically
labeled for 2 days under anaerobic conditions (created by a
Thermo Scientific Oxoid AnaeroGen Sachet in an airtight
container; 37 °C) using 0.1 mM Ac4GalNAz according to previous
literature.69 Cells were then harvested, washed with phosphate
buffered saline (PBS), and whole cells were analyzed by flow
Conflicts of interest
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PleaseCdhoemnoictaal Sdcjuiesntcme argins
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Varki, R. D. Cummings, J. D. Esko, P. Stanley, G. W. Hart, M.
There are no conflicts to declare.
View Article Online
Aebi, A. G. Darvill, T. Kinosita, N. H. PacDkeOrI:, 1J0. .H10.3P9r/eCs9tSeCg0a5r9d5,5RE.
L. Schnaar, P. H. Seeberger, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor (NY), 2009.
Acknowledgements
22 Y. Yuan, S. Fuse, B. Ostash, P. Sliz, D. Kahne and S. Walker, ACS
Chem. Biol., 2008, 3, 429-436.
We gratefully acknowledge insightful conversations with A.
McBride and B. Kohorn, the technical support provided by K.
Moulton, and members of our research laboratories for support
and guidance. Research reported in this publication was
supported by the National Institutes of Health under grant
number R15GM109397 to D.H.D., an Institutional Development
Award (IDeA) from the National Institute of General Medical
Sciences of the National Institutes of Health under grant
number P20GM103423, and by awards to D.A.W. from the
James Stacy Coles Fellowship. S.S.K. thanks the Science and
Engineering Research Board (grant No.CRG/2019/000025), the
ISF-University Grants Commission (UGC) joint research program
framework (grant No. 2253) and Department of Biotechnology
(BT/INF/22/SP23026/2017) for financial support. K.P. and A.P.
thank Indian Institute of Technology for fellowships.
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DOI: 10.1039/C9SC05955E
Metabolic inhibitors based on rare bacterial monosaccharide scaffolds interfere with bacterial glycan biosynthesis in a bacteria-
selective manner.