Effect of the lipid II sugar moiety on bacterial transglycosylase: the 4-hydroxy epimer of lipid II is a TGase inhibitor

Article abstract of DOI:10.1039/c6cc07871k

Lipid II analogues bearing major modifications on the second sugar (GlcNAc) were synthesized and evaluated for their substrate activity toward TGases. Unexpectedly, N-deacetyled lipid II decreased its activity dramatically, and the C4-axial OH lipid II became an inhibitor (IC₅₀ = 8 μM) with an approximately 14-fold increase in binding affinity toward TGase (25 vs. 27).

Full text of DOI:10.1039/c6cc07871k

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Effect of the Lipid II Sugar Moiety on Bacterial
Transglycosylase: the 4-Hydroxy Epimer of Lipid II is a TGase
Inhibitor
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Kuo-Ting Chen, Cheng-Kun Lin, Chih-Wei Guo, Yi-Fan Chang, Chia-Ming Hu, Hsiao-Han Lin, Yuting
Lai, Ting-Jen R. Cheng and Wei-Chieh Cheng*
www.rsc.org/
Lipid II analogues bearing major modifications on the second sugar moiety. Structure-activity relationship studies of Lipid II
(GlcNAc) were synthesized and evaluated for their substrate towards TGase are important to both establish minimum
activity toward TGases. Unexpectedly, N-deacetyled Lipid II structural requirements of Lipid II for transglycosylation, and
decreased its activity dramatically, and the C4-axial OH Lipid II inspire novel, substrate-based, TGase inhibitors.5-7 To date,
became an inhibitor (IC50 = 8 μM) with an approximately 14-fold only straightforward and easy structural modifications on Lipid
increase in binding affinity toward TGase (25 vs. 27).
II, including MurNAc (the first sugar), lipid chain, peptide
chains, have been studied previously by us and others.5,6,8,9
Their TGase substrate studies have shown that Lipid II
The rise of antibiotic drug resistant bacteria such as MRSA
(methicillin-resistant Staphylococcus aureus) has stimulated
the development of new antibiotics.1 Peptidoglycan, a major
component in the bacterial cell wall, is composed of long
glycan chains cross-linked by short peptides to form a mesh-
like structure, and is essential for bacterial shape and growth.2
Among those enzymes involved in bacterial cell-wall
(peptidoglycan) biosynthesis, transglycosylases (TGases)
catalyze the transfer of the sugar moiety from the activated
Lipid II or polymeric peptidoglycan such as Lipid IV (a glycosyl
donor) to the specific hydroxyl group (4-OH) of Lipid II (a
glycosyl acceptor), with concomitant release of an
undecaprenyl pyrophosphate moiety (Figure 1).3 These
enzymes are attractive antibiotic targets since their functions
are essential and; their structures are conserved; and they are
located on the external surface of bacterial membranes, which
should allow easy access by inhibitors.2 To date, moenomycin
A (MoeA) and its analogues are the only known naturally
occurring antibiotics that target the glycosyl donor site in
TGase and disable peptidoglycan biosynthesis, but its poor
pharmacokinetic properties preclude its clinical use.4
derivatives bearing a shorter peptide (only L-alanine) or a
shorter lipid chain containing a fluorophore tag can still be
recognized by substrates with reasonable activities.5,6 These
preliminary results suggest that not only the complex structure
of natural Lipid II is readily amenable to simplification, but also
we are able to use this simplified Lipid II as a molecule
template to investigate the difficult but important task: what is
the effect of GlcNAc (the second sugar moiety) in Lipid II
toward TGase?
Lipid II is a structurally unique and complex molecule,
Fig. 1 TGase-catalyzed formation of peptidoglycan from Lipid II.
consisting of
a
disaccharide (GlcNAc-MurNAc);
a
Bespoke Lipid II analogues are a prerequisite for the
investigation of TGase substrate specificity, but known chemo-
enzymatic approaches are still limited.10,11 Chemical synthesis,
in contrast, allows the systematic generation of analogues for
testing.11-13 We have a long-standing interest in developing the
synthetic methodology necessary to prepare peptidoglycan
fragments and precursors as well as investigating their
biological functions.5,14,15 Herein, we describe the design and
synthesis of a series of Lipid II analogues wherein the structure
of the second sugar (GlcNAc) moiety is varied. These modified
pyrophosphate; an undecaprenol lipid tail; and a pentapeptide
a.Genomics Research Center, Academia Sinica, 128, Section 2, Academia Road,
Taipei, 11529, Taiwan; Fax: (+886)2-2789-8771; E-mail:
wcheng@gate.sinica.edu.tw.
b.Department of Chemistry, National Cheng Kung University, 1, University Road,
Tainan City, 701, Taiwan.
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Lipid II derivatives were tested, to see if they still function as group in the subsequent transformations. Glycosylation
TGase substrates, and/or are suitable for the development of between donor 1719 and acceptor 15 gave disaccharide 18 in a
TGase probes or inhibitors.
reasonable yield (70%). Selective N-Troc deprotection of 18
, which consist of a was performed under mild conditions (Zn/acetic acid),20
Figure 2 depicts Lipid II analogues
1–
5
disaccharide (modified GlcNAc-MurNAc), a pyrophosphate, a followed by N-acetylation (TFAA in pyridine) to obtain
shorter C27 polyprenol (five cis-isoprene units) bearing a disaccharide in 82% over two steps. With N-TFA protected
fluorescent group (NBD) instead of an undecaprenyl lipid tail in hand, Lipid II analogue was smoothly prepared (35%, from
(C55)8, and the simplified peptide, -alanine alone.5 The
over six steps, Scheme 1).
9
9
5
L
9
fluorophore NBD was deliberately incorporated at the end of
the lipid to allow quantitative monitoring of substrate
consumption in our HPLC-based TGase activity assay.8,12
Fig. 2 Synthetic Lipid II analogues for this substrate activity study.
Scheme 2 Preparation of disaccharides 8 and 9. Reagents and conditions: (a)
NIS, TMSOTf, CH2Cl2, -78 oC, 2 h, 86% for 16, 70% for 18; (b) i. TFA, CH2Cl2, ii.
hydrazine acetate, MeOH, 60 oC, iii. Ac2O, pyridine, 53% over three steps; (c)
i. Zinc dust, HOAc, CH2Cl2, ii. TFAA, pyridine, 82% over two steps.
A general route to prepare Lipid II analogues
from the corresponding key intermediates was shown in
Scheme 1. Debenzylation of , followed by the phosphoryl
diester formation got 10 13, respectively. And, a sequence of
1, and 3–5
6–9
6–9
We next turned our attention to the preparation of azido-
–
debenzylation, pyrophosphate formation16,17 (conjugation with
Lipid II 2. As depicted in Scheme 3, 20 was converted to 21
with a sequence of steps including removal of the benzylidene
moiety, selective tosylation at the C6 position,21 de-
a fluorophore-labeled lipid phosphate (NBD-C27P), and global
deprotection by catalytic hydrogenation of 10
–
13 was
phthalimidation, and acetylation. After debenzylation of 21
,
performed to furnish the desired Lipid II analogues
(Scheme 1).
1
, and
3–5
followed by phosphitylation/oxidation, 22 was obtained in 67%
over three steps. To avoid azido reduction during catalytic
hydrogenation,21 installation of the azide moiety on 22 was
performed after debenzylation. The azido-disaccharide
intermediate was conjugated with NBD-C27P, and deprotected
under basic conditions to furnish 2.
Scheme 3 Preparation of Azido-Lipid II 2. Reagents and conditions: (a) i.
PTSA, MeOH, 60 °C, 0.5 h; ii. TsCl, pyridine, 0 °C, 8 h, iii. hydrazine acetate,
MeOH, 80 oC, 15 h; iv. Ac2O, pyridine, rt. 4 h, 43% over four steps; (b) i.
Pd(OH)2/C, H2, MeOH, ii. (i-Pr)2NP(OBn)2, 1H-tetrazole, CH2Cl2, iii. t-BuOOH, -
78 °C, 67% over three steps; (c) i. Pd(OH)2/C, H2, MeOH, 95%, ii. NaN3, DMF,
78%, iii. NBD-C27P, CDI, CH2Cl2, 1H-tetrazole, DMF, iv. LiOH, MeOH, 25%
over four steps.
Scheme 1 A General Route for the Synthesis of Lipid II Analogues 1, and 3–5.
Reagents and conditions: (a) i. Pd(OH)2/C, H2, MeOH, ii. (i-Pr)2NP(OBn)2, 1H-
tetrazole, CH2Cl2, iii. t-BuOOH, -78 °C, 62-87% over three steps; (b) i.
Pd(OH)2/C, H2, MeOH, 95%, ii. NBD-C27P, CDI, CH2Cl2, 1H-tetrazole, DMF, iii.
LiOH, MeOH, 41–47% over three steps.
Notably, key intermediates
-glucosamine following the similar approach previously
described.8 Preparation of other two disaccharides
and was
6 and 7 were synthesized from
D
8
9
clearly described in Scheme 2. Glycosylation between donor
1418 and acceptor 15 mediated by TMSOTf afforded the
corresponding disaccharide 16 (86%), followed by
deprotection of the benzylidene and N-phthalimide moiety
Fig. 3 Evaluation of the substrate activity of Lipid II analogues 1–5 toward
TGase. Time course of consumption of 1–5 (30 μM) by C. difficile PBP1b (2.5
μg/mL) without (A) or with (B) Lipid IV (20 μM).
and then global acetylation to give
8. To prepare disaccharide
9, the N-Troc protected donor 17 was applied instead of N-
Phth protected 19 in order to selectively manipulate the amino
2 | J. Name., 2012, 00, 1-3
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With fluorescent Lipid II analogues
1
–
5
in hand, we highly conserved in many bacterial species,25-27 the inhibition
26 against C. difficile TGase (PBP1b) were also
embarked on the study of their TGase (C. difficile PBP1b) study of 23
–
substrate activity using an HPLC-based activity assay to performed. Our results show that the lipid modification more
measure the initial rate of the consumption of Lipid II in the significantly affects inhibitory potency than the peptide part
absence or presence of Lipid IV. According to our previous (Table 1).
study, Lipid IV binds only at the TGase donor site (C. difficile
Table 1 Inhibition activity of Lipid II analogues against TGases.
PBP1b) and it alone cannot undergo self-elongation in
transglycosylation.12 In contrast, an enzymatic reaction occurs
OH
O
OH
O
HO
HO
only when the reaction mixture contains Lipid II alone or a
mixture of both Lipid II and Lipid IV.22 As expected, when the
reaction mixture contained a structurally simplified Lipid II
23, Lipid = undecaprenyl,
R = -iso-Glu- -Lys- -Ala-D-Ala
24, Lipid = undecaprenyl, R = OH
Lipid
25, Lipid = tetraprenyl,
O
D
L
D
O
R
O
P
O
P
AcHN
AcHN
O
O
O
OH OH
R =
D-iso-Glu-
L-Lys-D-Ala-
D-Ala
HN
analog
1 alone, it functioned as a TGase substrate, with an
26, Lipid = tetraprenyl, R = OH
initial consumption rate of 0.35 μM/min. The consumption
rate increased to 2.60 μM/min when Lipid IV was added (Lipid
O
IV /
1
= 1:1.5).
To our delight, analog
substrate with a consumption rate of 0.87 μM/min in the
presence of Lipid IV, which implies incorporates into
Compound
23
8
18
24
15
34
25
77
248
26
80
246
2
was also recognized as a TGase
E. coli TGase
C.difficile TGase
IC50 (µM)a
a Experiments were performed in 0.085% decyl-PEG, 50 mM Tris-HCl, pH 8.0,
10 mM CaCl2, 10% DMSO, 15% MeOH, 10 μM NBD-Lipid II and E. coli PBP1b
or C. difficile PBP1b at 37 °C.
2
peptidoglycan biosynthesis without affecting an oligopeptide
or cross-linking peptides.23 Analog
was significantly less active than
4, the C3-dehydroxylated
1,
1, even after a prolonged
To quantitatively measure the impact of C4-OH
epimerization on the second sugar in Lipid II analogues, a
surface plasmon resonance (SPR) analysis was used to
determine the difference in binding affinity resulting from
structural modifications. Inspired from our previous structural
studies, we planned to use MoeA to occupy the glycosyl donor
site and evaluate the relative binding affinity of the molecules
tested at the acceptor site.27,28 To verify our hypothesis, a
preliminary molecular competitive examination was
performed by a fluorescence polarization (FP) assay with using
FITC-labeled MonA analog as the probe against TGase.18 The
results show that FITC-moenomycin probe cannot be replaced
by 25 at high concentration (up to 1 mM), importantly
indicating that 25 binds to the acceptor site but not the donor
site of TGase.
period of time (2 h), or with prior incubation with Lipid IV.
Analog , without the N-acetyl group at C2 on GlcNAc of
5
1,
resulted in significant loss of its TGase substrate activity. N-
Deacetylated peptidoglycans have been reported to resist
degradation by the host lysozyme, but it is still unclear when
N-deacetylation of the second sugar (GlcNAc) occurs.24 Since
5,
the N-deacetylated Lipid II, is an unsuitable TGase substrate,
we reasoned that N-deacetylation should occur after the
transglycosylation stage in the peptidoglycan forming process.
Compound 3, the GalNAc-MurNAc typed analog, was not
itself a TGase substrate, even in the presence of Lipid IV (see SI,
Fig S1). To confirm this, the analog S4 (GalNAc-MurNAc type)
bearing a longer polyprenyl lipid chain was applied (see in SI,
Fig S3). Our result showed that it alone cannot be a TGase
substrate. Besides, it could not be consumed in the presence
of normal Lipid II (up to 1000 μM) in our HPLC-based TGase
activity analysis, suggesting it does not bind to the glycosyl
donor site during the peptidoglycan elongating process (see in
SI, Fig S3).
For the SPR study, to reduce non-specific hydrophobic
interactions, the shorter C20 lipid was employed,5 and 25
, 28,
29 (GalNAc-MurNAc type) and 27 (GlcNAc-MurNAc type) were
synthesized using the procedure above.29 Our results are
shown in Table 2 (also see in SI). Analog 25 with a C4 axial OH
(the C4 epimer of 27), had a KD value with 33 μM. The KD value
of 27 was 445 μM. The results show that changing the
orientation of the C4-hydroxy group on GalNAc in Lipid II not
only mechanically interrupts the transglycosylation progress,
but also significantly increases the binding affinity (14-fold
increase) to TGase. Analog 28, lacking the pyrophosphate-lipid
moiety, exhibited a very weak binding affinity (KD = 778 μM) to
Since
curious whether it could function as a TGase inhibitor. Four
GalNAc-MurNAc typed analogues 23 26 bearing different lipid
3 turned out not to be a TGase substrate, we were
–
chain lengths (C20 or C55) and oligopeptides (pentapeptide or
monopeptide) were synthesized (see detailed experiments in
SI) to examine their inhibitory activities against E. coli TGase
(Table 1). All four were found to be moderate to potent
inhibitors of TGase, with IC50 values ranging from 8 to 80 μM,
with inhibitors bearing longer lipid chains exhibiting the best
inhibitory potency. For example, 23 or 24 bearing a longer C55
lipid were more potent inhibitors than 25 or 26 bearing a
shorter C20 lipid. The IC50 value of 23 was 8 μM, about 9-fold
more potent than that of its cognate 25 (IC50 = 77 μM). Similar
activity relationships were also observed between 24 and 26.
In contrast, when the peptide chain of 23 or 25 was truncated
to give the corresponding 24 or 26, respectively, no significant
decrease in inhibitory activity was observed. Since TGases are
the enzyme. In addition, no binding signal was observed for 29
,
lacking both the pyrophosphate-lipid moiety and oligopeptide
of 25. This quantitative data demonstrates the importance of
the orientation of the hydroxyl group on the second sugar in
Lipid II for enzyme recognition. We also illuminated the
important role played by the pyrophosphate-lipid moiety in
Lipid II-enzyme binding, even though this information cannot
be inferred from the previous co-crystal structure.27
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Table 2 Dissociation constant (KD) values for Lipid II analogues toward C.
difficile TGase (PBP1b).
6
7
8
X.-Y. Ye, M.-C. Lo, L. Brunner, D. Walker, D. Kahne, S. Walker, J. Am.
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F.-C. Meng, K.-T. Chen, L.-Y. Huang, H.-W. Shih, H.-H. Chang, F.-Y.
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OH
O
OH
O
OH
O
OH
O
HO
HO
O
O
HO
HO
O
O
AcHN
O
AcHN
O
AcHN
AcHN
R1
R1
9
R2
R2
1
25
28
27
, R = tetraprenyl pyrophosphate;
, R1 = tetraprenyl pyrophosphate;
R2
1
=
-Ala- -iso-Glu- -Lys- -Ala- -Ala
R2
L
D
L
D
D
L D L D D
= -Ala- -iso-Glu- -Lys- -Ala- -Ala
, R = OH;
10 E. Breukink, H. E. van Heusden, P. J. Vollmerhaus, E. Swiezewska, L.
Brunner, S. Walker, A. J. R. Heck, B. de Kruijff, J. Biol. Chem. 2003,
278, 19898.
R2
=
L-Ala-
D
-iso-Glu-
-Ala
L-Lys-D-Ala-D-Ala
29, R1 = OH; R2
= L
11 K.-T. Chen, Y.-C. Kuan, W.-C. Fu, P.-H. Liang, T.-J. Cheng, C.-H. Wong
and W.-C. Cheng, Chem. Eur. J., 2013, 19, 834.
12 H.-W. Shih, K.-T. Chen, T.-J. Cheng, C.-H. Wong and W.-C. Cheng,
Org. Lett., 2011, 13, 4600.
13 M. S. VanNieuwenhze, S. C. Mauldin, M. Zia-Ebrahimi, B. E. Winger,
W. J. Hornback, S. L. Saha, J. A. Aikins, L. C. Blaszczak, J. Am. Chem.
Soc., 2002, 124, 3656.
Compound
25
33 ± 6.5
27
28
29
KD (μM)a
445 ± 19.9 778 ± 21.1
NBb
a Experiments were performed in the presence of 1 μM MonA (KD = 600
nM) in triplicate at 25 °C, and the KD values were determined by using
steady-state affinity. bNB refers no significant binding signal was
observed.
In summary, a series of Lipid II analogues were designed
and synthesized to investigate the effect of the GlcNAc moiety
of Lipid II on TGase. Special attention was given to structural
modifications at the second sugar GlcNAc. The capacity of
these molecules to serve as TGase substrates was tested, and
several important results were obtained. Firstly, the N-acetyl
group on GlcNAc was found to play an important role in
14 S.-H. Huang, W.-S. Wu, L.-Y. Huang, W.-F. Huang, W.-C. Fu, P.-T.
Chen, J.-M. Fang, W.-C. Cheng, T.-J. Cheng and C.-H. Wong, J. Am.
Chem. Soc., 2013, 135, 17078.
15 K.-T. Chen, P.-T. Chen, C.-K. Lin, L.-Y. Huang, C.-M. Hu, Y.-F. Chang,
H.-T. Hsu, T.-J. R. Cheng, Y.-T. Wu, W.-C. Cheng, Sci. Rep. 2016,
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16 V. Wittmann and C.-H. Wong, J. Org. Chem., 1997, 62, 2144.
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17 M. S. VanNieuwenhze, S. C. Mauldin, M. Zia-Ebrahimi, B. E. Winger,
W. J. Hornback, S. L. Saha, J. A. Aikins and L C. Blaszczak, J. Am. Chem.
Soc., 2002, 124, 3656.
substrate activity
– without it, activity was found to
dramatically decrease. Secondly, the Lipid II analog bearing a
modification at the C6-position of GlcNAc still can be
recognized as a TGase substrate. This finding suggests it could
be qualified as a chemical probe for further bacterial cell wall
studies. Thirdly, a C4-epimer of Lipid II analog alone is not a
TGase substrate. In contrast, it is identified as an inhibitor and
the epimerization of the C4-OH group from GlcNAc to GalNAc
in Lipid II results in a 14-fold increase of binding affinity in our
study. Fourthly, this study resulted in the discovery of 23, the
most potent TGase inhibitor with the IC50 value of 8 μM. Taken
together, these results not only illuminate the structure
activity relationships of Lipid II and TGase, but also provide a
direction for both probe and inhibitor design; and validate the
TGase acceptor site as a target for novel antibiotics. New
applications derived from molecules discovered in this work
are ongoing, and the results will be published in due course.
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Acknowledgements
We thank Academia Sinica and Ministry of Science and
Technology (MOST) for financial support.
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