Towards new antibiotics targeting bacterial transglycosylase: Synthesis of a Lipid II analog as stable transition-state mimic inhibitor

Article abstract of DOI:10.1016/j.bmcl.2018.03.035

Described here is the asymmetric synthesis of iminosugar 2b, a Lipid II analog, designed to mimic the transition state of transglycosylation catalyzed by the bacterial transglycosylase. The high density of functional groups, together with a rich stereochemistry, represents an extraordinary challenge for chemical synthesis. The key 2,6-anti- stereochemistry of the iminosugar ring was established through an iridium-catalyzed asymmetric allylic amination. The developed synthetic route is suitable for the synthesis of focused libraries to enable the structure–activity relationship study and late-stage modification of iminosugar scaffold with variable lipid, peptide and sugar substituents. Compound 2b showed 70% inhibition of transglycosylase from Acinetobacter baumannii, providing a basis for further improvement.

Full text of DOI:10.1016/j.bmcl.2018.03.035

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Towards new antibiotics targeting bacterial transglycosylase: Synthesis
of a Lipid II analog as stable transition-state mimic inhibitor
Xiaolei Wang a, Larissa Krasnova a, Kevin Binchia Wu a, Wei-Shen Wu b, Ting-Jen Cheng b,
Chi-Huey Wong a,b,
⇑
a The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92122, USA
b Genomics Research Center, Academia Sinica, 128 Sec 2 Academia Road, Taipei, Nankang 115, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Described here is the asymmetric synthesis of iminosugar 2b, a Lipid II analog, designed to mimic the
transition state of transglycosylation catalyzed by the bacterial transglycosylase. The high density of
functional groups, together with a rich stereochemistry, represents an extraordinary challenge for chem-
ical synthesis. The key 2,6-anti- stereochemistry of the iminosugar ring was established through an irid-
ium-catalyzed asymmetric allylic amination. The developed synthetic route is suitable for the synthesis
of focused libraries to enable the structure–activity relationship study and late-stage modification of imi-
nosugar scaffold with variable lipid, peptide and sugar substituents. Compound 2b showed 70% inhibition
of transglycosylase from Acinetobacter baumannii, providing a basis for further improvement.
Ó 2018 Elsevier Ltd. All rights reserved.
Received 23 January 2018
Revised 12 March 2018
Accepted 13 March 2018
Available online xxxx
Keywords:
Iminosugar
Inhibitor
Lipid II
Transglycosylase
Synthesis
Introduction
identified (e.g., vancomycin), the direct binders of TG with potent
inhibitory activities and pharmacological properties suitable for
The increasingly common occurrences of infections caused by
the drug-resistant bacteria represent a major threat to public
health.1 The urgent demand for novel antibacterials has led to
the search for underexploited drug targets. In the past, targeting
the assembly of peptidoglycan (PG), a polymer-like structure that
helps maintain the integrity of bacteria cell and protects it from
lysis, has proven to be a successful strategy for the discovery of
antibiotics. Our group has a long-standing interest in the enzyme
transglycosylase (TG) as target, which catalyzes the polymerization
of Lipid II (1, Fig. 1A) to generate a nascent PG before it is cross-
linked by transpeptidase (Fig. 1B). First reported 50 years ago,2a
TG is still viewed as a difficult,2b albeit an attractive target.2c,2d
Located on the external surface of the cytoplasmic membrane, TG
is accessible to potential inhibitors. As TG does not have any
mammalian counterpart, it is possible to design new antibiotics
that are specific against prokaryotic pathogens. In addition,
because TG recognizes an invariant carbohydrate backbone, it
may be less susceptible to the traditional mechanisms of
resistance development.3 Although, antibacterials that inhibit Lipid
II polymerization by sequestering its substrate have been
clinical use have yet to be developed. One major effort in this
direction has been the optimization of moenomycin structure,4
the only TG-specific inhibitor known to date.
Based on recent efforts towards finding the minimal required
features of Lipid II/Lipid IV,5–7 we designed structure 2 (Fig. 1A)
as a potential transition-state mimic of the TG-catalyzed reaction.5
Compound 2 consists of iminosugar ring connected to an addi-
tional ring of GlcNAc, a truncated peptide moiety with two essen-
tial methyl groups from the lactyl-alanine sequence,5a,6a,6d along
with
a
phosphono-phosphate linked lipid chain,6b which is
necessary for the proper recognition and binding. Towards
structure 2, our group has initially reported the synthesis of the
truncated analog 2a (Fig. 1A), which indeed showed inhibition of
TG function.6b The two drawbacks of compound 2a are of note.
First, because TG is a processive enzyme,7 it is highly unlikely that
this mono-sugar derivative 2a can reach the desired donor site of
TG due to the lack of the second GlcNAc, therefore preventing
enzyme to process 2a. Hence, the observed activity could be a
result of 2a binding to the acceptor site only, and its designation
as a transition-state analog inhibitor could not be fully realized.
Second, the synthetic strategy developed for the assembly of 2a
is not suitable for the preparation of the highly functionalized imi-
nosugar 2 and its derivatives,6b required for the detailed structure–
activity relationship (SAR) study of TG inhibition. To solve the
⇑
Corresponding author at: Genomics Research Center, Academia Sinica, 128 Sec
2 Academia Road, Taipei, Nankang 115, Taiwan.
E-mail address: chwong@gate.sinica.edu.tw (C.-H. Wong).
https://doi.org/10.1016/j.bmcl.2018.03.035
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang X., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.03.035

2
X. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 1. (A) The structure of Lipid II (1) and the target iminosugar derivatives. (B) Schematic representation for the inhibition of the donor site of TG with iminosugar 2.
drawbacks of compound 2a, herein we report an optimized
asymmetric synthesis of the iminosugar 2b as a core inhibitor of
TG. Since 2b is a pseudo-disaccharide derivative of Lipid II, we
envisioned that upon binding it could be processed by the
enzyme and pulled into the donor site, where it can block any
further transglycosylation reactions (Fig. 1B). The synthetic route
developed for the assembly of 2b can be further applied for the
preparation of other analogs, e.g., 2c and 2d (Scheme 1).
The retrosynthetic analysis of the target molecule is presented
in Scheme 1. The introduction of the sugar and lipid substituents
can take place at the late stage using conventional glycosylation
with GlcNAc donor (3) and a CDI-activated lipid phosphate (4).
Scheme 1. Retrosynthetic analysis of target molecule 2.
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X. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
HO
HO
O
O
c). Im-SO2N3
d). PhCH(OMe)2
NBoc
O
NH2
Ph
NH2
a). vinylMgBr
b). 3N HCl
O
H
e). NaBH4
34% (5 steps)
10
9
(R)-Garner's
one column
aldehyde
f). PMBOH
Amberlite
IR-120(H)
PMBO
PMBO
HO
g). red-Al
75%
OH h). ClCO2Me
86% (2 steps)
OH
8a
11
OCO2Me
12
Scheme 2. Synthesis of the amine 9 and carbonate 8a.
Table 1
Screen of substrates for the Ir-catalized allylic amination reactiona.
13
14
[Ir(dbcot)Cl]2 (2 mol%)
OR1
NH2
R3
H
N
O
P
n-BuNH2 (4 mol%)
R3
+
OEt
OCO2Me
OR1 OR2
OEt
R2O
O
Ph
Ph
8a R3=CH2OPMB
N
P
9
R1, R2=CHPh
10 R1=R2=H
F
F
8b R3=CH2PO(OEt)2
8c R3=CF2PO(OEt)2
H
N
O
OEt
OEt
R1O
R2O
P
O
12 (4 mol%)
15
Entry
Amine
8
Additive
Resultb
1
2
3
4
5
6
10.HCl
10
9
9
9
8a
8a
8a
8b
8b
8c
NaH2PO4
–
–
–
NaH2PO4
–
13 (trace)
13 (trace)
13 (>95%)c
14
14
15
9
a
b
c
Standard reaction conditions: 8 (1.1 equiv.), 9 or 10 (1.0 equiv.), [Ir(dbcot)Cl]2 (2 mol%), 12 (4 mol%), n-BuNH2 (4 mol%), DMSO, 50 °C, 12 h.
Determined by LCMS.
Isolated yield.
Functional groups at positions C-3 and C-4 can be installed via dou-
ble bond functionalization of 6. Although the proposed synthesis
leads to a mannosyl-like iminosugar, the hydroxyl group R2 is well
positioned for the inversion with azide to provide the NHAc sub-
stituent R3 if required. The most challenging part is the 2,6-anti
configuration of the key iminosugar core, which could be accessed
through a ligand-controlled asymmetric allylic amination.8 The
stereo centers at positions C-5 and C-6 of amine 9 can be obtained
from (R)-Garner’s aldehyde. The dense array of functional groups,
high polarity and rich stereochemistry of the target molecule sig-
nify a substantial synthetic challenge. The most difficult steps of
our synthesis, which we successfully solved, include installation
of the 2,6-anti configuration of iminosugar, the C-P bond formation
and pyrophosphate coupling of the complex pseudo-disaccharide
to a long hydrophobic lipid chain.
The starting amine 9 was prepared on a gram-scale through a 5-
step sequence from the (R)-Garner’s aldehyde requiring only one
purification step (Scheme 2). Carbonate 8a was obtained in a high
yield via a 3-step sequence. Compounds 8b and 8c were synthe-
sized in a similar manner according to the known procedures.9
Using chiral amines 9 and 10, and different carbonates 8a-c, we
tested the conditions for the asymmetric allylic amination reaction.
The results are summarized in Table 1. During the initial screen
using 10 (or its HCl salt) and carbonate 8a (entries 1–2, Table 1),8d
we observed a trace amount of the desired product 13 together
with unreacted starting material. Because the free hydroxyl groups
might influence reactivity, we then switched to the protected
amine 9, which provided 13 in an excellent yield and selectivity.
Since it is very difficult to introduce the phosphonate group into
the highly functionalized iminosugar at the late stage,10 we
decided to test esters 8b-c under the optimized reaction condi-
tions. Unfortunately, using 8b we could only observe the product
of elimination (14). The reaction with 8c, where the acidic protons
are replaced with fluorine atoms,11 gave only a linear diene (15).
Nevertheless, the availability of intermediate 13 allowed us to
pursue the synthesis of our target molecule.
Synthesis of amine 13 was scaled up without any impact on the
stereoselectivity or yield, and afforded gram-quantities of material.
Upon switching to inert atmosphere, the loading of iridium catalyst
can be lowered to 1 mol% (Scheme 3). With the key intermediate
13, we aimed to close the ring and to introduce substituents at
positions C-3 and C-4. For the optimization of ring-closing
metathesis conditions, we screened different salts of amine 13,
which however failed to provide the cyclized product. Trifluoroac-
etamide protection of amine (16),12 however, allowed a high-yield
preparation of ene-17 using the second-generation Grubbs cata-
lyst. Crystallization of the intermediate 17 resulted in compound
18 (Scheme 3),13a a crystal structure of which confirmed the
2,6-anti selectivity of the asymmetric allylic amination step.
Next, we performed an osmium-catalyzed dihydroxylation of
ene-17, which gave diol 19a. To verify the stereochemistry of the
last step, we carried out a global deprotection to obtain 19b,
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4
X. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
OPMB
TFAc
N
NH2
O
H
a). [Ir(dbcot)Cl]2, 12
N
b). TFAA
91%
n-BuNH2, 8a
O
O
OPMB
O
Gram-scale
95% yield
Ph
O
Ph
O
Ph
9
excellent selectivity
13
16
c). Grubbs II
98%
O
O
TFAc
N
N
crystallization
O
O
OPMB
OPMB
DCM, MeOH,
EtOAc, rt
Ph
O
Ph
O
17
18
d). OsO4, NMO
e). DDQ
TFAc
N
H
N
OH
OH
f). TFA
g). NaOMe
HO
HO
HO
OH
OH
O
64%
OH
HO
Ph
O
OH
N
H
(3 steps
OH
19b
OH
19a
from 17)
ref. 13b
Scheme 3. Synthesis of the iminosugar core and determination of the absolute stereochemistry.
R
N
R2
Cbz
N
O
OPMB
OH
O
f). P(OEt)3
c). NaH, BnBr
o
Ph
O
Ph
O
OBn
130 C
H
OH
OBn
21 R2 = OPMB
22 R2 = OH
23 R2 = I
19a R1 = TFAc
20 R1 = Cbz
a). NaOMe
b). CbzCl
71% (3 steps
from 17)
d). DDQ, 91%
e). I2/PPh3/Im, 90%
O
STol
NHTroc
BnO
O
OBn
O
Cbz
Cbz
N
BnO
N
O
P OEt
OEt
P OEt
OEt
.
g). BH3 NMe3
3
OBn
Ph
O
OBn
HO
OBn
BF3.Et2O
48% (2 steps
from 23)
h). TfOH, NIS
85%
OBn
OBn
24
25
Ph
O
Ph
O
OBn
OBn
Cbz
O
O
O
O
P OH
OH
Cbz
O
H
O
i). Zn/Ac2O
H
P OEt
OEt
N
N
BnO
BnO
j). TMSBr
77%
O
NHAc
O
NHTroc
BnO
OBn
BnO
OBn
27
26
HO
HO
O
OH
HO P O
O
P OH
OH
O
k). H2
OH
4
H
O
NH
HO
Pd(OH)2
77%
(2 steps)
3
2b
NHAc
l). 4, CDI, 46%
HO
OH
28
Scheme 4. Diversification of iminosugar scaffold and synthesis of target molecule 2b.
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X. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
which matched the NMR data reported for the iminosugar scaffold
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In conclusion, we have developed an efficient route towards the
Lipid II analog 2b from the commercially available (R)-Garner’s
aldehyde. The key step, installation of the 2,6-anti-stereochemistry
of iminosugar was achieved using the iridium-catalyzed asymmet-
ric allylic amination procedure, which was optimized to the gram-
scale process. The developed route could be used to access other
Lipid II mimics, particularly 2c and 2d, which are expected to have
better binding affinities towards TG, than 2b. These structures will
serve as a template for further SAR and structural studies, hence
accelerating the development of new antibiotics.
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Acknowledgments
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14. Other known methods (e.g., TFA/TES, or TfOH/TES) for the selective opening of
benzylidene ring afforded only fully deprotected diol in our hands.
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making it unsuitable for the assignment of the stereochemistry at the anomeric
center. Nevertheless, 1H NMR of the fully deprotected intermediate 28 confirms
formation of the glycosidic b-linkage.
16. The TMSBr/pyridine/acetonitrile procedure was found to be the only effective
method for the hydrolysis of phosphonate ester. Use of DCM instead of
acetonitrile or switching to triethylamine base has led to decomposition of
starting material, whereas bulky base, such as 2,6-lutidine, was not efficient at
promoting the desired transformation.
We thank Dr. Gembicky (UCSD crystallography facility) for the
X-ray diffraction analysis of 18. Professor Timor Baasov is acknowl-
edged for the help with preparation of this communication. This
work was supported by the National Institute of Health
(AI072155), Academia Sinica (Taiwan) and the Kwang Hua
Foundation.
A. Supplementary data
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Supplementary data (procedures and characterization of com-
pounds) associated with this article can be found, in the online ver-
sion, at https://doi.org/10.1016/j.bmcl.2018.03.03.
Please cite this article in press as: Wang X., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.03.035