Design, synthesis and biological activity of novel demethylvancomycin dimers against vancomycin-resistant enterococcus faecalis

Article abstract of DOI:10.1016/j.tet.2018.04.091

The emergence of resistance to vancomycin and other glycopeptide antibiotics is a serious concern in clinical practice and has prompted intensive efforts to develop analogues that may overcome the resistance. One of major strategies to enhancing anti-vancomycin-resistant enterococci (VRE) activity emerged in recent years was connecting two vancomycin molecules by covalent linkers. Herein, we reported the design and synthesis of three different covalently linked demethylvancomycin dimers 7a-c by applying click chemistry. Interestingly, these dimers restored their activities against VRE. Furthermore, the interactions of molecules with peptidoglycan were also investigated via computer modelling.

Full text of DOI:10.1016/j.tet.2018.04.091

Accepted Manuscript
Design, synthesis and biological activity of novel demethylvancomycin dimers against
vancomycin-resistant enterococcus faecalis
Yong-Wei Jiang, Liang Xu, Wei Fu, Hua Lin, Jian-Ming Yu, Xun Sun
PII:
S0040-4020(18)30503-9
10.1016/j.tet.2018.04.091
TET 29506
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 30 March 2018
Revised Date: 24 April 2018
Accepted Date: 28 April 2018
Please cite this article as: Jiang Y-W, Xu L, Fu W, Lin H, Yu J-M, Sun X, Design, synthesis and
biological activity of novel demethylvancomycin dimers against vancomycin-resistant enterococcus
faecalis, Tetrahedron (2018), doi: 10.1016/j.tet.2018.04.091.
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Design, Synthesis and biological activity of
novel demethylvancomycin dimers against
vancomycin-resistant enterococcus faecalis
Yong-Wei Jiang,a Liang Xu,b Wei Fu,c Hua Lin,a Jian-Ming Yu,a and Xun Sun*,a
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Tetrahedron
journal homepage: www.elsevier.com
Design, Synthesis and biological activity of novel demethylvancomycin dimers
against vancomycin-resistant enterococcus faecalis
Yong-Wei Jiang,a Liang Xu,b Wei Fu,c Hua Lin,a Jian-Ming Yu,a and Xun Sun*,a
a Department of Natural Products Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Department of Chemistry of Medicinal, Natural Products West China School of Pharmacy, Sichuan University Chengdu, Sichuan 610041, china
cDepartment of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The emergence of resistance to vancomycin and other glycopeptide antibiotics is a serious
concern in clinical practice and has prompted intensive efforts to develop analogues that may
overcome the resistance. One of major strategies to enhancing anti-vancomycin-resistant
enterococci (VRE) activity emerged in recent years was connecting two vancomycin molecules
by covalent linkers. Herein, we reported the design and synthesis of three different covalently
linked demethylvancomycin dimers 7a-c by applying click chemistry. Interestingly, these dimers
restored their activities against VRE. Furthermore, the interactions of molecules with
peptidoglycan were also investigated via computer modeling.
Available online
Keywords:
Antibiotics
Demethylvancomycin
Dimer
2009 Elsevier Ltd. All rights reserved
.
Synthesis
Design
Introduction
In order to regain activity against resistant bacterial, several
approaches have emerged in recent years. Among them, chemical
modifications on vancomycin core tended to be the promising
strategy to achieve the goal. The fruitful results have been
obtained through addition of hydrophobic group to the amino-
disaccharide moieties of vancomycin. Initially, Nagarajan et al. [8]
synthesized lipophilic N-alkyl vancomycin derivatives which
were actively against vancomycin resistant bacteria. After that,
the similar principle has been applied to prepare many other
Vancomycin has long functioned as an antibiotic of last resort
for treating life-threatening infections caused by Gram-positive
pathogens, many of which are resistant to most other antibiotics.
Vancomycin targets the bacterial cell wall and inhibit
peptidoglycan (PG) biosynthesis by forming complexes with PG
[1, 2]
precursors.
The cup-shaped undersurface of the vancomycin
forms five hydrogen bonds to the D-Ala-D-Ala dipeptide
terminus of peptidoglycan.
threatened by the emergence of resistant pathogens that are
spreading rapidly and becoming a major public health concern.
Thus, extensive efforts have been made to develop analogues that
may overcome the resistance.
[3]
[9]
However, its effectiveness is
vancomycin, chloroeremomycin, and teicoplanin derivatives.
Telavancin, Oritavancin, and Dalbavancin were demonstrated as
successful examples of this principle (Figure 1). A second
approach has sought to improve the affinity of vancomycin for
D-Ala-D-Lac by covalent linking of two vancomycin molecules
(vancomycin dimer). A series of studies have shown that
vancomycin can self-associate via hydrogen bonding and
hydrophobic interactions to form specific dimers. [10, 11] This non-
covalent dimerization of glycopeptide can be beneficial for both
peptide ligand binding and bacterial cell surface binding, which
enhances its in vitro antibacterial activity.[12] However,
vancomycin only self-associates weakly in solution, and this non-
covalent dimerization of vancomycin alone is insufficient to act
Vancomycin-resistant enterococcus (VRE) mutates its
terminal peptides from D-Ala-D-Ala to D-Ala-D-Lac. This
simple replacement of an amide NH group for an ester oxygen
(NH→O) has substantially lowered its affinity to vancomycin
due to the elimination of one of the hydrogen bonds in the
vancomycin-PG precursor’s interaction complex. [4, 5] The loss of
one hydrogen bond interaction has indeed reduced 1000-fold
binding affinity of vancomycin for the mutated terminal peptides
[6]
against VRE. This prompted us to examine the effectiveness of
(D-Ala-D-Lac),
which parallelly decreased the antibiotic
[13, 14]
effectiveness. [7]
covalent dimerization.
In vancomycin structure, at least
three functional sites are accessible for covalent modification,
including a primary amine group located on the vancomycin
disaccharide structure (V), a C-terminal carboxylic acid (C), and
a secondary amino group at the N-terminus (N).
———
* Corresponding authors. E-mail address: sunxunf@shmu.edu.cn

2
Tetrahedron
ACCEPTED MANUSCRIPT
Figure 1. Structures of Vancomycin, Chloroeremomycin, Teicoplanin, Telavancin, Oritavancin and Dalbavancin
[15]
Griffin et al.
reported the covalent dimerization of
reported the study of covalent dimerizing of glycopeptide
antibiotics demethylvancomycin 1 which shows similar activity
and mode of action to vancomycin against Gram-positive
bacteria. We aimed to investigate N-N linked dimeric
demethylvancomycin derivatives by covalently linked at N-
terminal. Considering the linker rigidity and length, compounds
7a-c were designed to confirm our hypothesis. As the first time,
we reported the synthesis of 1, 4-disubstituted [1, 2, 3]-triazole
N-linked dimers 7a-c via an expedient click chemistry (Figure 2)
vancomycin by preparing a series of dimeric vancomycin
carboxamide derivatives. Interestingly, all compounds
demonstrated improved in vitro potency against strains of
enterococci which was highly resistant to vancomycin and other
glycopeptides. Sharpless et al.
3]-triazole vancomycin dimer derivatives by applying click
chemistry at C-terminal. Some derivatives showed good or better
activity against VRE compared with vancomycin. Herein, we
[16]
also synthesized several [1, 2,
.
a
b
Figure 2. a) Three functional sites for covalent modifications; b) General dimer structures.

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: a) DIEA (2.0 equiv), FmocCl (1.1 equiv), Dixane:H2O (v:v = 1:1), rt, 2.5h; b) 7-phenylheptanal (5.0 equiv),
NaCNBH3 (3.0 equiv), DIEA (2.0 equiv), DMF, rt, 2h; c) piperidine (15 equiv) in DMF, rt, 15min; d) trifluoromethanesulfonyl azide (3.0 equiv), DIEA
(2.0 equiv), CuSO4·5H2O (0.1 equiv), DMF, 0 0C, 8h.
and high yield. Worth to mention, the transformation of the
Results and Discussion
Synthesis
For the syntheses of dimers 7a-c, demethylvancomycin was
chosen as the starting material. Following previous reported
Figure 3. Structures of diazo transfer reagents: a) benzotriazol-1-yl-
[17, 18]
procedures
with slight modifications, intermediate 4 was
sulfonyl azide; b) imidazole-1-sulfonyl azide; c) trifyl azide
prepared as follows in gram scale. The N-terminal free amino
group of demethylvancomycin was protected with N-
fluorenylmethoxycarbonyl (Fmoc-Cl) to obtain intermediate 2 by
reductive alkylation with 7-phenylheptanal in the presence of
diisopropylethylamine (DIEA) and NaBH3CN. After removing
the Fmoc protecting group with piperidine in DMF at room
temperature, the crude compound 4 was obtained in 23% overall
yield. Then, click reaction [19, 20] was used to construct the dimeric
demethylvancomycin derivatives. Initially, the conversion of
amino group to azide group was problematic for this complex
amino group at V-terminal to azide was unsuccessful under these
conditions. Thus, 4 was subjected to the freshly prepared trifyl
azide (TfN3) in DMF in the presence of a catalytic amount of
[24]
Cu2SO4·H2O, the reaction was completed in 8h.
After poured
the reaction mixture into dichloromethane, the resulting
precipitate was centrifuged and then washed with a small amount
of dichloromethane to afford crude compound 5 in 80%-90%
yield (Scheme 1). This crude 5 was used directly for the next
step click reaction without further purification. As shown in
Scheme 2., monomers 6a-c were synthesized by mixing the
appropriate compound 5 with corresponding alkyne in DMF in
the presence of CuI (0.1 equiv).
molecule. Several diazo transfer reagents were explored, such as
[21]
benzotriazol-1-yl-sulfonyl azide,
imidazole-1-sulfonyl azide
[22]
[23]
and trifyl azide (TfN3) (Figure 3).
The best result was
achieved by using trifyl azide condition in terms of mild reaction
Scheme 2. Reagents and conditions for the preparation of 6a-6c: 5 (1.0 equiv), alkyne (3.0 equiv), CuI (0.1 equiv), N2, 8h

4
Tetrahedron
ACCEPTED MANUSCRIPT
OH
OH
OH
NH
5
HO
H
O
O
O
O
Cl
O
O
O
HO
OH
Cl
O
O
N
N
H
H
N
N
N
N
N
N
H
O
H
H
5
O
O
O
HO
O
NH
NH2
OH
OH
HO
OH
OH
OH
R2
NH
5
HO
H
O
O
O
O
Cl
O
O
O
7a
7b
R2=
R2=
O
O
O
HO
OH
O
Cl
O
N
N
H
H
N
N
N
O
N
N
N
O
H
H
H
O
O
O
HO
O
NH
NH2
O
O
O
7c R2=
OH
OH
HO
7
Scheme 3. Reagents and conditions for the preparation of dimers 7a-7c: 5 (2.0 equiv), alkyne (1.0 equiv), CuI (0.1 equiv), N2, 3-5d
Biological assays
After stirred at room temperature for 8h, the reaction mixture
was poured into EtOAc. The resulting precipitate was isolated by
centrifugation and then washed with EtOAc to remove the excess
alkyne. The crude product was further purified by HPLC to
obtain pure 6a-c. Surprisingly, 6a-c could not be converted to 7a-
c after numerous experiments, only trace of desired product was
detected. Thus, we turned to use 5 (2 equiv) directly for this click
reaction. As outlined in Scheme 3, a solution of compound 5
with corresponding alkyne in DMF was stirred for 3-5 days at
room temperature. The reaction progress was monitored by
HPLC on an 18C column. As illustrated in Figure 4 for the
synthesis of 7a, the starting material 5 was initially converted to
monomer 6a which subsequently cyclized with N3-group of
another compound 5 to form dimer 7a. Finally, all pure final
compounds 7a-c were obtained by using RP-HPLC eluted with
43% of acetonitrile in water (with 0.1% trifluoroacetic acid).
The antibacterial activities of analogues 6a-c and 7a-c were
obtained with a broth microdilution assay against methicillin-
resistant Staphylococcus aureus (MRSA) vancomycin-resistant
Enterococcus faecalis (VRE) and Pneumococcus, as shown in
table 1. Monomers 6a-c demonstrated slightly decreased
activities against MRSA and with no activity against VRE,
expects 6c had an MIC value of 25 µg/mL for VRE2. Though 7a-
c exhibited less potent activity (higher MIC values) than
demethylvancomycin for both clinical isolates MRSA and
Pneumococcus, all of them were more potent against VRE. This
was consists with previous study that the activity of N-N dimers
was less potent than that of demethylvancomycin against MRSA.
[25]
Both 7b and 7c were two times more potent than 7a against
VRE. Possibly, more bulky and rigid substituent on the linker did
not favour the activities against VREs.
Table 1. MIC Values of monomers 6a-6c and dimers 7a-7c (µg/mL)
Pneumococcus
Compd.
6a
MRSA1
50
MRSA2
50
VRE1
>50
VRE2
>50
50
6b
6c
>50
25
>50
12.5
12.5
12.5
12.5
1.56
>50
>50
50
>50
25
>50
25
7a
12.5
12.5
6.25
1.56
25
25
7b
25
12.5
12.5
>50
25
7c
25
25
Devan
>50
3.13
Molecular modelling
Molecular modelling was used to investigate the feasibility of
simultaneous binding of the two parts (A and B). The 3D
structure of peptidoglycan was constructed with the Sybyl 6.9
[26, 27, 28]
suite.
The docking result of dimer 7c with peptidoglycan
indicates that 7c forms ten interactions with the peptidoglycan
(Figure 5). The peptide backbone in Part A (dimer 7c) forms five
hydrogen bonds with the residues in the C-terminal and one
hydrogen bond with (gly)5. The peptide backbone of Part B forms
three hydrogen bonds with the residues in the C-terminal and one
hydrogen bond with (gly)5. It was possible that the restoration of
activity against VRE of vancomycin dimer may be due to the
increase of interactions between the ligand and the dimer.
Figure 4. a) The retention time of compound 5; b) HPLC
monitor of reaction mixture of 7a after stirring for 1.5 days; c)
HPLC monitor of reaction mixture of 7a after 5 days; d) HPLC
monitor of crude compound 6a after work-up the reaction
mixture

splitting patterns are designated as s (singlet), d (doublet), t
ACCEPTED MANUSCRIPT
(triplet), q (quartet), m (multiplet), or br (broad). A Shimadzu
LCMS-2012EV was used for low-resolution mass spectra (ESI)
and IonSpec 4.7 Tesla FTMS (MALDI) or Bruker Daltonics
APEXIII7.0 TESLA FMS (ESI) for high-resolution mass spectra.
Synthesis of 5:
Compound 4 (200 mg, 0.12 mmol) was dissolved in DMF (2 mL),
then DIEA (30.2 ꢀL, 0.24 mmol) and CuSO4·5H2O (2.5 mg, 0.01
0
mmol) were added under N2 at 0 C. To this mixture, freshly
prepared TfN3 (43.6 mg, 0.36 mmol) in DCM solution was added
0
dropwise. The reaction mixture was stirred at 0 C for 8h before
the addition of DCM (20 mL). The resulting precipitate was
filtered, washed with DCM, and dried in vacuo to give compound
5 as a white solid which was used directly for the next step
without further purification. ESI-MS m/z: 1633.5 [M+H]+.
General procedure for the preparation of 6a-c: compound 5
(0.12 mmol) and corresponding alkyne (0.36 mmol) were
dissolved in DMF (4 mL), then CuI (0.01 mmol) was added
under N2. The reaction mixture was stirred at room temperature
for 8 h before the addition of EtOAc (20 mL). The resulting
precipitate was filtered, washed with EtOAc, and dried in vacuo
to give crude compounds 6a-c. The residue was further purified
by HPLC to provide the desired pure fractions (gradient eluent:
CH3CN/H2O, 20-55% in 0.1% TFA). The combined fractions
were concentrated to a small volume (15 mL), which was
lyophilized to give corresponding pure compounds 6a-c as an
off- white solid.
Figure 5. Molecular docking studies of 7c with the peptidoglycan of
VRE. The docking complex is shown in a stick format peptidoglycan
is colored in cyan and dimer 7c is colored in white. The 7c-
peptidoglycan complex shows ten possible interactions comprising
six hydrogen bonds in part A and four hydrogen bonds in part B.
1
Compound 5: 89.8%; H NMR (600 MHz, DMSO) δ: 9.44 (s,
1H), 9.18 (s, 1H), 9.07 (s, 1H), 8.97 (s, 1H), 8.66 (s, 1H), 8.52 (s,
1H), 8.53 (s, 1H), 8.41 (s, 1H), 7.80 (s, 1H), 7.53-7.48 (m, 2H),
7.42-7.40 (m, 2H), 7.30-7.27 (m, 1H), 7.14-7.10 (m, 2H), 7.02 (s,
1H), 6.76-6.73 (m, 1H), 6.70-6.69 (m, 2H), 6.36 (s, 1H), 6.21 (s,
1H), 5.95 (s, 1H), 5.75-5.71 (m, 1H), 5.64-5.56 (s, 1H), 5.29-5.23
(m, 2H), 5.14 (s, 3H), 5.07 (s, 1H), 4.89 (s, 1H), 4.67-4.60 (m,
1H), 4.38-4.40 (m, 2H), 4.20-4.07 (m, 2H), 3.94-3.88 (m, 1H),
3.55-3.22 (m, 6H), 2.85 (s, 2H), 2.69 (s, 1H), 2.64-2.58 (s, 3H),
2.56 (s, 1H), 2.20-2.16 (m, 1H), 2.14 (s, 1H), 2.02 (m, 1H), 1.98-
1.90 (m, 3H), 1.42-1.34 (m, 4H), 1.08-1.06 (m, 2H), 1.00-0.98
(m, 2H), 0.88-0.87 (m, 3H), 0.83-0.82 (m, 3H); ESI-MS m/z:
1462.2 [M+H]+.
Conclusion
Since the dimerization of vancomycin could potentially
enhance biological activity, dimers 7a-c were designed and
synthesized to search for potent antibacterial agents through
multivalent interactions. Compounds 7a-c was synthesized by
click chemistry and their antibacterial activities were evaluated
for clinical isolates of MRSA, VRE and Pneumococcus. All three
different covalently linked dimers 7a-c were more potent than
their corresponding monomers 6a-c, except 6c had a similar
activity against MRSA. Compared with Devan, 7a-c showed
more potent activities against VRE, though slightly weaker
activities against MRSA and Pneumococcus were observed.
More potent activities of 7b-c than 7a against VRE may
contribute from their flexible linkers which could allow 7b-c to
adopt into more favourite conformations. This information
provides us the insight which can guide future structural
modifications to design more potent novel dimeric derivatives.
Monomer 6a: 34.6%; 1H NMR (600 MHz, DMSO) δ: 9.45 (s,
1H), 9.17 (s, 1H), 9.09 (s, 1H), 8.69 (s, 1H), 8.54 (s, 1H), 8.40 (s,
1H), 8.23 (s, 1H), 7.85 (s, 2H), 7.57 (s, 2H), 7.56 (s, 2H), 7.49-
7.46 (m, 2H), 7.31 (d, J = 7.8 Hz, 1H), 7.28-7.26 (m, 3H), 7.21-
7.20 (m, 1H), 7.19-7.17 (m, 2H), 7.13 (s, 1H), 7.11 (s, 1H), 7.06-
7.04 (m, 2H), 6.79 (d, J = 8.4Hz, 1H), 6.73 (d, J = 8.4Hz, 1H),
6.70-6.69 (m, 2H), 6.41 (s, 1H), 6.26 (s, 1H), 5.96 (s, 1H), 5.79 (s,
1H), 5.76-5.74 (m, 2H), 5.66 (s, 1H), 5.61 (s, 1H), 5.36-5.33 (m,
2H), 5.30 (m, 1H), 5.19 (s, 2H), 5.12 (s, 3H), 4.84-4.82 (m, 2H),
4.76 (s, 1H), 4.63 (d, J = 6.6Hz,1H), 4.46-4.44 (m, 2H), 4.29 (s,
1H), 4.21-4.19 (m, 1H), 4.10 (s, 1H), 3.59-3.56 (m, 6H), 3.27-
3.25 (m, 2H), 2.77 (s, 1H), 2.70 (s, 1H), 2.58-2.55 (m, 4H), 2.17
(s, 1H), 2.08-1.98 (m, 4H), 1.91-1.90 (m, 1H), 1.81-1.79 (m, 1H),
1.76 (s, 2H), 1.57-1.55 (m, 4H), 1.36 (s, 2H), 1.30-1.26 (m, 6H),
1.20-1.16 (m, 2H), 1.10-1.09 (m, 3H), 0.90-0.89 (m, 3H), 0.87-
0.85 (m, 2H), 0.83-0.82 (m, 3H); 13C NMR (150 MHz, DMSO)
δ:174.3, 173.0, 171.9, 170.9, 169.6, 169.0, 168.3, 157.7, 157.6,
157.0, 156.8, 155.5, 152.6, 151.7, 148.7, 142.9, 142.6, 142.2,
142.2, 140.0, 135.8, 135.1, 132.5, 132.3, 130.1, 129.9, 129.1,
128.7, 127.5, 127.0, 126.4, 126.1, 125.6, 124.2, 123.8, 123.2,
Experiment Section
Chemistry
General: Reagents were purchased from Sigma-Aldrich and TCI
Chemical companies. All solvents were purified and dried in
accordance with standard procedures, unless otherwise indicated.
Reactions were monitored by TLC (Yantai China GF254 silica gel
plates 5×10cm) and HPLC (Waters e2695). H NMR and 13C
1
NMR spectra were recorded on Bruker DRX-400 (400MHz) and
Bruker DRX-600. Chemical shifts were recorded in ppm and

6
Tetrahedron
ACCEPTED MANUSCRIPT
122.0, 118.5, 117.8, 116.6, 115.1, 108.1, 106.1, 105.2, 102.3,
with EtOAc, and dried in vacuo to give crude compounds 7a-c.
101.3, 96.9, 78.7, 77.4, 77.3, 76.8, 76.4, 72.0, 70.6, 69.1, 68.4,
63.6, 62.2, 61.6, 59.1, 57.1, 56.4, 54.1, 53.6, 51.5, 45.5, 35.5,
33.7, 31.3, 30.7, 30.7, 29.0, 28.8, 28.8, 28.7, 28.5, 28.4, 28.1,
26.5, 25.5, 25.0, 24.7, 24.5, 22.5, 21.8, 19.0, 17.3. ESI-MS m/z:
1898.65 [M+H]+; HRMS m/z: calcd for C96H103Cl2N11O26 [M+H]+:
1898.6543, found 1898.6554 [M+H]+.
The residue was further purified by HPLC to provide the desired
pure fractions (isocratic eluent: CH3CN/H2O, 43% in 0.1% TFA)
with Waters SunFire Prep C18 (5 ꢀm 10×250 mm) column. The
combined fractions were concentrated to a small volume (15 mL)
which was lyophilized to give corresponding pure compounds
7a-c as a white solid.
1
1
Monomer 6b: 32.5%; H NMR (600 MHz, DMSO) δ: 9.47 (s,
Dimer 7a: 18.6%, H NMR (600 MHz, DMSO) δ: 9.45 (s, 2H),
1H), 9.20 (s, 1H), 9.13 (s, 1H), 8.66 (s, 1H), 8.52 (s, 1H), 8.17 (s,
1H), 7.81 (s, 2H), 7.44-7.42 (m, 2H), 7.39-7.37 (m, 2H), 7.30-
7.28 (m, 3H), 7.26 (s, 2H), 7.24 (s, 2H), 7.22-7.21 (m, 1H), 7.15
(s, 4H), 7.14 (s, 4H), 6.76-6.73 (m, 1H), 6.70-6.67 (m, 3H), 6.38
(s, 1H), 6.22 (s, 1H), 5.98-5.95 (m, 1H), 5.78 (s, 1H), 5.71-5.67
(m, 3H), 5.55 (s, 1H), 5.37-5.36 (m, 1H), 5.32-5.29 (m, 2H), 5.25
(s, 1H), 5.14-5.13 (m, 2H), 5.08-5.07 (m, 2H), 4.71 (s, 1H), 4.59-
4.58 (m, 1H), 4.46 (s, 2H), 4.40 (d, J = 7.8Hz, 2H), 4.18-4.07 (m,
2H), 4.06-4.05 (m, 1H), 3.67-3.64 (m, 2H), 3.55-3.22 (m, 6H),
3.07-3.03 (m, 1H), 2.74 (s, 1H), 2.64 (s, 1H), 2.15-2.10 (m, 1H),
2.00-1.94 (m, 4H), 1.73 (s, 4H), 1.54-1.50 (m, 6H), 1.46-1.42 (m,
6H), 1.32 (s, 3H), 1.15-1.13 (m, 4H), 1.07-1.05 (m, 3H), 0.85-
0.84 (m, 3H), 0.82-0.80 (m, 2H), 0.89-0.78 (m, 3H); 13C NMR
(150 MHz, DMSO) δ: 173.0, 171.9, 170.9, 169.6, 169.0, 168.3,
157.7, 157.6, 157.0, 156.8, 155.5, 152.6, 151.7, 150.4, 148.7,
144.5, 142.7, 142.9, 141.6, 136.5, 135.4, 132.9, 130.1, 129.1,
128.7, 127.8, 127.5, 126.9, 126.6, 126.1, 124.6, 123.4, 122.0,
118.5, 116.7, 106.1, 102.8, 96.9, 80.8, 78.3, 77.6, 77.4, 72.0, 71.2,
70.6, 70.1, 69.9, 69.4, 68.9, 63.9, 63.6, 62.2, 61.6, 61.5, 59.6,
59.1, 57.9, 57.1, 55.4, 51.5, 46.0, 35.5, 31.7, 31.3, 30.2, 29.5,
29.4,29.4, 29.3, 29.2, 29.1,29.0, 28.8, 27.0, 26.5, 25.9, 23.0, 22.5,
21.8, 19.4, 19.0, 17.3. ESI-MS m/z: 1830.4 [M+H]+; HRMS m/z:
calcd for C90H107Cl2N11O26 [M+H]+: 1830.6853, found 1830.6773
[M+H]+.
9.17 (s, 2H), 9.10 (s, 2H), 8.66 (s, 2H), 8.52 (s, 2H), 8.38 (s, 2H),
8.14 (s, 2H), 7.83 (s, 4H), 7.68 (d, J = 12 Hz, 2H), 7.56-7.54 (m,
4H), 7.50-7.48 (m, 2H), 7.46-7,44 (m, 4H), 7.41 (s, 2H), 7.31-
7.28 (m, 2H), 7.29-7.24 (m, 6H), 7.25 (d, J = 8.6 Hz, 6H), 7.17-
7.15 (m, 4H), 7.11-7.08 (m, 3H), 7.09 (s, 2H), 6.75 (d, J = 12 Hz,
2H), 6.71 (d, J = 12 Hz, 2H), 6.40 (s, 2H), 6.24 (s, 2H), 5.96 (s,
2H), 5.81 (s, 2H), 5.76-5.74 (m, 4H), 5.57 (s, 2H), 5.34-5.32 (m,
2H), 5.27 (s, 2H), 5.17 (s, 4H), 5.10 (s, 4H), 4.91 (s, 2H), 4.85 (s,
2H), 4.82 (s, 3H), 4.75 (s, 3H), 4.65-4.61 (m, 2H), 4.50-4.51 (m,
6H), 4.42-4.41 (m, 4H), 4.28(s, 4H), 4.20-4.17 (m, 4H), 4,12-
4.10 (m, 2H), 3.86-3.06 (m, 12H), 2.77-2.73 (m, 4H), 2.67-2.65
(m, 4H), 2.18-2.15 (m, 6H), 1.99-1.96 (m, 6H), 1.53-1.47 (m,
6H), 1.48-1.44 (m, 6H), 1.35 (s, 6H), 1.24-1.17 (m, 6H), 1.08-
1.07 (m, 6H), 0.88-0.86 (m, 6H), 0.85-0.84 (m, 4H), 0.81-0.79
(m, 6H); 13C NMR (150 MHz, DMSO) δ: 174.4, 172.4, 171.8,
170.4, 169.1, 168.5, 167.7, 157.8, 157.6, 157.4, 157.2, 156.5,
155.0, 152.1, 151.2, 148.2, 142.6, 142.4, 142.2, 142.1, 140.4,
135.9, 135.6, 132.5, 132.3, 131.8, 129.9, 129.5, 128.1, 127.2,
127.0, 126.3, 126.1, 125.5, 124.1, 123.7, 123.2, 121.5, 118.2,
117.9, 116.2, 115.0, 107.5, 105.6, 104.6, 102.3, 100.8, 96.4, 78.5,
77.7, 76.9, 76.8, 72.4, 70.1, 68.5, 67.6, 63.0, 61.7, 61.1, 59.1,
58.5, 56.6, 54.8, 53.5, 51.0, 45.5, 35.0, 33.5, 31.2, 30.8, 30.7,
29.0, 28.9, 28.7, 28.5, 28.3, 28.3, 28.2, 26.4, 25.9, 25.0, 24.3,
24.2, 22.5, 21.2, 18.8, 16.8. ESI-MS m/z: 1767.5 [M+2H]2+;
HRMS m/ z: calcd for C174H192Cl4N22O50 [M+2H]2+: 1766.6043,
found 1766.6071 [M+2H]2+.
1
Monomer 6c: 21.7%; H NMR (600 MHz, DMSO) δ: 9.45 (s,
1H), 9.18 (s, 1H), 9.11 (s, 1H), 8.67 (s, 1H), 8.52 (s, 1H), 8.20 (s,
1H), 7.85-7.82 (m, 2H), 7.46-7.42 (m, 2H), 7.41-7.39 (m, 2H),
7.29 (s, 2H), 7.28 (s, 1H), 7.27 (s, 1H), 7.25 (s, 1H), 7.23 (s, 1H),
7.16 (s, 4H), 7.14 (s, 4H), 7.04 (s, 2H), 6.76-6.74 (m, 2H), 6.70-
6.68 (m, 2H), 6.40-6.38 (m, 1H), 6.23-6.22 (m, 1H), 5.96-5.94
(m, 1H), 5.76 (s, 1H), 5.73-5.70 (m, 2H), 5.66-5.65 (m, 1H), 5.56
(s, 1H), 5.36-5.30 (m, 3H), 5.26 (s, 1H), 5.15 (s, 1H), 5.13 (s,
1H), 5.09-5.07 (m, 2H), 4.71 (s, 1H), 4.64-4.54 (m, 1H), 4.51 (s,
2H), 4.42-4.39 (m, 2H), 4.14-4.03 (m, 3H), 3.70-3.64 (m, 1H),
3.54-3.49 (m, 10H), 3.40-3.22 (m, 6H), 2.65 (s, 1H), 2.55-2.54
(m, 3H), 2.13 (s, 1H), 2.00-1.94 (m, 2H), 1.86-1.77 (m, 1H), 1.73
(s, 1H), 1.55-1.51 (m, 4H), 1.33 (s, 2H), 1.16-1.12 (m, 3H), 1.07-
1.06 (m, 3H), 0.85-0.87 (m, 3H), 0.80-0.83 (m, 2H), 0.79-0.81
(m, 3H); 13C NMR (150 MHz, DMSO) δ: 173.0, 171.9,
170.9,169.6, 169.0, 168.3, 157.7,157.6, 157.0, 156.8, 155.5,
152.6, 151.7, 150.4,148.7, 144.5, 142.9, 142.7, 141.6, 140.9,
136.5, 135.4, 132.9, 132.3, 130.1, 129.1, 128.7, 127.8, 127.5,
126.9, 126.6, 126.1, 124.6, 123.8, 123.4, 122.0, 118.5, 116.7,
106.1, 102.8, 96.9, 80.8, 78.3, 77.6, 77.4, 72.0, 70.6, 70.1, 69.9,
69.4, 68.9, 68.7, 63.9, 63.6, 62.2, 61.6, 61.5, 59.6, 59.1, 57.9,
57.1, 56.4, 55.4, 54.1, 51.5, 46.0, 35.5, 31.7, 31.3, 29.5, 29.4,
29.3, 29.0, 28.8, 27.0, 26.5, 25.5, 24.7, 23.0, 22.5, 21.8, 19.4,
19.0, 17.3. ESI-MS m/z: 1818.6 [M+H]+; HRMS m/z: calcd for
C88H103Cl2N11O27 [M+H]+: 1818.6489, found 1818.6475 [M+H]+.
1
Dimer 7b: 9.8% H NMR (600 MHz, DMSO) δ: 9.46 (s, 2H),
9.21 (s, 2H), 9.12 (s, 2H), 8.69 (s, 2H), 8.52 (s, 2H), 8.19 (s, 2H),
7.95 (s, 3H), 7.88-7.84 (m, 3H), 7.47-7.46 (m, 2H), 7.45-7.38 (m,
4H), 7.31 (d, J = 7.8 Hz, 2H), 7.27-7.26 (m, 4H), 7.22 (s, 2H),
7.18 (s, 4H), 7.17 (s, 4H), 6.79-6.77 (m, 2H), 6.73 (d, J = 8.4 Hz,
2H), 6.70-6.66 (m, 4H), 6.40 (s, 2H), 6.26 (s, 2H), 5.98-5.97 (m,
2H), 5.75-5.73 (m, 4H), 5.67-5.65 (m, 4H), 5.59 (s, 2H), 5.36-
5.34 (m, 4H), 5.33-5.31 (m, 2H), 5.29 (s, 2H), 5.18 (s, 2H)), 5.11
(s, 6H), 4.75 (s, 2H), 4.62-4.61 (m, 2H), 4.49 (s, 4H), 4.46-4.45
(m, 2H), 4.44-4.43 (m, 2H), 4.41-4.39 (m, 4H), 4.20-4.19 (m,
4H), 4.13-4.11 (m, 4H), 4.09-4.08 (m, 4H), 3.22-3.45 (m, 12H),
2.89 (s, 6H), 2.73 (s, 4H), 2.62 (s, 2H), 2.17-2.15 (m, 2H), 2.03-
2.01 (m, 2H), 1.98-1.96 (m, 8H), 1.91-1.88 (m, 2H), 1.80-1.76
(m, 2H), 1.56-1.53 (m, 6H), 1.49-1.45 (m, 6H), 1.37-1.35 (m,
6H), 1.10-1.09 (m, 6H), 1.07-1.05 (m, 8H), 0.89-0.87 (m, 6H),
0.85-0.84 (m, 4H), 0.83-0.82 (m, 6H); 13C NMR (150 MHz,
DMSO) δ: 173.0, 171.9, 170.9, 169.6, 169.0, 168.3, 157.7, 157.6,
157.0, 156.8, 155.5, 152.6, 151.7, 150.4, 148.7, 143.3, 142.9,
142.7, 136.5, 136.1, 135.1, 133.3, 133.0, 132.3, 130.1, 129.1,
128.7, 127.8, 127.5, 126.9, 126.6, 126.1, 124.6, 124.2, 123.8,
122.0, 118.5, 115.7, 115.6, 105.2, 106.1, 105.2, 102.8, 101.3,
96.9, 79.8, 78.7, 78.3, 77.5, 77.4, 72.0, 70.6, 69.1, 62.2, 61.6,
59.6, 59.1, 57.1, 56.5, 55.9, 55.4, 54.1, 51.5, 35.5, 33.2, 31.7,
31.3, 29.5, 29.4, 29.2, 29.2, 29.1, 29.0, 28.8, 28.8, 27.0, 26.5,
25.5, 24.7, 23.0, 22.5, 21.8, 19.4, 17.3. ESI-MS m/z: 1733.0
[M+2H]2+; HRMS m/ z: calcd for C168H196Cl4N22O50 [M+2H]2+:
1732.0907, found 1732.0916 [M+2H]2+
General procedure for the preparation of 7a-c: Compound 5
(0.18 mmol) and corresponding alkyne (0.09 mmol) were
dissolved in DMF (5 mL), then CuI (0.036 mmol) and ascorbate
(0.045 mmol) were added under N2. The reaction mixture was
stirred at room temperature for 3-5 d before the addition of
EtOAc (25 mL). The resulting precipitate was filtered, washed
1
Dimer 7c: 15.7%, H NMR (600 MHz, DMSO) δ: 9.46 (s, 2H),
9.19 (s, 2H), 9.12 (s, 2H), 8.66 (s, 2H), 8.52 (s, 2H), 8.36 (s, 2H),

8.20 (s, 2H), 7.82 (s, 2H), 7.42-7.44 (m, 2H), 7.39-7.41 (m, 4H),
7.30-7.32 (m, 6H), 7.28 (s, 2H), 7.24 (d, J = 8.6 Hz, 4H), 7.22 (s,
Acknowledgment
ACCEPTED MANUSCRIPT
4H), 7.19 (s, 4H), 7.16 (s, 4H), 7.14 (s, 4H), 7.07 (s, 6H), 6.76 (d,
J = 12 Hz, 2H), 6.70 (m, 4H), 6.38 (s, 2H), 6.22 (s, 2H), 5.97-
5.92 (m, 2H), 5.77 (s, 1H), 5.73-5.67 (m, 4H), 5.55 (s, 1H), 5.37-
5.36 (m, 2H), 5.31-5.30 (m, 2H), 5.26 (s, 2H), 5.15-5.12 (m, 4H),
5.07 (s, 4H), 4.69 (s, 1H), 4.60-4.58 (m, 2H), 4.51 (s, 3H), 4.43-
4.39 (m, 4H), 4.17-4.11 (m, 4H), 3.42-3.22 (m, 12H), 3.04- 3.02
(m, 4H), 2.73-2.64 (m, 2H), 2.55-2.51 (m, 4H) 2.14-2.10 (m, 2H),
1.98-1.96 (m, 8H), 1.84-1.77 (m, 4H), 1.73 (s, 4H), 1.54-1.51 (m,
10H), 1.33 (s, 6H), 1.17-1.14 (m, 6H), 1.07-1.05 (m, 6H), 0.86-
0.84 (m, 6H), 0.84-0.82 (m, 4H), 0.80-0.78 (m, 6H); 13C NMR
(150 MHz, DMSO) δ: 173.0, 171.9, 171.0, 169.6, 169.0, 168.3,
158.2, 158.0, 157.6, 157.0, 156.8, 155.6, 152.6, 151.7, 150.4,
148.7, 144.5, 142.9, 142.7, 136.5, 136.1, 135.1, 133.3, 133.0,
132.3, 130.1, 129.1, 128.7, 127.8, 127.5, 126.9, 126.7, 126.1,
124.6, 124.6, 124.2, 123.8, 123.5, 122.0, 118.5, 116.6, 115.7,
115.6, 106.1, 105.0, 102.8, 101.3, 96.9, 79.8, 18.7, 78.3, 77.6,
77.3, 72.0, 70.6, 69.9, 68.9, 62.2, 61.6, 59.0, 57.9, 57.1, 54.1,
51.5, 35.5, 33.2, 31.7, 31.3, 29.5, 29.3, 29.1, 29.0, 28.8, 28.8,
27.0, 26.5, 25.5, 24.7, 23.0, 22.5, 21.8, 19.3, 17.3. ESI-MS m/z:
1727.4 [M+2H]2+; HRMS m/ z: calcd for C166H192Cl4N22O51
[M+2H]2+: 1725.5947, found 1725.5961 [M+2H]2+.
These investigations were supported by the National Natural
Science Foundation of China (No. 81673297), Science and
Technology Developing Foundation of Shanghai (No.
17JC1400200), Shanghai Municipal Committee of Science and
Technology (No. 17431902500). Prof. L. Liu (Shanghai Institute
of Pharmaceutical Industry) is gratefully acknowledged for
antibacterial assays. We thank Prof. W. Fu for assistance with
computer modelling.
References and notes
[1] Patti, G. J.; Kim, S. J.; Schaefer, J. Biochemistry 2008, 47, 8378-8385.
[2] Kishimoto, K.; Manning, J. M. J. Protein Chem. 2001, 20, 455-461.
[3] Reynolds, P. E. Eur. J. Clin. Microbiol. 1989, 8, 943-950.
[4] Walsh, C. Nature 2000, 406, 775-781.
[5] Popieniek, P. H.; Pratt, R. F. Anal. Biochem. 1987, 165, 108-113.
[6] Bugg, T. D. H.; Wright, G. D.; Dutka-Malen, S.; Arthur, M.;
Courvalin, P.; Walsh, C. T. Biochemistry 1991, 30, 10408-10415.
[7] Arthur, M.; Courvalin, P. Antimicrob. Agents Chemother. 1993, 37,
1563-1571.
[8] Nagarajan, R.; Schabel, A. A.; Occolowitz, J. L.; Counter, F. T.; Ott, J.
L.; Felty-Duckworth, A. M. J. Antibiot. 1989, 42, 63-72.
[9] Ashford, P.-A.; Bew, S. P. Chem. Soc. Rev. 2012, 41, 957-978.
[10] Mackay, J. P.; Gerhard, U.; Beauregard, D. A.; Maplestone, R. A.;
Williams, D. H. J. Am. Chem. Soc. 1994, 116, 4573-4580.
[11] Mackay, J. P.; Gerhard, U.; Beauregard, D. A.; Williams, D. H.;
Westwell, M. S.; Searle, M. S. J. Am. Chem. Soc. 1994, 116, 4581-
4590.
[12] Allen, N. E.; LeTourneau, D. L.; Hobbs, J. N.; Thompson, R. C.
Antimicrob. Agents Chemother. 2002, 46, 2344-2348.
[13] Rao, J.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 10286-10290.
[14] Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Labischinski,
H.; Endermann, R. Chem. Eur. J. 2001, 7, 3824-3843.
[15] Sundram, U. N.; Griffin, J. H.; Nicas, T. I. J. Am. Chem. Soc. 1996,
118, 13107-13108.
Biological assays
The minimun inhibitory concentration (MIC) values were
measured to investigate the antibacterial activity of the test
compounds against MRSA, VRE, and Pneumococcus.
Demethylvancomycin (Devan) was used as a positive control.
MIC values were investigated using an agar dilution method
according to the methods of CLSI. Compound stock solutions
(320 ꢀg ml-1) were prepared in DMSO/H2O (50%). Serial
twofold dilutions prepared from the stock solutions with sterile
H2O were diluted ten-fold with Mueller-Hinton (MH) agar
medium to obtain a concentration range of 0.024-50 ꢀg ml-1. The
test organisms were grown in MH broth medium at 35 for 8 h
and were adjusted to a turbidity of 0.5 using the McFarland
standard. The bacterial suspensions were inoculated onto the
drug-supplemented MH agar plates with a multipoint inoculator
and incubated at 35 for 16 h.
[16] Silverman, S. M.; Moses, J. E.; Sharpless, K. B. Chem. Eur. J. 2017,
23, 79-83.
[17] Zhang, S.-J.; Yang, Q.; Xu, L.; Chang, J.; Sun, X. Bioorg. Med. Chem.
Lett. 2012, 22, 4942-4945.
[18] Chang, J.; Zhang, S. J.; Jiang, Y. W.; Xu, L.; Yu, J. M.; Zhou, W. J.;
Sun, X. ChemMedChem 2013, 8, 976-984.
[19] Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001,
40, 2004–2021.
[20] Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Chem. Rev. 2013, 113,
4905-4979.
[21] Katritzky, A. R.; El Khatib, M.; Bol’shakov, O.; Khelashvili, L.; Steel,
P. J. J. Org. Chem. 2010, 75, 6532-6539.
Molecular docking and modelling
[22] Ye, H.; Liu, R.; Li, D.; Liu, Y.; Yuan, H.; Guo, W.; Zhou, L.; Cao, X.;
Tian, H.; Shen, J.; Wang, P. G. Org. Lett. 2013, 15, 18-21.
[23] Liu, Q.; Tor, Y. Org. Lett. 2003, 5, 2571-2572.
[24] Pintér, G.; Batta, G.; Kéki, S.; Mándi, A.; Komáromi, I.; Takács-
Novák, K.; Sztaricskai, F.; Röth, E.; Ostorházi, E.; Rozgonyi, F.;
Naesens, L.; Herczegh, P. J. Med. Chem. 2009, 52, 6053-6061.
[25] Griffin, J. H.; Linsell, M. S.; Nodwell, M. B.; Chen, Q.; Pace, J. L.;
Quast, K. L.; Krause, K. M.; Farrington, L.; Wu, T. X.; Higgins, D. L.;
Jenkins, T. E.; Christensen, B. G.; Judice, J. K. J. Am. Chem. Soc.
2003, 125, 6517-6531.
[26] Kim, S. J.; Cegelski, L.; Preobrazhenskaya, M.; Schaefer, J.
Biochemistry 2006, 45, 5235-5250.
[27] Meroueh, S. O.; Bencze, K. Z.; Hesek, D.; Lee, M.; Fisher, J. F.;
Stemmler, T. L.; Mobashery, S. Proc. Natl. Acad. Sci. U. S. A. 2006,
103, 4404-4409.
The 3D structure of 7c was constructed with the Sybyl 6.9 suite
using the crystal structure of vancomycin as a template. Energy
minimization was conducted by using the Powell method with
Tripos force field and MMFF94 charge for 1000 steps iterations.
3D structure of the receptor (peptidoglycan) was constructed and
optimized using the AMBER force field. Three pairs of
glycosidic dihedral angles defined the tetrasaccharide
conformation in the receptor. Docking procedure was conducted
with AutoDock4.2, during the docking process, the number of
generation, energy evaluation, and docking runs were set to
380000, 1500000, and 10 respectively. The atom type, generation,
and the number of runs for the LGA algorithm were edited and
assigned according to the requirement of the AMBER force field.
The best estimated binding conformations of dimer 7c were
selected considering the low binding energy and geometrical
complementarity.
[28] Rekharsky, M.; Hesek, D.; Lee, M.; Meroueh, S. O.; Inoue, Y.;
Mobashery, S. J. Am. Chem. Soc. 2006, 128, 7736-7737.