Structure-based design of bacterial transglycosylase inhibitors incorporating biphenyl, amine linker and 2-alkoxy-3-phosphorylpropanoate moieties

Article abstract of DOI:10.1016/j.ejmech.2018.03.034

Transglycosylase (TGase) is essential to biosynthesis of peptidoglycan for formation of bacterial cell wall. Moenomycin is a potent TGase inhibitor, but not used in clinic treatment due to its poor pharmacokinetics. The E?F disaccharide, phosphoglycerate and lipid tail in moenomycin are crucial elements for TGase inhibition and antibacterial activity. Based on this scaffold, a series of truncated mimics comprising biphenyl, amine linker and 2-alkoxy-3-phosphorylpropanoate moieties were designed to test their TGase inhibitory activity. In this design, the phosphorylpropanoate group is a surrogate of phosphoglycerate with improved stability. A library of lipid tails can be constructed by a straightforward approach using Cu(I)-catalyzed (3 + 2) cycloaddition reactions, and the as-synthesized triazole ring can provide additional hydrogen bonds in the TGase active site. Our molecular docking experiments reveal that the biphenyl group provides π–π and π–cation interactions to act as a simplified alternative of the C–E disaccharide in moenomycin. To play the role of the oxonium transition state in transglycosylation, the amine linker exists as a positively charged species in physiological condition to attain electrostatic interactions with acidic residues. In this study, two biphenyl-linked 2-alkoxy-3-phosphorylpropanoate compounds (8 and 10) are found to exhibit modest inhibitory activity (IC₅₀ ≈ 150 μM) against the TGase of Acinetobacter baumannii and good antibacterial activity against Staphylococcus aureus (MIC = 6.3 μM).

Full text of DOI:10.1016/j.ejmech.2018.03.034

Accepted Manuscript
Structure-based design of bacterial transglycosylase inhibitors incorporating biphenyl,
amine linker and 2-alkoxy-3-phosphorylpropanoate moieties
Jui-Yin Yu, Hsiu-Jung Cheng, Huei-Ru Wu, Wei-Shen Wu, Jui-Wen Lu, Ting-Jen
Cheng, Ying-Ta Wu, Jim-Min Fang
PII:
S0223-5234(18)30277-0
10.1016/j.ejmech.2018.03.034
EJMECH 10299
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 December 2017
Revised Date: 2 March 2018
Accepted Date: 12 March 2018
Please cite this article as: J.-Y. Yu, H.-J. Cheng, H.-R. Wu, W.-S. Wu, J.-W. Lu, T.-J. Cheng, Y.-T. Wu,
J.-M. Fang, Structure-based design of bacterial transglycosylase inhibitors incorporating biphenyl, amine
linker and 2-alkoxy-3-phosphorylpropanoate moieties, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2018.03.034.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Structure-Based Design of Bacterial Transglycosylase Inhibitors Incorporating Biphenyl,
Amine Linker and 2-Alkoxy-3-Phosphorylpropanoate Moieties
a
a
a
b
b
b
Jui-Yin Yu, Hsiu-Jung Cheng, Huei-Ru Wu, Wei-Shen Wu, Jui-Wen Lu, Ting-Jen Cheng,
b
, a, b
Ying-Ta Wu, and Jim-Min Fang*
a
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan.
The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
b
_
*
_________________________
Corresponding author. J.-M.F.: Department of Chemistry, National Taiwan University,
Taipei, 106, Taiwan. Tel.: (8862)33661663; fax: (8862)2367812. E-mail address:
jmfang@ntu.edu.tw.
1

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Abstract
Transglycosylase (TGase) is essential to biosynthesis of peptidoglycan for formation of
bacterial cell wall. Moenomycin is a potent TGase inhibitor, but not used in clinic treatment
due to its poor pharmacokinetics. The E−F disaccharide, phosphoglycerate and lipid tail in
moenomycin are crucial elements for TGase inhibition and antibacterial activity. Based on
this scaffold, a series of truncated mimics comprising biphenyl, amine linker and
2
-alkoxy-3-phosphorylpropanoate moieties were designed to test their TGase inhibitory
activity. In this design, the phosphorylpropanoate group is a surrogate of phosphoglycerate
with improved stability. A library of lipid tails can be constructed by a straightforward
approach using Cu(I)-catalyzed (3+2) cycloaddition reactions, and the as-synthesized triazole
ring can provide additional hydrogen bonds in the TGase active site. Our molecular docking
experiments reveal that the biphenyl group provides π–π and π–cation interactions to act as a
simplified alternative of the C–E disaccharide in moenomycin. To play the role of the
oxonium transition state in transglycosylation, the amine linker exists as a positively charged
species in physiological condition to attain electrostatic interactions with acidic residues. In
this study, two biphenyl-linked 2-alkoxy-3-phosphorylpropanoate compounds (8 and 10) are
found to exhibit modest inhibitory activity (IC50 ≈ 150 µM) against the TGase of
Acinetobacter baumannii and good antibacterial activity against Staphylococcus aureus (MIC
=
6.3 µM).
2

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Keywords:
Transglycosylase; moenomycin; inhibitor; biphenyl; phosphorylpropanoate; amine linker.
Research highlights
•
•
•
•
•
Biphenyl-linked 2-alkoxy-3-phosphorylpropanoate is a truncated mimic of moenomycin.
Phosphorylpropanoate is a phosphoglycerate surrogate with improved stability.
Alternative to disaccharide, biphenyl provides hydrophobic interactions with TGase.
Positively charged amine linker provides electrostatic interactions with TGase.
Lipid tail comprising triazole shows hydrophobic interactions and H-bonds with TGase.
Abbreviations:
A. baumannii, Acinetobacter baumannii; Arg, arginine; Asn, asparagine; Asp, aspartic acid;
ATCC, american type culture collection; DMF, N,N-dimethylformamide; DMSO,
dimethylsulfoxide, EDCI, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; ESI,
electrospray
ionization;
Fmoc,
9-fluorenylmethoxycarbonyl;
GlcNAc,
N-acetylglucosamine; Glu, glutamic acid; HOBt, 1-hydroxybenzotriazole; Ile, isoleucine;
IR, infrared; Leu, leucine; Lys, lysine; MIC, minimal inhibitory concentration; MS,
molecular sieves; MTG, monofunctional transglycosylase; MurNAc, N-acetylmuramic
3

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acid; NBD, 7-nitrobenz-2-oxa-1,3-diazole; NCCLS, National Committee for Clinical
Laboratory Standards; OPLC, optimized potentials for liquid simulations; PBP,
penicillin-binding protein; PEG, polyethyleneglycol; PDB, Protein Data Bank; PG,
peptidoglycan; PGM, peptidoglycan monomer; S. aureus, Staphylococcus aureus; SD,
standard deviation; TGase, transglycosylase; THF, tetrahydrofuran; TMS, trimethylsilyl;
TPase, transpeptidase; Troc, 2,2,2-trichloroethoxycarbonyl; Tyr, tyrosine; Val, valine.
4

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1
. Introduction
Pathogenic bacteria have caused serious problems to humans. Although many antibiotics
have been developed for treatment of bacterial infections, abuse and over-reliance of
antibiotics have led to emergence of multidrug-resistant bacteria, which become an even more
serious threat to humans’ health. [1, 2] Bacterial cell wall is a very attractive target for
development of antibiotics because cell wall is absent in mammalian cells but essential for
bacteria to preserve cell integrity to overcome internal osmotic pressure. [3, 4] Bacteria cell
wall is constituted by the mesh-like peptidoglycan, which consists of repeating β-1,4-linked
N-acetylglucosaminyl-N-acetylmuramyl (GlcNAc−MurNAc) units cross-linked by peptide
chains. [5, 6] Transglycosylase (TGase) and transpeptidase (TPase) are two important
enzymes to catalyze the last two important steps, transglycosylation and transpeptidation, in
bacteria cell wall biosynthesis. [7–10] By extensive structure–activity relationship studies,
many effective antibiotics targeting TPase have been developed. [11, 12] However, more and
more drug-resistant bacteria are found partly due to extensive uses of TPase inhibitors. [2, 13]
As an outer-membrane enzyme, TGase has become another attractive target for development
of new antibiotics because of its indispensable function and ready accessibility. [14]
5

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Fig. 1. Chemical structures of moenomycin A (1) and lipid II (2)
Moenomycin A (1, Fig.1) is the most prominent TGase binder in the moenomycin family.
[
15–17] Moenomycin A shows better affinity to TGase than the growing glycan chain, thus
moenomycin A can directly interact with the active site of TGase to inhibit the formation of
peptidoglycan. Moenomycin A consists of three components, including a pentasaccharide
with A-ring chromophore, a phosphoglycerate and moenocinol, a C25 lipid tail. [18]
Moenomycin A is a potent antibacterial agent with low minimal inhibitory concentration
(MIC). [19] However, the poor pharmacokinetics renders moenomycin A inadequate for clinic
treatment. [20] The systematic degradation studies indicate that the E−F disaccharide,
phosphoglycerate and moenocinol in moenomycin are essential elements for TGase inhibition
and antibacterial activity. [21] The synthetic analogs containing chemically modified E−F
disaccharide, phosphoglycerate and lipid chain can still possess TGase inhibition and
antibacterial activity, though their physiological effects may differ from that of moenomycin.
[
19, 22]
6

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Fig. 2. Design of potential TGase inhibitors. Biphenyl group (blue) mimics the C–E or D–E
disaccharide, and phosphorylpropanoate (red) mimics phosphoglycerate. The C25 lipid,
moenocinol, is replaced by an alkyl-substituted triazole. An amine or amide linker is placed at
the position of F ring. Different Y groups (e.g. Troc, Fmoc, methyl and biphenyl-4-methyl are
attached on the amine linker to explore more possible interactions in TGase active site.
In this study, the potential TGase inhibitors were designed primarily based on the scaffold
of the disaccharide–phosphoglycerate–lipid part of moenomycin (Fig. 2). [23, 24] Because the
phosphoglycerate containing a phosphate group is unstable under acidic conditions, it was
replaced by a phosphorylpropanoate moiety in the designed TGase inhibitors. [25] The lipid
chain, such as undecaprenyl in lipid II (2, Fig. 1) and moenocinol in moenomycin A, is crucial
for transglycosylation. [20, 26] It was desirable to establish a library of lipid chain by click
chemistry. [27] For this purpose, a propargyl moiety could be incorporated into the substituent
for the subsequent (3+2) cycloaddition reactions with various lipids containing a terminal
7

ACCEPTED MANUSCRIPT
azido group (e.g. 12-azidododecane in this study).
In addition to the phosphoglycerate−lipid part, we also consider possible use of a less
complicated non-carbohydrate component as the surrogate of the C−E or D−E disaccharide
moiety in moenomycin. The transition state of transglycosylation is known to involve an
oxocarbenium ion, [23] which has been mimicked by aminocyclitol in design of TGase
inhibitors. [28] In another aspect, oxocarbenium ion has a relatively flat half-chair
conformation that may be mimicked by an (oxa)cyclohexene ring, as evidence of the
influenza neuraminidase inhibitors zanamivir [29] and oseltamivir [30], or by an aromatic
ring. Indeed, a benzene-containing C1-phosphonate analogue has been utilized as the inhibitor
of O-GlcNAc transferase by mimicking the oxocarbenium transition state in the enzymatic
reaction. [31] There are also many precedents of economical synthesis using substituted
benzoic acids as a template to develop neuraminidase inhibitors. [32–36] The similar concept
has been applied to using other aromatic core structure, such as pyridine [37] and thiazole, [38]
to mimic the oxonium intermediate derived from sialic acid. Inspired by these studies, we
chose biphenyl as a surrogate of disaccharide to establish a library of potential TGase
inhibitors.
In search of TGase inhibitors with simplified structures, Vederas and coworkers have
previously utilized a three-carbon chain to connect GlcNAc (the E-ring in moenomycin) and
the C-phosphonate group (as mimic of phosphoglycerate). [25] This linker is assumed to take
8

ACCEPTED MANUSCRIPT
the place of the F-ring in moenomycin. Taking together all the above-mentioned elements in
design of TGase inhibitors, a series of aryl-linked 2-alkoxy-3-phosphorylpropanoate
derivatives 3−10 were set as our synthetic targets (Fig. 3). In comparison, the amine linker in
compounds 3, 3a and 7−10 will be protonated in physiological condition (pH ≈ 7.4), whereas
the amide and carbamate linkers in compounds 4−6 will remain as neutral species. The
aminium moiety was expected to mimic the positively-charged oxonium transition state in
transglycosylation. In one case to monitor the effect of linker length, compound 10 was
devised to have the amine linker of one-carbon shorter than other target molecules.
Compounds 3−10 were synthesized, and their TGase inhibitory activities were evaluated.
O
P
O
N
H
O
HO
OR
N
N
N
O
C12H25
3
3
R = Et
a R = H
O
O
O
O
N
H
O
P
OR
OR
HO
N
N
N
N
O
O
C12H25
4
4
R = Et
a R = H
O
N
Y
O
HO
P
N
N
C12H25
R =
Y =
5
Et
H
Troc
Troc
5a
6
8
9
Et
Et
Et
Fmoc
Me
BiPh-4-CH2
O
P
O
N
O
HO
OR
H
N
N
N
O
C12H25
7
7
R = Et
R = H
a
O
O
O
HO
P
OEt
N
N
N
H
N
O
C12H25
10
Fig. 3. The designed TGase inhibitors in this study
9

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2
. Results and Discussion
2
.1. Chemistry
According to the previously reported method, [39] ethyl glycidate 17 was prepared from
-serine. Using Lewis acid ZnCl as a promoter to activate the epoxide 17, [40] the S 2
L
2
N
o
reaction with
a
phosphite reagent proceeded at 50−70
C
to give
2
-hydroxy-3-phosphorylpropanoate 18, which underwent O-alkylation with propargyl
bromide to afford compound 19 (Scheme 1). The Cu(I)-catalyzed (3+2) cycloaddition [41, 42]
of alkyne 19 with 1-azidododecane was carried out to give a triazole product. On treatment
with trimethylsilyl bromide at room temperature, the dimethyl phosphonate group was
converted to phosphonic acid, [43] giving 20 in a high overall yield. The ethyl ester remained
intact under such reaction conditions.
Scheme 1. Synthesis of 2-oxy-3-phosphorylpropanoate derivative 20 bearing a triazole-dodecyl lipid chain.
o
Reagents and conditions: (a) TMSOP(OMe) , ZnCl , THF, 50−70 C, 42 h; 55%. (b) HC≡C-CH Br, NaH, DMF,
2
2
2
o
0
C, 0.5 h; 57%. (c) 1-azidododecane, CuSO , sodium ascorbate, t-BuOH, H O, rt, 2 h; 90%; (d) TMSBr,
4 2
CH Cl , rt, 1.5 h; 81%. DMF, N,N-dimethylformamide; THF = tetrahydrofuran; TMS = trimethylsilyl.
2
2
1
0

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Scheme 2. Synthesis of (arylmethylamino)propyl phosphonate esters 3−7. Reagents and conditions: (a)
H N(CH ) OH, EDCI, HOBt, NEt , CH Cl , rt, 22 h; 89%. (b) H N(CH ) OH, NaBH(OAc) , ClCH CH Cl, rt,
2
2 3
3
2
2
2
2 3
3
2
2
1
9–27 h; 24a, 99%; 24b, 99%. (c) TrocCl, NaHCO3(aq), THF, rt, 1.5 h; 25a, 67%. (d) FmocCl, NaHCO3(aq),
o
CH Cl , rt, 22–25 h; 25b, 84%; 25c, 69% (e) CCl CN, pyridine, 70 C, 45 h; 4, 97%; 5, 80%; 6, 63%; 26, 69%.
2
2
3
(
f) NaOH, H O, rt, 30 min; 3a, 91%; 4a, 81%; 5a, 97%; 7a, 94%. (g) piperidine, CH Cl , rt, 2–3 h; 3, 71%; 7,
2 2 2
6
9
2
9%.
EDCI
=
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride;
Fmoc
=
=
-fluorenylmethoxycarbonyl; HOBt
,2,2-trichloroethoxycarbonyl
=
1-hydroxybenzotriazole; THF
= tetrahydrofuran; Troc
Scheme 2 shows the synthetic routes to compounds 3−7. The condensation reaction of
11

ACCEPTED MANUSCRIPT
3
-amino-1-propanol with (1,1′-biphenyl)-4-carboxylic acid (21) was performed in the
presence of EDCI, HOBt and Et N to give the amide compound 22. [44] Alternatively, the
3
linker 3-amino-1-propanol was reacted with 1,1′-biphenyl-4-carbaldehyde (23a) using sodium
triacetoxyborohydride as the reducing agent to produce the amine compound 24a. [45] The
Troc and Fmoc derivatives of 24a were obtained by treatments with 2,2,2-trichloroethyl
chloroformate and fluorenylmethyloxycarbonyl chloride, respectively, in the presence of
NaHCO base. Similarly, the reductive amination of naphthalene-2-carbaldehyde (23b) with
3
3
-amino-1-propanol afforded amine 24b, which was subsequently decorated to the Fmoc
derivative 25c.
To synthesize (biphenylamido)propyl phosphonate ester 4, the coupling reaction between
phosphonic acid 20 and alcohol 22 was carried out using trichloroacetonitrile as the promoter.
46] Saponification of ester 4 gave the corresponding acid 4a. By a similar procedure, the
[
coupling reactions of 20 with 25a, 25b and 25c were promoted by Cl CCN to give
3
phosphonate esters 5, 6 and 26, respectively. The Fmoc group in 6 and 26 were readily
removed by piperidine to yield the target compounds 3 and 7. The carboxylate esters 3, 5 and
7
were subjected to saponification to give the corresponding carboxylic acids 3a, 5a and 7a.
Compound 3 containing a secondary amine was subjected to reductive amination with
formaldehyde and 1,1′-biphenyl-4-cabaldehyde to give compounds 8 and 9, respectively
Scheme 3). As an extra electron-donating substituent (methyl or biphenyl-4-methyl) was
(
1
2

ACCEPTED MANUSCRIPT
introduced, it was expected that formation of aminium ions from the tertiary amine groups of
8
and 9 would enhance at physiological pH to facilitate the binding in TGase active site.
To synthesize compound 10, which is an analog of 3 with a shorter linker, ethanolamine
was utilized to react with 1,1′-biphenyl-4-cabaldehyde in the presence of NaBH(AcO) to give
3
2
7. Using Cl CCN as the activator, the Fmoc derivative of 27 underwent a condensation
3
reaction with phosphonic acid 20 to give the desired product 10 after removal of the Fmoc
protecting group.
O
P
O
O
a
N
O
OEt
3
3
+ H CO
2
HO
N
N
CH3
N
O
O
C12H25
8
O
P
a
N
O
HO
OEt
+ 23a
N
N
N
C12H25
9
OH
a
N
Y
20
2
3a + H N(CH ) OH
2 2 2
c, d
2
7 Y = H
8 Y = Fmoc
b
2
O
P
O
O
HO
OEt
N
H
N
N
N
O
C12H25
1
0
Scheme 3. Synthesis of N-alkyl-(arylmethylamino)propyl phosphonate esters 8 and 9 as well as
arylmethylamino)ethyl phosphonate ester 10. Reagents and conditions: (a) NaBH(OAc) , 3Å MS, ClCH CH Cl,
(
3
2
2
rt, 18–22 h; 8, 98%; 9, 92%. (b) FmocCl, NaHCO3(aq), CH Cl , rt, 17 h; 28, 59% from 23a. (c) CCl CN, pyridine,
2
2
3
o
7
0 C, 46 h; (d) piperidine, CH Cl , rt, 2.5 h; 44% from 20. Fmoc = 9-fluorenylmethoxycarbonyl; MS =
2 2
molecular sieves.
1
3

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2
.2. Biological activity
In this study, the TGase activity assay was performed using the bifunctional penicillin
binding protein (PBP) isolated from Acinetobacter baumannii and the substrate NBD-lipid II,
[
47, 48] which is a lipid II analog having a fluorophore of 7-nitrobenz-2-oxa-1,3-diazole
NBD) attached to the pentapeptide moiety (Fig. 4). By the catalysis of TGase, the fluorescent
(
substrate NBD-lipid II will undergo polymerization to give NBD-peptidoglycan (NBD-PG),
which can be degraded by muramidase to give NBD-peptidoglycan monomer (NBD-PGM).
[
47] When the test compound is an effective TGase inhibitor, the peak of NBD-lipid II will
retain; otherwise, the peak of NBD-PGM will occur. The degree of transglycosylation can be
determined by HPLC analysis on a Supelcosil LC-SAX1 column (anion exchange type) based
on the relative fluorescence intensity (λem = 535 nm) of the peaks for NBD-PGM product (less
retained on the column) and NBD-lipid II substrate.
Fig. 4. Transglycosylase activity assay using a fluorescent substrate NBD-lipid II. After
1
4

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TGase-catalyzed polymerization, the resulting NBD-peptidoglycan (NBD-PG) is cleaved by
muramidase to give NBD-peptidoglycan monomer (NBD-PGM) for HPLC analysis.
For the antibacterial assay, the synthesized compounds were diluted to a series of
different concentrations (100, 50, 25, 12.5 and 6.3 µM, etc.) and incubated with
Staphylococcus aureus to measure the MIC values. If the compound inhibits bacteria at a
certain concentration, no growth of the bacteria culture will be detected. Conversely, if the
compound is ineffective, the bacteria will grow to form colony.
2
.2.1. TGase inhibitory activities of the synthetic compounds.
The TGase inhibitory activities of synthetic compounds 3−10 are listed in Table 1.
Compound 3 with an amine linker showed 90% TGase inhibition at 500 µM, whereas the
derivatives 5 and 6 bearing Troc or Fmoc carbamate linker displayed inferior inhibitory
activities. The analogous compound 4 with an amide linker was inactive to TGase even at 500
µM. Compounds 5 and 6 showed better inhibitory activity than compound 4, presumably
because the Troc and Fmoc carbamate groups were available for more hydrogen-bonds and
hydrophobic interactions than the amide group. Unlike amide and carbamate, the amine linker
of 3 could form positively charged aminium ion in the assay buffer (Tris pH 8.0) to provide
electrostatic interactions with certain acidic residues in the TGase active site. This speculation
1
5

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was supported by the increased potency of compounds 8 and 9, in which formation of tertiary
aminium ion would be enhanced by the electron-donating methyl or biphenylmethyl
substituent. These assay results are consistent with the finding of a previous study, [28] which
demonstrates that the tertiary iminocyclitol-based compounds have better TGase inhibition
than the analogs with secondary iminocyclitol counterpart.
Compound 10 having a one-carbon shorter linker than that of 3 showed much higher
TGase inhibitory activity (99%) than 3 (34%) at 200 µM. The enhanced TGase inhibition of
1
0 was presumably rendered by proximity effect because its aminium ion could be at a closer
position to mimic the role of oxonium transition state. On the other hand, changing the
biphenyl group in 3 to a more compact naphthyl group, compound 7 exhibited a decreased
TGase inhibitory activity.
Table 1
a
Inhibitory activity against TGase of A. baumannii.
Compound TGase Inhibition Compound TGase Inhibition
(
500 µM)
(%)
90
(200 µM)
(%)
34
3
3
8
9
3a
13
99
4
< 5
99
1
6

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4
a
< 5
70
10
99
5
5
a
< 5
33
6
7
55
7a
48
a
Moenomycin was used as positive control, showing 100% inhibition at 1 µM.
In our original design (Figure 2), the moiety of phosphorylpropanoic acid such as in
compounds 3a, 4a, 5a and 7a were assumed to mimic the moiety of phosphoglyceric acid as a
2
+
chelator of Mg ion involved in transglycosylation. Unexpectedly, compounds 3a, 4a and 5a
showed low TGase inhibitory activity at 500 µM, and the acid compound 7a had an inhibitory
activity slightly inferior to its corresponding ester 7.
2
.2.2. Half maximum inhibitory concentration and antibacterial activity of selected
compounds
For those compounds (3, 8, 9 and 10) showed relatively good TGase inhibitory activity
Table 1), their IC50 values against the TGase isolated from A. baumannii were determined
Table 2). In comparison with compound 3 (IC = 232 µM), compounds 8 and 9 containing
(
(
5
0
1
7

ACCEPTED MANUSCRIPT
tertiary amine linker and compound 10 having a shorter linker were better TGase inhibitors
with the IC50 values in a range of 87−157 µM. The antibacterial assay indicated that
compounds 3, 7, 8 and 10 were effective to suppress the growth of S. aureus at MIC = 6.3 µM,
though not so potent as ampicillin (MIC = 1.4 µM). In addition to TGase inhibition, it is
possible that these compounds may also possess other functions to attain good antibacterial
activities. For unknown reason, compound 9 did not exhibit appreciable antibacterial activity,
even though it had the highest TGase inhibitory activity among this series of compounds.
Table 2
Inhibitory activity (IC ) against TGase of A. baumannii and antibacterial activity (MIC)
5
0
against S. aureus.
a
b
Compound
IC (µM)
MIC (µM)
5
0
c
Ampicillin
ND
1.4
3
7
8
9
232 ± 49
6.3
c
ND
6.3
153 ± 6
87 ± 46
157 ± 4
6.3
> 400
6.3
1
0
a
Half maximal inhibitory concentration against TGase of A. baumannii. Data are average of triplicate
1
8

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b
independent experiments. Errors are given as standard deviation (SD). Lowest concentration of test compound
c
which prevents visible growth of S. aureus. ND is not determined.
2
.3. Computer modeling
The crystal structure (PDB code: 3VMR) of S. aureus monofunctional TGase (MTG)
with moenomycin bound to its donor site was used as a model for molecular docking
experiments to reveal the possible structures of bacterial TGase complexes with inhibitors 3
and 10 that are designed to mimic moenomycin. In addition to the TGase−donor complex
(3VMR), there are known X-ray structures of TGase apoprotein (3VMQ) and acceptor
complex (3VMT for TGase−lipid II analog) having better resolution than 3VMR. [23]
However, several key residues in 3VMR, 3VMQ and 3VMT have shown delicate regional
variations that are presumably caused by the substrate induced-fit of these protein structures.
Before our docking experiments (Fig. 5), we meticulously investigated the variations, and
carried out a pre-screening by using a blind docking mode to a hybrid model based on these
three structures. Afterwards, the donor site derived from 3VMR was applied to describe the
interactions between TGase and the inhibitor by refined docking experiments.
As illustrated in Figure 5a, the internal benzene ring of the biphenyl moiety may induce
a π–π stacking interaction with Tyr-187, while the terminal benzene ring can reach the
hydrophobic pocket, namely 76-pocket, surrounded by Lys-75, Ile-76 and Lys-79, to form a
1
9

ACCEPTED MANUSCRIPT
π−cation interaction with the ε-aminium group of Lys-79. Instead of the biphenyl moiety,
compound 7 has a more compact naphthyl ring that cannot effectively approach to the
7
6-pocket; thus the TGase inhibitory activity of compound 7 is inferior to compound 3. As the
amino group of compound 9 contains an additional biphenylmethyl substituent that may reach
Leu-73 and Tyr-142 to gain extra hydrophobic and π–π stacking interactions, compound 9
shows a higher affinity to TGase than compound 3.
Taking the combined structural features of lipid II and moenomycin, Vederas and
coworkers have synthesized a series of 2-alkoxy-3-phosphorylpropanoic acids carrying either
one or two saccharides. [25] They found the compound having two GlcNAc units displays
better TGase inhibitory activity than the analog having only one GlcNAc. In a related study,
[
49] we also found that the potency of TGase inhibitor could be improved by extending the
terminal phenyl to a biphenyl group. It appears that the π–π stacking and π−cation interactions
of biphenyl group as shown in Figure 5a can trade for the hydrogen bonding exerted by the
disaccharide moiety of lipid II (or moenomycin) in the TGase active site.
According to the transition state in transglycosylation, [23] the carboxylate group of
Glu-100 acts as a general base to abstract the proton from 4-hydroxyl group of GlcNAc in the
elongating peptidoglycan chain (the glycosyl acceptor), allowing a simultaneous S 2-like
N
reaction at the anomeric center (C-1) of α-linked MurNAc (the glycosyl donor). However, our
modeled molecular complex shows that the amino group of 3 is not in the proximity of
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Glu-100, a result straying from our assumption in design of TGase inhibitors (Fig. 2). Instead,
this molecular modeling reveals that the aminium group of 3 has appreciable electrostatic
interactions with the carboxylate group of Asp-145.
(a)
(b)
Fig. 5. Modeling analysis of (a) compound 3 and (b) compound 10 in complexation with the
monofunctional TGase (PDB code: 3VMR) of S. aureus. Protein residues are illustrated in
stick shape and colored in dark grey. The backbone of both compounds are colored in green
with nitrogen atom in blue, oxygen in red, phosphorus in orange and hydrogen in white.
2
1

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Hydrogen bond and electrostatic interaction are depicted in dash lines with estimated
distances. The carboxylate group of Glu-100 is estimated 14.2 Å away from the amino group
of 3.
In the previous studies, [24, 50] the carboxylate group of moenomycin was found to bind
with Lys-153 in S. aureus MTG. However, no such electrostatic interaction was found in our
model. Interestingly, the ester moiety of 3 not only utilizes its carbonyl group to exert
hydrogen bonding with Lys-140 but also places its ethoxy group into a hydrophobic pocket
surrounded by Ile-180 and Val-223. This disposition of ethyl ester is thus accounted for the
better TGase inhibitory activity of 3 than its corresponding carboxylic acid 3a. The similar
effects are also attributable to higher TGase inhibitory activities of the ester compounds 4, 5
and 7 over their carboxylic acid counterparts 4a, 5a and 7a.
In the complex of moenomycin A with S. aureus MTG, the 2-alkoxy and
3
-phosphoryloxy groups exhibit electrostatic interactions with Lys-140 and Arg-148, while
the carboxylate group forms ionic interactions with Lys153 and Arg-148. [50] Figure 5a
shows that the phosphoryl group of 3 not only displays electrostatic interactions with Lys-140
and Arg-148, but also forms a hydrogen bond with Asn-141. Interestingly, the Arg-148 residue
also favors to form hydrogen bonds with the triazole ring in compound 3. The oxygen atom at
2
-alkoxy group of 3 also forms a hydrogen bond with Asn-224.
Compound 10 has a shorter linker in comparison with 3. The moiety of
2
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2
-alkoxy-3-phosphorylpropanoate ester in either 3 or 10 binds with TGase in a similar manner.
As illustrated in Figure 5b, the biphenyl rings are also close to Tyr-187 and Lys-79 to attain
the π–π stacking and π−cation interactions. However, the molecular model shows that even
one-carbon curtail may cause an appreciable change of the binding strength with TGase. The
aminium ion in 10 is not only situated in the proximity of Asp-145 to exhibit ionic interaction
but also near Asn-141 to form an additional hydrogen bond. Collectively, the molecular
docking experiments support that compound 10 has higher TGase inhibitory activity than 3 as
shown in Tables 1 and 2.
3
. Conclusion
In this study, we have designed and synthesized a series of biphenyl-linked
-alkoxy-3-phosphorylpropanoate compounds 3–10, which were evaluated as TGase
2
inhibitors to prevent the formation of bacterial cell wall. The phosphorylpropanoate group is
considered as a surrogate with improved stability for the phosphoglycerate moiety in
moenomycin. Unlike the previous report, [27] the carboxylate group in our designed
compounds (3a−5a and 7a) does not reside in the proximity of Lys-153 to gain the critical
electrostatic interaction. Alternatively, the ethyl esters (3−5 and 7) can form hydrogen
bonding with Lys-140 and render hydrophobic interactions in a pocket surrounded by Ile-180
and Val-223 to attain higher TGase inhibition than their corresponding carboxylic acids.
2
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It is known that the lipid tail at 2-position is essential for the function of TGase substrate
(
lipid II) and inhibitor (moenomycin). We demonstrate in this study that using click chemistry
41] is a feasible approach to construct a library of lipid tails. Moreover, the as-formed
[
triazole ring can provide additional hydrogen bonds in the TGase active site.
Our molecular docking model indicates that the positively charged aminium linker in
the designed compounds can form ionic interaction with residue Asp-145. Thus, the TGase
inhibitory activity is enhanced in the compounds (e.g. 8 and 9) having tertiary aminium linker
but reduced in the compounds (e.g. 4−6) having amide or carbamate linker. Compound 10
bearing a shorter aminium linker can attain extra hydrogen bonding with Asn-141, in addition
to the electrostatic interaction with Asp-145.
The TGase inhibitory activity of our designed TGase inhibitors (e.g. 3 and 8−10) was
partly attributable to the biphenyl group, which provides the π–π and π–cation interactions in
TGase active site, thus compensating the hydrogen bonding of disaccharide units that are
often incorporated into the TGase inhibitors to mimic the structural feature of lipid II and
moenomycin. From the viewpoint of synthesis, introduction of biphenyl group is easier than
disaccharide. It will be desirable to elaborate the biphenyl group with appropriate substituents
to improve the TGase inhibitory activity.
Taking together all the favorable structural elements, the synthetic compounds 8 and 10
exhibit modest inhibitory activity (IC ≈ 150 µM) against the TGase of A. baumannii and
5
0
2
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good antibacterial activity against S. aureus (MIC = 6.3 µM). Our present study sheds light on
designing simplified molecules to target bacterial TGase.
4
. Experimental section
4
.1. General Part
All the reagents and solvents were reagent grade and used without further purification
unless otherwise specified. Dichloromethane (CH Cl ) was distilled from CaH . All air or
2
2
2
moisture sensitive experiments were performed under an atmosphere of nitrogen. Wild-type
Staphylococcus aureus (ATCC 29213) and Acinetobacter baumannii (ATCC 19606) were
obtained from American Type Culture Collection (ATCC). Ampicillin was purchased from
Bioshop (Taiwan). Moenomycin was isolated according to the procedure described previously.
[
47, 51]
E. Merck silica gel 60 (0.040–0.063 mm particle size) and LiChroprep RP-18 (0.040–
.063 mm particle size) were used for column chromatography. Melting points were recorded
0
on a Yanaco micro apparatus and are uncorrected. Infrared (IR) spectra were recorded on
Thermo Nicolet iS5. Optical rotations were measured on digital polarimeter of Japan JASCO
–
1
2
–1
Co. DIP-1000. [α] values are given in the unit of 10 deg cm g . Nuclear magnetic
D
resonance (NMR) spectra were obtained on Varian Unity Plus-400 (400 MHz) or Bruker A
VIII (500 MHz) NMR. Chemical shifts (δ) are given in parts per million (ppm) relative to
2
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internal standards: CHCl (δ = 7.24), CDCl (δ = 77.0 for the central line of triplet),
3
H
3
C
CD HOD (δ = 3.31), CD OD (δ = 49.0), DHO (δ = 4.81), (CD H) CO (δ = 2.05),
2
H
3
C
H
2
2
H
(
(
(
CD ) CO (δ = 29.9), and (CD ) SO (δ = 39.5). Coupling constants (J) are given in hertz
3 2 C 3 2 C
Hz) and the splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m
multiplet), and br (broad). The ESI–HRMS experiments were conducted on a Bruker
Daltonics BioTOF III high-resolution mass spectrometer. High-performance liquid
chromatography (HPLC) was conducted on a Hitachi L-2130 instrument equipped with
Hitachi L-2485 fluorescence detector.
.
4
.2. PBP1b Transglycosylase Inhibition Assay
Nitrobenzoxadiazole-lipid II (NBD-lipid II) was prepared according to previously
reported procedure. [52] The enzymatic assay comprised NBD-lipid II (10 µM),
Acinetobacter baumannii PBP1b (90 nM), test compound (1 mM, 500 µM, 200 µM, or 100
µM as indicated) and muramidase (10 µg/µL) in a buffer solution (50 mM Tris at pH 8.0)
containing 10 mM CaCl , 0.085% Decyl PEG, 10% DMSO and 15% MeOH. The mixture
2
o
was incubated at 37 C and shaken at 1000 rpm in dark for 2 h. Moenomycin (10 µM in
ddH O) was added to stop reaction, and the mixture was centrifuged (16,000 × g). The
2
supernatant was taken for HPLC analysis on a Supelcosil LC-SAX1 column (anion exchange
type, 250 mm × 4.6 mm, 5 µm), using a gradient elution (100% A, 3 min; A/B, gradient over
2
3 min; 100% B, 3 min; 100% A, 4 min; where A is 20 mM NH OAc in MeOH and B is 1 M
4
2
6

ACCEPTED MANUSCRIPT
NH OAc in MeOH) at a flow rate of 1 min/mL. The detection was set at excitation and
4
emission wavelengths of 466 and 535 nm, respectively.
4
.3. Determination of Minimal Inhibition Concentration (MIC)
The minimal inhibitory concentration of tested compounds was determined following the
NCCLS standard. The experiments were conducted in 96-well microtiter plates using 16-point
2
-fold dilutions in Mueller-Hinton broth to 100, 50, 25, 12.5, and 6.3 µM, etc. Staphylococcus
o
aureus culture (100 µL) was added to Mueller-Hinton broth (1900 µL), and incubated at 37 C
–
5
for 2–4 h in shaker. The bacterial culture (1 mL) was taken, diluted to OD600 of 5 × 10 , and
dispensed to all wells of an assay plate containing test compounds at different concentrations.
o
After incubation at 37 C for 16–18 h, the minimal concentration of the compound that
prevent at least 95% bacterial growth was determined as the MIC value.
4
.4. Molecular Modeling
The molecular modeling was conducted by using Schrödinger Suit (Schrödinger, LLC,
New York). Initially, compound structures and the corresponding low-energy 3D structures
with possible ionization states at pH 7.0 were generated by using LigPrep. The crystal
structure of S. aureus monofunctional TGase (MTG) was obtained from RCSB Protein Data
Bank (PDB coded 3VMR) and subjected to energy refinement by Prime after addition of
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hydrogen atoms. The OPLS_2005 force field was applied in all works. The refined TGase
structure was applied as the target for molecular docking. A grid box (22×22×22 Å in xyz
directions) centralizing the moenomycin in the donor site of the TGase molecule was
produced by using Glide. The van der Waals radii of the protein atoms were scaled by 0.80
with a partial atomic charge cutoff of 0.25 to soften the potential for nonpolar parts of the
protein structure. During docking experiments, the SP mode of Glide was applied, such that
compounds were sampled flexibly while the target protein was kept in rigid. Plausible
docking modes were selected and refined further by using Prime with a VSGB solvation
model to obtain the final protein−compound complexes.
4
.5. Synthetic Procedures and Characterization of Compounds
.5.1.
4
Ethyl (S)-3-(dimethoxylphosphoryl)-2-hydroxypropanoate (18)
To a solution of (R)-epoxide 17 (6 g, 52 mmol) in anhydrous THF (11 mL) were added
dimethyl trimethylsilylphosphite (35 mL, 18 mmol) and ZnCl (2.3 mL of 1.0 M solution in
2
o
Et O, 2.3 mmol). The mixture was stirred at 50 C for 18 h, another portion of dimethyl
2
trimethylsilylphosphite (10 mL, 52 mmol) was added, and the reaction temperature was raised
o
to 70 C. The mixture was stirred for another 24 h, and then quenched with saturated
NaHCO3(aq) solution. The mixture was concentrated under reduced pressure to remove excess
2
8