Staphylococcus aureus penicillin-binding protein 2 can use depsi-lipid ii derived from vancomycin-resistant strains for cell wall synthesis

Article abstract of DOI:10.1002/chem.201301074

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin. Copyright

Full text of DOI:10.1002/chem.201301074

DOI: 10.1002/chem.201301074
Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II
Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis
Jun Nakamura,[a] Hidenori Yamashiro,[b] Hiroto Miya,[a] Kenzo Nishiguchi,[b]
Hideki Maki,*[b] and Hirokazu Arimoto*[a]
Abstract: Vancomycin-resistant Staphy-
lococcus aureus (S. aureus) (VRSA)
uses depsipeptide-containing modified
cell-wall precursors for the biosynthesis
of peptidoglycan. Transglycosylase is
responsible for the polymerization of
the peptidoglycan, and the penicillin-
binding protein 2 (PBP2) plays a major
role in the polymerization among sev-
eral transglycosylases of wild-type S.
aureus. However, it is unclear whether
VRSA processes the depsipeptide-con-
taining peptidoglycan precursor by
using PBP2. Here, we describe the
total synthesis of depsi-lipid I, a cell-
wall precursor of VRSA. By using this
chemistry, we prepared a depsi-lipid II
analogue as substrate for a cell-free
transglycosylation system. The reconsti-
tuted system revealed that the PBP2 of
S. aureus is able to process a depsi-lipi-
d II intermediate as efficiently as its
normal substrate. Moreover, the system
was successfully used to demonstrate
the difference in the mode of action of
the two antibiotics moenomycin and
vancomycin.
Keywords: antibiotics · enzymes ·
lipids · peptides · vancomycin
Introduction
serious threat because of the more harmful nature of S.
aureus as a nosocomial pathogen.[8]
Drug resistance is an unavoidable consequence of the clini-
cal use of antibiotics. Particularly, vancomycin-resistant
strains among enterococci and Staphylococcous aureus (S.
aureus) are of serious concern, because vancomycin is some-
times the only available antibiotic left for severe infections
caused by multi-resistant gram-positive bacteria. Vancomy-
cin resistance was initially identified in enterococci in the
1980s[1] and is currently widely spread all over the world.[2]
For example, a report indicated that 76% of clinically isolat-
ed Enterococcus faecium in intensive care units in the USA
were vancomycin resistant.[3] Although much less frequent
than enterococci, horizontal transfer of the resistant gene
from enterococci to Staphylococcus aureus,[4] as reported in
the USA,[5] India,[6] and Iran,[7] may become an even more
Vancomycin owes its characteristic antibacterial action to
the binding with the d-alanyl-d-alanine terminal (d-Ala-d-
Ala terminal, Figure 1) of bacterial cell wall intermediates.[9]
However, in the most resilient class of vancomycin-resistant
bacteria (VanA and VanB phenotypes), the d-Ala-d-Ala ter-
minal is replaced with d-alanyl-d-lactate (d-Ala-d-Lac).[10]
Vancomycin lacks the ability to bind to this ester-containing
peptide (depsipeptide), and thus does not inhibit the bacte-
rial cell wall biosynthesis. Under these circumstances, efforts
have been made to develop novel antibacterial agents
against vancomycin-resistant strains.[11]
In vitro reconstitution of the bacterial biosynthesis of
macromolecules can serve as a valuable tool for the rational
design of novel antibacterial candidates. Peptidoglycan is
the major constituent of bacterial cell wall, and its biosyn-
thesis involves a number of successive enzymatic transfor-
mations. We have recently reported a cell-free assay that re-
constitutes the late stage of the biosynthesis of peptidogly-
can in vancomycin-resistant S. aureus (VRSA).[12]
[a] Dr. J. Nakamura, H. Miya, Prof. Dr. H. Arimoto
Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira
Aoba-ku, Sendai, 980-8577 (Japan)
Fax : (+81)0-22-217-6204
E-mail: arimoto@biochem.tohoku.ac.jp
This in vitro assay uses cell membrane of S. aureus that
contains enzymes involved in the transformations shown in
Figure 2. It provides nascent peptidoglycan from UDP-
MurNAc-pentapeptide (vancomycin-susceptible model;
UDP=uridine diphosphate, MurNAc=N-acetylmuramic
acid) or UDP-MurNAc-pentadepsipeptide (resistant model).
The modes of action of the vancomycin derivatives were an-
alyzed by using this assay, and differences were successfully
shown for a lipoglycopeptide[12] and vancomycin dimers in
the resistant model.[13] However, a limitation of this assay is
that inhibition by antibacterials is provided as a general
[b] H. Yamashiro, Dr. K. Nishiguchi, Dr. H. Maki
Medicinal Research Laboratories, Shionogi & Co., Ltd., 3-1-1
Futaba-cho
Toyonaka, Osaka, 561-0825 (Japan)
E-mail: hideki.maki@shionogi.co.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301074.
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of Creative
Commons the Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial and no modifica-
tions or adaptations are made.
12104
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Chem. Eur. J. 2013, 19, 12104 – 12112

FULL PAPER
scant evidence for the contribution of each transglycosylase
to the cell wall synthesis of vancomycin-resistant strains.
Identification of the transglycosylase(s) involved in cell wall
synthesis of VRSA is thus an important step for the discov-
ery of novel antibacterial agents against VRSA.
Here, we describe a reconstitution of PBP2 reaction by
using a recombinant S. aureus enzyme and its substrates, lip-
id II (wild-type) and depsi-lipid II analogues (VRSA).
Depsi-lipid I (3) and its analogue (4, Figure 3) were pre-
pared by total synthesis. The analogue 4 was converted into
a depsi-lipid II analogue by enzymatic addition of an N-ace-
tylglucosamine. Kinetic analysis of the transglycosylation re-
vealed that S. aureus PBP2 can process a depsi substrate
with an efficiency similar to that for processing a normal
substrate. The results of this study also show that cell-free
peptidoglycan polymerization with a depsi substrate can
shed light on the mode of action of cell wall-targeting antibi-
otics.
Results and Discussion
Total synthesis of depsi-lipid I and its analogue: The first
major obstacle in this study was the supply of depsi-lipid in-
termediates,[10a] that is, depsi-lipid I (Figure 3) and depsi-lipi-
d II (Figure 1), as mandatory substrates for MurG and PBP2
reactions (Figure 2). These depsi-lipid intermediates are
common to VRSA and vancomycin-resistant enterococci
(VRE). Although, as previously reported,[12] isolation of
cell-wall precursors in the early
Figure 1. Structure of bacterial cell wall building blocks, wild-type lipid II
(1) and depsi-lipid II (2), and their mode of interaction with vancomycin.
stage of the biosynthesis of
peptidoglycan is possible, all
precursors are lipidated with
undecaprenyl-pyrophosphate
(C55) in the late stage of the
synthesis of peptidoglycan. Pu-
rification of these cell-wall pre-
cursors from natural resources
has been proven to be difficult
(even for wild-type lipid inter-
mediates),[16] and chemical syn-
thesis is currently the only way
to obtain these precursors.[17]
The total synthesis of wild-type
Figure 2. Late-stage cell wall synthesis of S. aureus. In vancomycin-resistant S. aureus. The d-Ala-d-Ala-termi-
nal of the pentapeptide moiety is replaced with d-Ala-d-Lac. M=MurNAc; G=N-acetylglucosamine
(GlcNAc); MurG=N-acetylglucosamyl transferase; FemA, FemB, and FemX: peptidyltransferases.
effect without information on the specific action at each en-
zymatic step.
lipid I with a d-Ala-d-Ala terminal was reported by Van-
Nieuwenhze et al.,[18] and that of lipid II has been reported
by Schwartz et al.[19] and VanNieuwenhze et al.[20] Further-
more, Kahne et al. synthesized a series of lipid intermediate
analogues carrying a truncated polyisoprenyl chain and
demonstrated that the analogue with a heptaprenyl subunit
(C35) had excellent physical properties for application in
cell-free transglycosylation catalyzed by the PBP enzyme.[21]
Chemical synthesis of cell-wall precursors derived from
vancomycin-resistant bacteria has rather been limited. Wong
et al. synthesized an early precursor, UDP-MurNAc-depsi-
pentapeptide (UDP-N-acetylmuramyl-l-Ala-d-Glu-l-Lys-d-
Ala-d-Lac), which was then converted to nascent peptido-
S. aureus penicillin-binding protein 2 (PBP2) is a bifunc-
tional enzyme that catalyzes the peptidoglycan polymeriza-
tion (transglycosylation, Figure 2) and its subsequent cross-
linking (transpeptidation). Inhibitors of the transglycosyla-
tion are therefore considered as promising antibacterial can-
didates. Although PBP2 is reported to play a major role in
transglycosylation of wild-type S. aureus, this strain has sev-
eral other enzymes containing a transglycosylase domain,
such as PBP1,[14] Mgt, and SgtA.[15] Moreover, all isolated
VRSA are methicillin resistant and have the PBP2a isozyme
required for methicillin resistance. Currently, there is only
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H. Maki, H. Arimoto et al.
Figure 3. Retrosynthetic analysis for depsi-lipid I and its analogue. Bn=benzyl, Teoc=2-(trimethylsilyl)ethoxycarbonyl, TMSE=2-(trimethylsilyl)ethyl,
Ac=acyl.
glycan by using crude bacterial enzymes.[22] Because lipidat-
ed depsipeptide intermediates could not be easily isolated in
the enzymatic transformation, we initiated our study by
chemically synthesizing depsi-lipid I (3) and its analogue 4
with a shorter isoprenyl unit. Our retrosynthetic strategy is
outlined in Figure 3. As the depsi intermediate involves a
hydolytically labile carboxyl ester linkage, the synthesis[18] of
the wild-type lipid I by using base-cleavable protecting
groups could not be directly applied to the synthesis of
depsi-lipid I. Instead, the trimethylsilylethyl ester was used.
This mildly cleavable protecting group was used by Walker
et al. in the synthesis of lipid intermediate analogues,[21a] and
then by Wong et al. in the synthesis of UDP-MurNAc-depsi-
pentapetide.[22]
There are two approaches to the preparation of wild-type
lipid intermediates. One connects the protected MurNAc
Figure 4. Synthesis of the phosphomuramyl peptide 8. Reagents and con-
ditions: a) l-alanine-2-(trimethylsilyl)ethyl ester, diisopropylethylamine
(DIPEA), 1-hydroxybenzotirazole (HOBt), benzotriazolyl-1-oxy-tripyrro-
lidinophosphonium hexafluorophosphate (PyBOP), THF/CH2Cl2, RT,
with the pentapeptide,[18,23] and the other connects the pro-
tected MurNAc-l-Ala with the remaining tetrapeptide.[19,20]
In this study, we adopted the latter strategy by using a tetra-
depsipeptide.
59%; b) 10% Pd/C, H2 gas, MeOH/EtOAc, RT, 83%; c) 1H-tetrazole, di-
benzyl-N,N-diisopropylphosphoramidite, À40 to À108C, then meta-chloro-
perbenzoic acid (m-CPBA), À408C, CH2Cl2, 56%; d) TBAF, THF, 08C
Synthesis of phosphoMurNAc-depsipeptide: Our synthesis
began with the addition of l-alanine trimethylsilylethyl
ester[24] to the known MurNAc derivative 10 (Figure 4).[19]
Selective deprotection of the benzyl ether in the presence of
benzylidene acetal was achieved by catalytic hydrogenation
in EtOAc/MeOH (1:1). This modification significantly facili-
tated our synthesis. The protected glycosyl monophosphate
was introduced by using the phosphoramidite method. Com-
to RT, 92%.
LysACTHUNRTGNEUNG(Teoc)-OH (Cbz=carbobenzyloxy) was prepared from
Cbz-l-Lys by using N-[2-(trimethylsilyl)ethoxycarbonyloxy]-
succinimide. Cbz-d-Glu-OTMSE was synthesized from Boc-
d-GluACTHNURGTNE(NUG OBn)-OH in four steps. These protected units were
assembled into the tetradepsipeptide 9 by using standard
solution conditions (Figure S2 in the Supporting Informa-
tion).
The carboxylate terminal of the muramic acid derivative 8
was first activated by HATU and the resulting mixture was
reacted with the depsitetrapeptide 9 to give the muramyl
pentadepsipeptide 7[22] in 81% yield (Figure 5). Reductive
removal of the two benzyl groups at the phosphate moiety,
followed by acidic hydrolysis of the benzylidene acetal pro-
vided compound 13.
1
parison of H and 13C NMR data of 12 was in good agree-
ment with the results reported by Schwartz et al.[19] Removal
of the 2-trimethylsilylethyl ester by tetrabutylammonium
fluoride (TBAF) provided the carboxylic acid 8.
The tetradepsipeptide 9 (Figure 5) was prepared by solu-
tion-phase synthesis by using protected amino acids and lac-
tate (Figure S1 in the Supporting Information). The trime-
thylsilylethyl lactate (d-Lac-OTMSE) was synthesized from
the methyl lactate by using a patent procedure.[25] Cbz-l-
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Cell Wall Synthesis of Staphylococcus aureus
FULL PAPER
mercially available, we synthe-
sized it from nerol and farnesol
in eighteen longest linear steps
(Figure S3 in the Supporting
Information) according to the
methods of Walker et al.[23] and
Sato et al.[28]
Preparation of the depsi-
lipid II analogue as substrate
for the transglycosylase reac-
tion: MurG is a membrane-as-
sociated enzyme that converts
wild-type lipid I to lipid II
(Figure 2). In this study, Es-
cherichia coli (E. coli) MurG
was purified according to the
method described in a previous
report by Walker et al.[23]
Briefly, MurG was overpro-
duced in E. coli and purified
by Ni2+-affinity column chro-
matography. After further pu-
rification by size exclusion
chromatography, sodium do-
decyl sulfate polyacrylamide
Figure 5. Synthesis of the depsi-lipid I and its C35 analogue. Reagents and conditions: a) compound 9, O-(7-
azabenzotriazol-1-yl)-tetramethyluroium hexafluorophosphate (HATU), DIPEA, DMF, 08C, 1 h, 81 %;
b) 10% Pd/C, H2 gas, MeOH, RT, 30 min, 94%; c) 80% AcOH(aq), RT, 4 d, 91%; d) pyridine, N,N’-carbonyl-
diimidazole (CDI), THF/DMF (4:1), RT, 3 h, then MeOH; undecaprenylphosphate ammonium salt 5 or 6,
1H-tetrazole, THF/DMF (4:1), RT, 2 d, (78%); f) TBAF, THF/DMF, 08C to RT, 2 d, 23% (depsi-lipid I, 3) or
34% (depsi-lipid I analogue 4).
gel
electrophoresis
(SDS-
PAGE) analysis showed
a
single protein band at about
40 kDa. Walker et al. reported
a reconstitution of the cell wall
Construction of pyrophosphate linkage: Connection of the
phosphomuramylpeptide to the polyisoprenyl phosphate re-
quires strict anhydrous conditions, and the yields are often
moderate. In the proceeding synthetic studies of wild-type
lipid intermediates, either a phosphosugar[18–20] or a polyiso-
prenyl phosphate[21a,26,27] could be activated with CDI and
the remaining part was added for coupling. We decided to
activate the phosphosugar 13 with CDI in the presence of
pyridine. After activation, excess CDI was quenched by
methanol and the solvents were removed to dryness under
reduced pressure. A solution of undecaprenyl phosphate
bis-ammonium salt and 1H-tetrazole in DMF was added to
the activated phosphosugar and the mixture was left to react
at room temperature for two days. Progress of the reaction
was monitored by LC-MS. The desired protected depsi-
lipid I was separated by gel filtration chromatography (LH-
20, MeOH), and the crude product was treated with TBAF
at 08C to eliminate the silyl protecting groups. The reaction
mixture was evaporated to dryness, and gel filtration chro-
matography (LH-20, MeOH) and lyophilization provided
the semi-purified depsi-lipid I, which was further purified by
synthesis by using C35-lipid I and E. coli MurG, because
wild-type lipid I with a C55-isoprenyl chain aggregates in a
manner that precludes the reaction in solution.[21a] Thus, we
used the C35-depsi-lipid I 4 in this study. The MurG reac-
tion was carried out in a buffer containing the C35 analogue
and UDP-(14C)-GlcNAc as substrates. Progress of the reac-
tion was monitored by incorporation of radiolabeled
GlcNAc into the depsi-lipid II analogue. Purification with
high-performance liquid chromatography provided the radi-
olabeled depsi-lipid II analogue (ꢀ2 mg, Figure S5 in the
Supporting Information). The C35-lipid II with a d-Ala-d-
Ala terminal (Figure S5 in the Supporting Information) was
prepared as reference substrate for the enzyme assay by
chemical synthesis according to a reported procedure.[21a]
Transglycosylation of the radiolabeled depsi-lipid II by S.
aureus PBP2: By using the radiolabeled product of the
MurG reaction, we next investigated the polymerization of
peptidoglycan with purified S. aureus PBP2 enzyme. A full-
length PBP2 of S. aureus RN4220 strain[29,30] was prepared
as previously described. The C35-lipid II containing a d-Ala-
d-Ala terminal was used for the vancomycin-susceptible
model, and the C35-depsi-lipid II with a d-Ala-d-Lac termi-
nal was used for the resistant model. The activity of PBP2
with each substrate was evaluated by measurement of the
peptidoglycan incorporated radioactivity by using thin-layer
HPLC by using a C8 column (MeOH/ACTHNUGTRNEG(UN NH4)HCO3(aq)) to
give the desired depsi-lipid I (3) (2.2 mg, 96% purity).
By using a similar protocol, the depsi-lipid I analogue 4
with a heptaprenyl (C35) group was prepared. Because the
heptaprenyl monophosphite bis-ammonium salt is not com-
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12107

H. Maki, H. Arimoto et al.
chromatography (TLC). The obtained kinetic parameters
Km, Vmax, and kcat of the PBP2 reaction are shown in Table 1.
The kinetic parameters for the S. aureus PBP2 reaction
were previously determined by Walker et al. at pH 5.0 by
Table 1. Kinetic parameters of the PBP2[a] reaction.[b]
Susceptible model
Resistant model
Vmax [mmsÀ1
Km [mm]
]
(0.91Æ0.16)
(11.0Æ1.6)
(1.2Æ0.56)
(6.0Æ2.2)
kcat [10À3 sÀ1
]
(2.5Æ0.4)
(3.2Æ1.5)
[a] S. aureus PBP2. [b] C35-lipid II was used.
using the same C35-lipid II, which corresponds to that of
our susceptible model.[30] In our susceptible model, the reac-
tion was slightly slower at pH 7.0, but the obtained parame-
ters were within 6-fold those obtained by Walker et al. at
pH 5.0. This may be rationalized by considering the reported
optimum pH value of S. aureus PBP2 for wild-type lipid II
as substrate (pH 4.5–5.5).
With the C35-depsi-lipid II substrate, transglycosylation at
pH 7.0 was faster and had a Km value comparable to that of
C35-lipid II, which indicates that depsi-lipid II can also be
an excellent substrate of PBP2.
Characterization of modes of action of the antibiotics within
the reconstituted PBP2 reaction: We next examined wheth-
er the in vitro PBP2 reaction can be used to show differen-
ces in the action of the antibiotics. Among all enzymatic
transformations in the late-stage cell wall biosynthesis, PBP2
is unique in that it works extracellularly. Some antibiotics,
such as vancomycin do not penetrate the bacterial mem-
brane and are effective only at the outer surface of the bac-
terial membrane. This indicates that PBP is a good target
for antibacterial drugs. In our susceptible and resistant
models of the PBP2 reactions, two cell-wall-targeting antibi-
otics, that is, vancomycin and moenomycin, exhibited dis-
tinct inhibitory patterns (Figure 6A and Table 2). Moeno-
mycin suppressed the peptidoglycan polymerization regard-
less of the use of normal or depsi-lipid II analogues. Vanco-
mycin, on the other hand, suppressed the peptidoglycan pol-
ymerization only when normal lipid II analogue was used.
This is because, as described earlier, vancomycin is a sub-
strate-binding antibiotics that cannot bind to the d-Ala-d-
Lac terminal of depsi-lipid II. The d-Ala-d-Ala terminal of
the normal lipid intermediate is a crucial motif for the activ-
ity of vancomycin. In contrast, moenomycin inhibits the
PBP2 reaction by directly binding to the enzyme,[31] and
thus is able to suppress in vitro transglycosylation regardless
of the substrate used.[32]
Figure 6. Inhibitory effect of vancomycin and moenomycin on the PBP2
~
~
*
*
reaction. and =lipid II,
and =depsi-lipid II. PG: peptidoglycan.
Table 2. Inhibitory effect of vancomycin and moenomycin on the PBP2[a]
reaction.
IC50 [mm]
susceptible model[b]
resistant model[c]
vancomycin
moenomycin
(1.52Æ0.52)
(0.16Æ0.08)
>64
(0.19Æ0.03)
[a] S. aureus PBP2. [b] C35-lipid II was used. [c] C35-depsi-lipid II was
used.
Chemical synthesis of depsi-lipid I and its analogue estab-
lished the basis of this study.
The clinically important classes of vancomycin-resistant
bacteria (VanA and VanB phenotypes) induce expression of
resistance-related enzymes that build the UDP-MurNAc-
pentadepsipeptide with the d-alanyl-d-lactate terminus as a
modified peptidoglycan precursor. Enzymes responsible for
the conversion of this precursor into the peptidoglycan are
unaltered and seem to accept modified substrates. However,
VRSA has three PBPs (PBP1, PBP2, and PBP2a) with a
transglycosylase domain and monofunctional transglycosy-
lases (e.g., Mgt and SgtA). There has only been scant evi-
dence for the contribution of each transglycosylase to the
Conclusion
We have shown in this study that PBP2 is able to catalyze
the transglycosylation of a VRSA peptidoglycan precursor.
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Cell Wall Synthesis of Staphylococcus aureus
FULL PAPER
thylsilane (0 ppm), CHCl3 (7.26 ppm), CHD2OD (3.31 ppm), (CHD2)-
AHCTUNGTREN(GNUN CD3)SO (2.5 ppm), and water (3.33 ppm in [D6]DMSO), and in the
polymerization of the abnormal depsipeptide-containing
precursor. The current study indicates that depsi-lipid II can
also be an excellent substrate of PBP2. Nonetheless, we do
not eliminate the possibility that other transglycosylases
could also use depsi-lipid II. Actually, Pinho et al. reported
that monofunctional transglycosylases are not essential for
the cell-wall synthesis of vancomycin-susceptible S. aureus,
whereas in the absence of PBP2 activity, the monofunctional
transglycosylase Mgt can be the sole enzyme for the pepti-
doglycan polymerization.[15] These observations indicate the
need for additional studies with depsi-lipid II to examine
whether Mgt and other transglycosylases are able to process
the VRSA cell-wall precursor. If monofunctional transglyco-
sylases were able to use the precursor, dual inhibitors of
PBP2 and the monofunctional transglycosylases might be re-
quired for an efficient suppression of the cell wall synthesis
of VRSA.
We also demonstrated in this study that the reconstituted
PBP2 reaction with a depsi substrate is an indispensible tool
in the search for antibacterials against VRSA. Antibiotics
that require d-Ala-d-Ala binding (i.e., vancomycin) did not
suppress the PBP2 reaction with depsi-lipid II, whereas anti-
biotics with direct interaction with the PBP2 enzyme did.
Reconstitution of the PBP2 reaction with a d-Ala-d-Ala-
containing normal substrate has actually been reported.[33]
However, the use of a depsipeptide-containing substrate
allows detailed evaluation of the transglycosylase inhibitor–
PBP2 interaction without interference of the binding be-
tween the inhibitor and the PBP2 substrate containing d-
Ala-d-Ala.[34]
13C NMR spectra relative to the residual solvent peak of CHCl3
(77.16 ppm). 31P NMR spectra were recorded using 85% H3PO4 (0 ppm)
as an external standard. Coupling constants are given in Hertz [Hz].
Thin-layer chromatography (TLC) was performed by using Merck silica
gel 60 F254 precoated plates (0.25 mm). Flash chromatography was car-
ried out by using 60–230 mesh silica gel (Kanto Chemical, silica gel 60N).
The purity of the compounds was assessed by HPLC under the following
conditions: method I: Cosmosil 5C18-AR-II (150ꢁ4.6 mm, Nacalai
Tesque), 15 to 100% acetonitrile/water containing 0.1% trifluoroacetic
acid (TFA), flow rate=1.0 mLminÀ1, duration=10 min, temperature=
308C, detection at l=280 nm; method II: Unison UK-C8 (150ꢁ4.6 mm,
particle size 3 mm, Imtakt), 80 to 95% acetonitrile/water containing 0.1%
NH4HCO3, flow rate=1.0 mLminÀ1, duration=10 min, room tempera-
ture, UV detection at l=214 nm).
Synthesis compound 11: l-Ala-OTMSE (0.312 g, 0.1.65 mmol), DIPEA
(0.6 mL, 3.4 mmol), PyBOP (0.98 g, 1.9 mmol), and HOBt (0.23 g,
1.7 mmol) were added to a solution of benzyl-4,6-O-bezylidene-MurNAc
(10) (0.81 g, 1.7 mmol) in dry THF/dry dichloromethane (1:1) (20 mL).
The mixture was stirred at room temperature for 5 h and partitioned be-
tween a water layer (50 mL) and an organic layer (ethyl acetate, 5ꢁ
50 mL). The combined organic layers were washed with brine (100 mL),
dried over anhydrous magnesium sulfate, and evaporated to give the
crude compound 11 as an oil. Purification by silica gel column chroma-
tography (chloroform/methanol 40:1) yielded 11 as a colorless solid
(0.64 g, 59%).
Rf =0.68
(chloroform/methanol=9:1);
1H NMR
(500 MHz, CDCl3, 208C): d=7.47–7.32 (complex multiplet (comp.),
10H), 6.93 (d, J=7 Hz, 1H), 6.28 (d, J=8.5 Hz, 1H), 5.56 (s, 1H), 4.98
(s, 1H), 4.72 (d, J=12 Hz, 1H), 4.50 (d, J=12 Hz, 1H), 4.46 (m, 1H),
4.28 (dt, J=9.5, 5.5 Hz, 1H), 4.27–4.19 (comp., 3H), 4.13 (q, J=7 Hz,
1H), 3.88 (dt, J=9, 4.5 Hz, 1H), 3.79–3.67 (comp., 3H), 1.94 (s, 3H),
1.42 (d, J=7.5 Hz, 3H), 1.39 (d, J=7 Hz, 3H), 1.01ACHTNUTRGNEUNG(m, 2H), 0.03 ppm (s,
9H); 13C NMR (125 MHz, CDCl3, 208C): d=172.8, 172.6, 170.4, 137,
136.8, 128.9, 128.6, 128.3, 128.1, 125.1, 101.4, 97.5, 81.7, 78.2, 77.5, 70.1,
68.8, 63.7, 63.1, 53, 48, 23.4, 19.4, 17.9, 17.2, À1.55 ppm; HRMS (FAB)
calcd for C33H47N2O9Si+: 643.3051 [M+H]+; found: 643.3055 [M+H]+.
In conclusion, we performed in this study the first chemi-
cal synthesis of the modified cell-wall precursors depsi-
lipid I (3) and its analogue 4. These precursors enabled the
reconstitution of the very last stage of the biosynthesis of
peptidoglycan. The in vitro PBP2 reaction by using both
normal and modified peptidoglycan precursors would open
a new way for understanding the mode of action of anti-
VRSA/VRE compounds.
Synthesis of compound 12: Methanol (13 mL) and 10% Pd/C (1.5 g,
150% w/w) were added to benzyl-4,6-O-benzylidene-MurNAc-l-Ala-
OTMSE (11) (1.0 g, 1.6 mmol) dissolved in ethyl acetate (13 mL). Selec-
tive removal of the benzyl group was succeeded by catalytic reduction
under a hydrogen atmosphere. After stirring at room temperature for
12 h, the reaction mixture was filtered to remove the Pd/C catalyst and
then evaporated to give the crude hemiacetal product. Purification of the
crude material by silica gel column chromatography (chloroform/metha-
nol 20:1 to 5:1) provided the hemiacetal as a colorless amorphous
(0.73 g, 83%). Rf =0.37 (chloroform/methanol 9:1); 1H NMR (500 MHz,
CDCl3, 208C): d=7.46–7.36 (comp., 5H), 7.01 (d, J=7 Hz, 1H), 6.87 (d,
J=6.5 Hz, 1H), 5.56 (s, 1H), 5.37 (s, 1H), 4.51 (quin., J=7 Hz, 1H),
4.27–4.13 (comp., 5H), 4.04 (dt, J=9.5, 4.5 Hz, 1H), 3.75 (m, 2H), 3.63
(t, J=9.5 Hz, 1H), 2.72 (s, 1H), 2.02 (s, 3H), 1.84 (s, 1H), 1.43 (d, J=
7 Hz, 3H), 1.41 (d, J=7 Hz, 3H), 1.03 (m, 2H), 0.039 ppm (s, 9H);
13C NMR (125 MHz, CDCl3, 208C): d=173.3, 172.7, 171.3, 137.1, 128.9,
128.1, 125.9, 101.2, 91.1, 81.7, 77.8, 76.9, 68.8, 63.8, 62.5, 53.7, 48, 23.2,
19.3, 17.7, 17.1, À1.61 ppm; HRMS (FAB) calcd for C26H41N2O9Si+:
553.2581 [M+H]+; found: 553.2579 [M+H]+.
Experimental Section
General methods: All reagents and solvents were used as purchased
from commercial suppliers. Undecaprenyl phosphate ammonium salt was
purchased from Larodan Fine Chemicals AB (Malmç, Sweden). [14C]-
UDP-GlcNAc was obtained from American Radiolabeled Chemicals,
Inc. (Saint Louis, USA). All reactions were carried out at room tempera-
ture unless otherwise noted. Mass spectra were obtained by using a
JEOL LMS-700 mass spectrometer (FAB MS) and a Bruker Daltonics
microTOF-TSfocus (LC-ESI-TOF MS). LC was performed on an Agilent
1100 Series, by using an Imtakt UNISON UK 3C8 column (150ꢁ2 mm)
under the following conditions: flow rate=0.2 mLminÀ1, 65 to 95% ace-
tonitrile/water containing 0.1% formic acid, duration=5 min, tempera-
ture=408C, and detection at l=214, 254, 280, and 190–400 nm. The
monoisotopic mass was used for calculation of the exact mass (C=
12.0000, H=1.0078, O=15.9949, N=14.0031, Cl=35.9689). Nuclear
The hemiacetal product of the above-described reaction (1.3 g, 2.4 mmol)
and 1H-tetrazole (1 g, 14 mmol) were azeotroped with toluene/dichloro-
methane (1:1) (2ꢁ30 mL), and then placed under vacuum. Dry dichloro-
methane (120 mL) was then added and the mixture was cooled to
À688C. After dropwise addition of dibenzyl-N,N-diisopropylphosphora-
midite (2.4 mL, 7.2 mmol) over 5 min, the reaction mixture was warmed
to À408C over 2 h, at which point the starting material was no longer evi-
dent. The reaction mixture was then cooled to À658C and m-CPBA
(3.6 g, 22 mmol) was added. The mixture was allowed to warm to À408C
over 1 h, and poured into ethyl acetate (0.4 L). The organic layer was
successively washed with 50% sodium sulfite(aq) (0.4 L) and brine
(0.3 L), dried over anhydrous magnesium sulfate, and concentrated under
magnetic resonance spectra were recorded on
a JEOL ECA600 at
600 MHz, a Varian Unity INOVA 500 at 500 MHz, and an INOVA 600
at 600 MHz. Chemical shifts (d) are reported in parts per million [ppm]
in the 1H NMR spectra relative to the residual solvent peak of tetrame-
Chem. Eur. J. 2013, 19, 12104 – 12112 ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
12109

H. Maki, H. Arimoto et al.
vacuum to yield the crude 12 (7.8 g). Purification of the crude 12 by silica
gel column chromatography (chloroform/methanol 20:1), followed by
alumina column chromatography (Al2O3 activated, Nacalai Tesque,
chloroform/methanol 20:1) gave compound 12 as a colorless amorphous
(1.1 g, 56%). Rf =0.57 (chloroform/methanol 9:1); 1H NMR (500 MHz,
CDCl3, 208C): d=7.47–7.34 (comp., 15H), 6.79 (d, J=7.5 Hz, 1H), 6.69
(d, J=8 Hz, 1H), 5.79 (dd, J=5, 4.3 Hz, 1H), 5.53 (s, 1H), 5.12 (m, 4H),
4.5 (quin., J=7.5 Hz, 1H), 4.28–4.19 (comp., 3H), 4.15 (q, J=6.5 Hz,
1H), 4.09 (dd, J=11, 7.8 Hz, 1H), 3.91 (dt, J=10, 5 Hz, 1H), 3.67 (q, J=
9.6 Hz, 2H), 3.61 (t, J=10 Hz, 1H), 1.82 (s, 3H), 1.42 (d, J=7.5 Hz, 3H),
1.39 (d, J=6.5 Hz, 3H), 1.01 (m, 2H), 0.036 ppm (s, 9H); 13C NMR
(125 MHz, CDCl3, 208C): d=172.7, 172.6, 170.8, 136.9, 135.47, 135.42,
135.25, 129.1, 128.8, 128.68, 128.66, 128.3, 127.99, 127.88, 125.9, 101.4,
96.8, 81, 77.8, 75.9, 69.8, 68.3, 64.6, 63.8, 53.2, 48, 22.9, 19.3, 18, 17.2,
À1.59 ppm; HRMS (FAB) calcd for C40H53N2NaO12PSi+: 835.3003
[M+Na]+; found: 835.3007 [M+Na]+.
1H), 4.68 (q, J=4.4 Hz, 1H), 4.58–4.50 (comp., 4H), 4.47–4.42 (comp.,
4H), 4.35 (t, J=4.7 Hz, 2H), 4.11 (brs, 2H), 3.75 (brs, 1H), 3.31 (s, 2H),
2.54–2.45 (comp., 3H), 2.29–2.17 (comp., 5H), 2.01 (m, 1H), 1.91 (m,
1H), 1.71–1.65 (comp., 18H), 1.28–1.18 (comp., 6H), 0.10 ppm (m, 27H);
13C NMR (150 MHz, CD3OD, 208C): d=175.9, 174.8, 174.5, 173.5, 172.8,
172, 159.2, 109, 80.7, 78.4, 74.9, 70.6, 70.5, 64.73, 64.69, 63.6, 62.5, 54.9,
54.7, 52.9, 50.6, 41.3, 32.6, 32.4, 30.5, 28.2, 23.9, 23.1, 19.7, 18.6, 18.3, 18.2,
18.1, 17.1, 17.1, À1.39, À1.48 ppm; HRMS (LC-ESI-TOF) calcd for
C47H88N6O21PSi3À: 1187.5053 [MÀH]À; found: 1187.5019 [MÀH]À.
Synthesis of the depsi-lipid I analogue 4: Dry pyridine (3.5 mL) was
added to the phospho-MurNAc-silyl-protected-pentadepsipeptide-TMSE
13 (25 mg, 21 mmol) and excess pyridine was removed under reduced
pressure to give the pyridinium salt. This salt was azeotropically dried
with toluene/benzene (1:1, 2ꢁ1 mL) under vacuum, and the residue was
dissolved in dry THF/DMF (4:1, 0.25 mL) and then added to a flask con-
taining a solution of CDI (10 mg, 63 mmol) in dry THF/DMF (1:1, 1 mL)
at 08C (ice/water bath) through a cannula. The whole was finally rinsed
with dry THF/dry DMF (1:1, 3ꢁ0.4 mL). The reaction mixture was first
allowed to warm to room temperature and then stirred for further 3 h at
which point the reaction was complete. Dry methanol (3 mL) was added
to the reaction mixture and the whole was evaporated to remove THF.
The residue was azeotropically dried with toluene/benzene (1:1, 1 mL) to
yield a solution of the activated phosphoimidazolide of 13 in DMF.
Synthesis of compound 8: A solution of tetrabutylammonium fluoride
(1.0m) in THF (0.65 mL) was added to a solution of benzylphospho-4,6-
O-benzylidene-MurNAc-l-Ala-OTMSE (12) (0.45 g, 0.56 mmol) in dry
THF (28 mL) at 08C. After stirring at 08C for 1 h, the reaction mixture
was warmed to room temperature and stirred for additional 2 h. The mix-
ture was then evaporated under reduced pressure and the resulting resi-
due was subjected to silica gel column chromatography (chloroform/
methanol 4:1) to yield 8 as a colorless oil (0.39 mg, 99%). Rf =0.47
(chloroform/methanol 9:1); 1H NMR (500 MHz, CD3OD, 208C): d=
7.45–7.28 (comp., 15H), 5.83 (dd, J=6, 4.8 Hz, 1H), 5.57 (s, 1H), 5.07–
5.03 (comp., 4H), 4.31–4.26 (comp., 2H), 4.05 (m, 1H), 3.98 (dd, J=8,
4 Hz, 1H), 3.78–3.66 (comp., 4H), 1.8 (s, 3H), 1.34 (d, J=7 Hz, 3H),
1.28 ppm (d, J=7 Hz, 3H); 13C NMR (150 MHz, CD3OD, 208C): d=
176.3, 175.5, 173.9, 138.8, 136.98, 136.96, 136.9, 130, 129.89, 129.83,
129.77, 129.75, 129.2, 129.1, 127.3, 102.8, 97.8, 82.5, 78.3, 76.2, 71.1, 69.1,
66, 55.1, 55, 22.7, 19.7, 17.7 ppm; HRMS (FAB) calcd for C35H42N2O12P+:
713.2475 [M+H]+; found: 713.2479 [M+H]+.
The heptaprenyl phosphate diammonium salt 6 (13 mg, 19 mmol) was
azeotropically dried with toluene/benzene (1:1, 2ꢁ1 mL), and the flask
was purged with argon gas. The solution of the activated phosphoimida-
zolide was transferred to the flask containing 6 through a cannula, and
rinsed with dry THF/DMF (1:1, 3ꢁ0.3 mL). The mixture was supple-
mented with 1H-tetrazole (6.0 mg, 83 mmol) and stirred at room temper-
ature for 2.5 d. After removal of THF under reduced pressure, the resi-
due was purified by gel permeation chromatography (Sephadex LH-20,
GE Healthcare, 260ꢁ15 mm, methanol) to yield the silyl-protected
depsi-lipid I analogue (38 mg, <21 mmol).
Synthesis of compound 7: The protected tetradepsipeptide-OTMSE 9
(0.15 g, 0.20 mmol), DIPEA (1.1 mL, 6.3 mmol), and HATU (0.24 g,
0.63 mmol) were added to a solution of compound 8 (0.15 g, 0.21 mmol)
in dry DMF (6 mL). The reaction mixture was stirred at room tempera-
ture for 1 h and then evaporated to give the crude product 7, which was
purified by silica gel column chromatography (chloroform/methanol
40:1) to yield 7 as a white solid (0.24 g, 81%). 1H NMR (500 MHz,
CD3OD, 208C): d=8.47 (d, J=7 Hz, 1H), 8.33 (d, J=6.5 Hz, 1H), 8.26
(d, J=8.5 Hz, 1H), 8.01 (m, 2H), 7.49–7.36 (comp., 15H), 5.86 (dd, J=6,
4 Hz, 1H), 5.63 (s, 1H), 5.13–5.09 (comp., 4H), 5.06 (q, J=7 Hz, 1H),
4.45 (t, J=7.5 Hz, 1H), 4.38–4.28 (comp., 5H), 4.2–4.15 (comp., 6H),
4.12 (t, J=8.5 Hz, 2H), 4.05 (dd, J=11, 3.8 Hz, 1H), 3.84–3.73 (comp.,
5H), 3.08 (t, J=6.5 Hz, 2H), 2.26 (t, J=6.5 Hz, 2H), 2.21 (m, 1H), 1.86
(s, 3H), 1.77 (m, 1H), 1.65 (m, 1H), 1.47–1.43 (comp., 9H), 1.38 (d, J=
7 Hz, 3H), 1.35 (d, J=6 Hz, 3H), 1.03–0.95 (comp., 6H), 0.03 (s, 18H),
0.02 ppm (s, 9H); 13C NMR (150 MHz, CD3OD, 208C): d=175.4, 174.5,
174.38, 174.34, 173.6, 173.4, 172.8, 171.9, 159.1, 138.8, 136.93, 136.88,
136.84, 129.8, 129.2, 129.1, 127.3, 102.6, 97.8, 82, 78.4, 76.5, 71, 70.5, 69,
65.9, 64.7, 63.6, 58.3, 54.9, 54.7, 52.9, 50.3, 41.3, 32.6, 32.5, 30.5, 28.6, 23.9,
This analogue (25 mg) was azeotropically dried with toluene and dis-
solved in dry DMF (0.3 mL). TBAF (1.0m) in THF (0.43 mL) was then
added to the protected analogue at 08C and the reaction mixture was
stirred and allowed to warm to room temperature for 2 d, at which point
the reaction was complete as confirmed by LC-MS. The reaction mixture
was next evaporated to dryness under vacuum and the residue was puri-
fied by gel permeation chromatography (Sephadex LH-20, GE Health-
care, 260ꢁ15 mm, methanol). The fractions containing the desired com-
pound were combined, diluted with water, and then lyophilized overnight
to yield the semi-purified depsi-lipid I analogue as a pale yellow solid
(19 mg, <14 mmol). This analogue was further purified by reverse-phase
HPLC (Imtakt, Unison UK-C8, particle size 3 mm, 250ꢁ10 mm, 80 to
95% methanol in 0.1% NH4HCO3(aq), duration=20 min, flow rate=
3 mLminÀ1, detection at l=214 nm). The eluted fractions were combined
and then lyophilized overnight to give the depsi-lipid I analogue 4 as a
white solid (6.8 mg, 4.7 mmol, 34% yield, 96% HPLC purity). The purity
of this compound was assessed by analytical method II. Rt =5.8 min;
[a]2D3 = +338 (c=0.10 in MeOH); 1H NMR (600 MHz, D2O, 20 8C): d=
6.89 (dd, J=7.2, 3 Hz, 1H), 6.86 (t, J=6 Hz, 1H), 6.53–6.48 (comp., 6H),
5.92 (t, J=6.0 Hz, 2H), 5.79 (q, J=7.4 Hz, 1H), 5.64–5.59 (comp., 4H),
5.33 (m, 1H), 5.27 (d, J=12 Hz, 1H), 5.19 (dd, J=12, 5.4 Hz, 1H), 5.1
<0 (t, J=12.0 Hz, 1H), 4.95 (t, J=9.0 Hz, 1H), 4.31 (t, J=6.6 Hz, 2H),
3.75–3.74 (comp., 1H), 3.72 (t, J=6.3 Hz, 2H) 3.52–3.44 (comp., 20H),
3.42 (s, 3H), 3.39 (m, 4H), 3.21 (m, 3H), 3.13 (s, 3H), 3.07–3.06 (comp.,
14H), 3.01 (s, 3H), 2.99 (s, 6H), 2.86–2.84 (comp., 9H), 2.81 ppm (d, J=
6.6 Hz, 3H); 13C NMR (150 MHz, D2O, 20 8C): d=177.9, 176.9, 175.5,
175.4, 174.9, 173.8, 173.71, 173.69, 140.6, 136.4, 136.3, 136.2, 136, 132.1,
126.1, 126.2, 125.9, 12.5, 125.4, 123.6, 123.5, 100.1, 96.3, 81.8, 79.1, 75.3,
73.1, 70.2, 63.6, 62.6, 55.7, 54.9, 54.4, 50.4, 49.9, 40.88, 40.85, 40.5, 33.32,
33.26, 32.9, 32.8, 32.5, 30.4, 28.4, 27.8, 27.7, 27.6, 27.5, 25.96, 25.93, 23.85,
23.83, 23.79, 23.77, 23.75, 23.72, 23.68, 19.3, 18.8, 17.9, 17.8, 16.9, 16.16,
16.14 ppm; 31P NMR (500 MHz, CD3OD, 208C): d=À9.21 (d, J=54 Hz),
À11.6 ppm (d, J=53 Hz); HRMS (LC-ESI-TOF) calcd for
C66H109N6O22P2À: 1399.7076 [MÀH]À; found: 1399.7088 [MÀH]À.
22.9, 19.8, 18.6, 18.4, 18.14, 18.12, 18.1, 17.3, 17.2, À1.36. À1.43,
+
À1.45 ppm; HRMS (FAB) calcd for C68H106N6O21PSi3
[M+H]+; found: 1457.6453 [M+H]+.
: 1457.6456
Synthesis of compound 13: Benzylphospho-4,6-O-benzylidene-MurNAc-
silyl-protected-pentadepsipeptide-OTMSE 7 (0.20 g, 0.14 mmol) was dis-
solved in methanol (3 mL) and its benzyl groups were removed by cata-
lytic hydrogenation in the presence of 10% Pd/C at room temperature
for 30 min. The Pd/C catalyst was removed by filtration through celite
and the solution was evaporated to dryness to yield the corresponding
phosphoric acid (0.16 g, 94%) as a colorless solid, which was used for the
next reaction without further purification. Acetic acid in water (80%,
10 mL) was added the obtained phosphoric acid (0.12 g, 94 mmol) and the
reaction mixture was stirred at room temperature for 1.5 d at which point
the reaction was complete. The mixture was diluted with toluene
(100 mL) and evaporated to dryness to yield compound 13 as a colorless
solid (111 mg, 91%). 1H NMR (300 MHz, CD3OD, 208C): d=5.28 (m,
12110
www.chemeurj.org ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2013, 19, 12104 – 12112

Cell Wall Synthesis of Staphylococcus aureus
FULL PAPER
Synthesis of depsi-lipid I (3): The phospho-MurNAc-silyl-protected-pen-
tadepsipeptide-OTMSE 13 (12 mg, 9.5 mmol) was dissolved in dry pyri-
dine (2.5 mL) and excess pyridine was removed under reduced pressure
to give the pyridinium salt. This salt was azeotropically dried with tol-
uene/benzene (1:1, 2ꢁ1 mL) under vacuum and the residue was dissolved
in dry THF/DMF (1:1, 0.25 mL) and then added to a flask containing a
solution of CDI (5.5 mg, 34 mmol) in dry THF/DMF (1:1, 0.5 mL) at 08C
(ice/water bath) through a cannula. The whole was finally rinsed with dry
THF/DMF (1:1, 3ꢁ0.2 mL). The reaction mixture was first allowed to
warm to room temperature and then stirred at room temperature for 4 h
at which point the reaction was complete. The reaction was then supple-
mented with dry methanol (3 mL), evaporated under vacuum to remove
THF, and azeotropically dried with toluene/benzene (1:1, 1 mL) to yield
a solution of the activated phosphoimidazolide of 13 in DMF.
SP-550 column (TOYOPAERL). Bounded enzyme was eluted by using a
linear gradient of NaCl from 50 to 1000 mm. The eluted fraction was dia-
lyzed against buffer containing 50 mm Tris-HCl (pH 8.0), 500 mm NaCl,
1 mm DTT, 2% Triton X-100 (v/v), and 5 mm imidazole. The dialyzed
enzyme was then applied to a Ni2+ Sepharose 6 Fast Flow column (GE
Healthcare) and eluted by using a linear gradient of imidazole from 5 to
300 mm. The resulting fraction was dialyzed against a buffer containing
50 mm Tris-HCl (pH 8.0), 500 mm NaCl, 1 mm DTT, and 0.5% Triton-X
(v/v). The fraction was then applied to a HiLoad 26/60 Superdex 75
column (GE Healthcare), and the collected fractions were dialyzed
against a buffer containing 50 mm Tris-HCl (pH 8.0), 500 mm NaCl, 1 mm
DTT, 0.5% Triton X-100 (v/v), and 50% glycerol (v/v). SDS-PAGE anal-
ysis of the purified protein showed an intense band at molecular weight
around 40 kDa. The purity of MurG enzyme was estimated to be greater
than 95% from the Comassie blue-stained gel. The purified enzyme was
stored at À808C until use.
The undecaprenyl phosphate diammonium salt (6.3 mg, 7.1 mmol) was
azeotropically dried with toluene/benzene (1:1, 2ꢁ1 mL), and the flask
was purged with argon gas. The activated phosphoimidazolide solution
Preparation of the depsi-lipid II analogue by in vitro MurG reaction: The
C35-depsi-lipid II analogue was enzymatically synthesized by a procedure
similar to that used for the preparation of the C35-lipid II by Men
et al.[23] Briefly, to a solution containing Tris-HCl (50 mm, pH 8.0), MgCl2
(10 mm), decyl-polyethylene glycol (0.01%), DMSO (10%), UDP-
GlcNAc (10 mm), [14C]-UDP-GlcNAc (0.333 mm, 300 mCimmolÀ1), and
purified E. coli MurG (1.1 mgmLÀ1) the C35-depsi-lipid I analogue 4
(2 mgmLÀ1) was added. The enzymatic reaction was carried out at room
temperature for 60 min, and the resulting mixture was loaded onto a GL-
Pak cartridge column (GL Science Inc., Tokyo, Japan) and eluted by
using methanol. The resulting crude material was further purified by re-
verse-phase HPLC (Develosil ODS-UG-5, 4.6ꢁ150 mm, Nomura Chemi-
cal Co., Ltd., Aichi, Japan; 60% acetonitrile/water containing 0.1%
NH4HCO3; UV detection at l=210 nm). The eluate was dried under ni-
trogen, and then re-dissolved in Tris-HCl (50 mm, pH 8.0). The concen-
tration of the final product (C35-depsi-lipid II analogue) was 0.328 mm
(0.364 kBqmLÀ1) as ascertained by scintillation counting. The same reac-
tion conditions were used for the preparation of the C35-lipid II analogue
(0.348 mm, 0.385 kBqmLÀ1).
was then transferred to
a flask containing undecaprenyl phosphate
through a cannula and rinsed with dry THF/dry DMF (1:1, 2ꢁ0.2 mL).
1H-Tetrazole (3.0 mg, 41 mmol) was added to the mixture and the whole
was stirred at room temperature for 2.5 d. After removal of THF under
reduced pressure, the residue was purified by gel permeation chromatog-
raphy (Sephadex LH-20, GE Healthcare, 260ꢁ15 mm, methanol) to yield
the silyl-protected depsi-lipid I (12 mg, <5.8 mmol).
The silyl-protected depsi-lipid I (12 mg) was azeotropically dried with tol-
uene, and dissolved in dry DMF (0.3 mL). TBAF in THF (1.0m, 0.17 mL)
was then added to the protected depsi-lipid I at 08C, and the reaction
mixture was stirred and allowed to warm to room temperature for 2 d, at
which point the reaction was completed as confirmed by LC-MS. The re-
action mixture was then evaporated to dryness under vacuum, and the
residue was purified by gel permeation chromatography (Sephadex LH-
20, GE Healthcare, 260ꢁ15 mm, methanol). The fractions containing the
desired compound were combined, diluted with water, and then lyophi-
lized overnight to yield the semi-purified depsi-lipid I as a pale brown
solid (9.8 mg, <5.9 mmol). This solid was further purified by reverse-
phase HPLC (Imtakt, Unison UK-C8, particle size 3 mm, 250ꢁ10 mm, 80
to 95% methanol in 0.1% NH4HCO3(aq), duration=20 min, flow rate=
3 mLminÀ1, detection at l=214 nm). The eluted fractions were combined
and lyophilized overnight to give the depsi-lipid I (3) as a white solid
(2.2 mg, 1.3 mmol, 23% yield, 96% HPLC purity). The purity of this com-
pound was assessed by analytical method II. Rt =5.6 min; [a]2D3 = +288
(c=0.10 in MeOH)); 1H NMR (600 MHz, CD3OD, 228C): d=5.51 (dd,
J=7.2, 3.1 Hz, 1H), 5.45 (t, J=6.4 Hz, 1H), 5.14–5.07 (comp., 10H), 4.94
(q, J=7.1 Hz, 1H), 4.52 (t, J=6.3 Hz, 2H), 4.39 (q, J=7.3 Hz, 1H), 4.31–
4.19 (m, 4H), 3.95 (m, 1H), 3.86 (dd, J=12, 1.9 Hz, 1H), 3.78 (dd, J=12,
5.5 Hz, 1H), 3.70 (t, J=9.6 Hz, 1H), 3.54 (t, J=9.5 Hz, 1H), 2.91 (m,
Purification of S. aureus PBP2: The PBP2 gene was amplified from S.
aureus RN4220 genomic DNA as a template, and the PCR product was
cloned into the pET21b plasmid for expression with an N-terminal His6
fusion in E. coli BL21ACTHNUTRGNE(UNG DE3)pLysS cells. The obtained transformant was
grown in 1.5 L medium supplemented with 100 mgmLÀ1 ampicillin. After
incubation with shaking for 3 h at 378C, IPTG was added to make a final
concentration of 1 mm, and the growth was continued for an additional
4 h at 258C. The cells were harvested and re-suspended in a solution con-
taining Tris-HCl (20 mm, pH 8.0), NaCl (200 mm), and one tablet of Com-
plete protease inh ibitors (Roche). After disruption of the cells by sonica-
tion, the cellular debris were collected at 75000 g for 30 min at 48C, and
re-suspended in a solution containing Tris-HCl (20 mm, pH 8.0), NaCl
(200 mm), sodium N-lauroylsarcosinate, and one tablet of Complete pro-
tease inbibitors. Cells disruption was again carried out by sonication. The
supernatant was collected at 75000 g for 30 min at 48C and applied to a
HisPrep FF Ni-NTA column (GE Healthcare). Bounded enzyme was
eluted by using a linear gradient of imidazole from 20 to 500 mm. The
eluted fraction was further applied to a HiLoad 26/60 Superdex 200pg
column (GE Healthcare) and eluted by using a buffer containing Tris-
HCl (100 mm, pH 8.0), K2HPO4 (400 mm), and DTT (1 mm). The result-
ing eluate was concentrated with a 10 kDa-cut off centrifugal ultrafiltra-
tion membrane, and was added to 1 vol. of glycerol. The purity of the
PBP2 enzyme was estimated to be greater than 95% from the Comassie
blue-stained gel. The purified enzyme was stored at À808C until use.
2H), 2.38ACHTUNGTRENNUNG(m, 1H), 2.33 (m, 1H), 2.14–2.02 (comp., 40H), 1.99 (comp.,
6H), 1.81 (m, 1H), 1.72 (s, 3H), 1.66–1.65 (comp., 24H), 1.60 (s, 3H),
1.58 (s, 6H), 1.47 (m, 9H), 1.41 ppm (d, J=6.8 Hz, 3H); 13C NMR
(150 MHz, CD3OD, 228C): d=175.6, 175.4, 173.9, 173.7, 140.5, 136.4,
136.3, 136.24, 136.19, 136, 135.8, 132, 126.2, 125.9, 125.53, 125.5, 125.46,
123.6, 123.5, 75.2, 72.7, 70.4, 63.7, 63.6, 55.7, 54.9, 54.8, 50.5, 49.8, 49.6,
40.9, 40.88, 40.8, 40.5, 39.9, 33.34, 33.29, 33.26, 32.9, 27.9, 27.8, 27.69,
27.66, 27.6, 27.5, 25.9, 23.9, 23.83, 23.78, 23.73, 23.6, 19.3, 18.5, 17.9, 17.8,
17, 16.2 ppm; HRMS (LC-ESI-TOF) calcd for C86H143N6O22P2: 1671.9608
[MÀH]À; found: 1671.958 [MÀH]À.
Purification of E. coli MurG: E. coli MurG was purified as previously
described by Ha et al.[35] Briefly, E. coli Rosetta 2 (DE3) cells (Novagen)
harboring pET21b with murG gene derived from E. coli were grown in
5 L medium supplemented with 100 mgmLÀ1 ampicillin at 378C. When
the OD600 nm reached 0.6, isopropyl b-d-1-thiogalactopyranoside (IPTG)
was added to achieve a final concentration of 1 mm. The induced cell cul-
ture was grown for another 12 h at 158C and the cells were harvested
and re-suspended in lysis buffer containing 25 mm 2-(N-morpholino)etha-
nesulfonic acid (MES) (pH 6.5), 50 mm NaCl, 1 mm dithiothreitol (DTT),
1 mm ethylenediaminetetraacetic acid (EDTA), and 2% Triton X-100 (v/
v). The cells were disrupted by a French press. The supernatant was col-
lected at 100000 g for 30 min at 48C, filtered (0.45 mm), and applied to a
Initial rate measurements for the PBP2 reaction: The enzymatic activity
of the purified S. aureus PBP2 was evaluated by monitoring the incorpo-
ration of the [14C]-C35-lipid II and [14C]-C35-depsi-lipid II analogues into
nascent peptidoglycan. The enzymatic reactions were initiated by adding
ACTHNUTRGNEUNG
each concentration of [14C]-C35-lipid II/[14C]-C35-depsi-lipid II (range
from 25 to 100 mm) into the reaction mixture containing 50 mm 4-(2-hy-
droxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.0), MnCl2
(18 mm), DMSO (10%), and purified PBP2 (30 mgmLÀ1). The reaction
was performed at 308C for 4 min and terminated by addition of 1/7 vol.
Chem. Eur. J. 2013, 19, 12104 – 12112 ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
12111

H. Maki, H. Arimoto et al.
of 0.25% sodium dodecyl sulfate solution. The reaction mixture was then
spotted onto a cellulose thin-layer chromatography plate (TLC plate; cel-
lulose F precoated, Merck, Darmstadt, Germany) and developed at room
temperature for 17 h with isobutyric acid/1m ammonia (5:3) as solvent.
The TLC plate was dried and then exposed to an imaging plate for 24 h
in a dark room. Radioactive signals on the imaging plate were quantified
with a fluorescent image analyzer (FLA3000). Data were analyzed with
the MultiGuage software (GE Healthcare, Tokyo, Japan) to obtain the
kinetic parameters (Vmax, Km, kcat) of S. aureus PBP2 against each sub-
strate.
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Acknowledgements
This study was supported in part by Grants-in-Aid for Scientific Re-
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Uehara Memorial Foundation, and by the Mochida Foundation. We are
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Received: March 20, 2013
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Published online: July 19, 2013
12112
www.chemeurj.org ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2013, 19, 12104 – 12112