Forming cross-linked peptidoglycan from synthetic gram-negative lipid II

Article abstract of DOI:10.1021/ja312510m

The bacterial cell wall precursor, Lipid II, has a highly conserved structure among different organisms except for differences in the amino acid sequence of the peptide side chain. Here, we report an efficient and flexible synthesis of the canonical Lipid II precursor required for the assembly of Gram-negative peptidoglycan (PG). We use a rapid LC/MS assay to analyze PG glycosyltransfer (PGT) and transpeptidase (TP) activities of Escherichia coli penicillin binding proteins PBP1A and PBP1B and show that the native m-DAP residue in the peptide side chain of Lipid II is required in order for TP-catalyzed peptide cross-linking to occur in vitro. Comparison of PG produced from synthetic canonical E. coli Lipid II with PG isolated from E. coli cells demonstrates that we can produce PG in vitro that resembles native structure. This work provides the tools necessary for reconstituting cell wall synthesis, an essential cellular process and major antibiotic target, in a purified system.

Full text of DOI:10.1021/ja312510m

Communication
pubs.acs.org/JACS
Forming Cross-Linked Peptidoglycan from Synthetic Gram-Negative
Lipid II
Matthew D. Lebar,† Tania J. Lupoli,†,‡ Hirokazu Tsukamoto,†,§ Janine M. May,† Suzanne Walker,*,‡
and Daniel Kahne*,†
†Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
‡Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: The bacterial cell wall precursor, Lipid II,
has a highly conserved structure among different
organisms except for differences in the amino acid
sequence of the peptide side chain. Here, we report an
efficient and flexible synthesis of the canonical Lipid II
precursor required for the assembly of Gram-negative
peptidoglycan (PG). We use a rapid LC/MS assay to
analyze PG glycosyltransfer (PGT) and transpeptidase
(TP) activities of Escherichia coli penicillin binding
proteins PBP1A and PBP1B and show that the native m-
DAP residue in the peptide side chain of Lipid II is
required in order for TP-catalyzed peptide cross-linking to
occur in vitro. Comparison of PG produced from synthetic
canonical E. coli Lipid II with PG isolated from E. coli cells
demonstrates that we can produce PG in vitro that
resembles native structure. This work provides the tools
necessary for reconstituting cell wall synthesis, an essential
cellular process and major antibiotic target, in a purified
system.
he most important antibiotics in clinical use target the
bacterial cell wall (Figure 1a), a rigid polymer that is
T
Figure 1. Bacterial cell wall is assembled from the precursor Lipid II
(1). (a) Schematic of an E. coli cell and the chemical structure of its
cell wall, composed of alternating GlcNAc (blue) and MurNAc
(green) residues with attached peptide side chains that can be cross-
linked. Purified E. coli cell wall typically lacks terminal D-Ala residues
(red). (b) Chemical structure of Gram-positive L-Lys Lipid II (1a) and
Gram-negative m-DAP Lipid II (1b),8 which differ at the third residue
of the peptide side chain. Because R varies depending on the organism,
a flexible route to 1 is needed to study the PG synthesis of various
organisms.
essential for survival under osmotic stress.1 The cell wall
precursor, Lipid II (1, Figure 1b), is synthesized in the
cytoplasm and then assembled into peptidoglycan (PG) outside
the cytoplasmic membrane by high molecular weight, bifunc-
tional penicillin binding proteins (PBPs) that contain PG
glycosyltransferase (PGT) and transpeptidase (TP) domains.
The PGT and TP domains catalyze polymerization of Lipid II
and cross-linking of the resulting glycan strands, respectively
(Figure 1a). The study of high molecular weight PBPs is
important because these enzymes are the lethal targets of the β-
lactam antibiotics;1 however, the required substrate, Lipid II, is
difficult to obtain.2−4 Lipid II is a β-(1,4)-N-acetylglucosamine
(GlcNAc)-N-acetylmuramic acid (MurNAc) disaccharide con-
taining a diphospholipid at the reducing end. A pentapeptide is
attached to the lactyl moiety of the MurNAc sugar. The
structure of Lipid II is conserved in bacteria except for
modifications at the third position of the peptide chain,5 which
contains a nucleophilic amine that forms a cross-link with a D-
Ala residue on another peptide side chain during the TP
reaction (Figure 1a).
reported previously2b,3,4 and has been useful for studying PG
polymerization.3c−f,6 Although TP-mediated hydrolysis of D-
Ala-D-Ala peptide bonds was detected, no evidence for cross-
linking was observed.3d,7a
We report the first chemical synthesis of the canonical Lipid
II precursor for Gram-negative organisms (1b), which contains
meso-diaminopimelic acid (m-DAP) as the third peptide
residue. We show using a newly developed LC/MS assay
capable of detecting both glycosyltransfer and transpeptidation
Synthesis of a Lipid II precursor containing L-Lys in the third
position of the peptide side chain (1a, Figure 1b) has been
Received: January 3, 2013
Published: March 12, 2013
© 2013 American Chemical Society
4632
dx.doi.org/10.1021/ja312510m | J. Am. Chem. Soc. 2013, 135, 4632−4635

Journal of the American Chemical Society
Communication
products that 1b is converted to cross-linked PG by the two
primary bifunctional PBPs in Escherichia coli, PBP1A and
PBP1B. We further show that PG produced in vitro from
purified E. coli enzymes resembles the structure of native PG
isolated from E. coli cells. These developments will enable
mechanistic studies of PG transpeptidation and the production
of cross-linked material for studies of other bacterial cell wall-
modifying enzymes.
modified method improved sensitivity and enabled direct
product identification.14 We initially tested two substrates that
varied only at the third peptide residue, L-Lys Lipid II (1a),
which is the precursor for most Gram-positive PG, and m-DAP
Lipid II (1b), which is the precursor for most Gram-negative
PG. Each substrate was incubated with E. coli PBP1A, one of
the organism’s two bifunctional PBPs.6k The reactions were
then treated with mutanolysin, which cleaves MurNAc-GlcNAc
bonds, to digest polymeric products, followed by sodium
borohydride, which reduces the MurNAc termini in order to
simplify the mixture by converging anomers (Figure 2a).
a
Scheme 1. Synthesis of m-DAP Lipid II (1b)
a
Reagents and conditions: (a) i. Grubbs second gen. catalyst, DCM,
12h, reflux (50%); ii. H2/Pd, MeOH, 1 h, rt (90%); (b) i. NH2-D-Ala-
D-Ala-OMe, EDC, HOBt, DIEA, DCM, 2h, rt; ii. 4 M HCl in dioxane,
15 min, rt; iii. Boc-L-Ala-γ-D-Glu(OH)-OMe, EDC, HOBt, DIEA,
DCM, 2h, rt; iv. 4 M HCl in dioxane, 15 min, rt (54%); (c) i. 3,
DMTNMM, DIEPA, MeOH, rt, 2h; ii. NaOH, H2O, dioxane, 45 min,
rt (48%); (d) UDP-GlcNAc, MurG, buffer, 1h, rt.
We developed a flexible route to generate large quantities of
m-DAP Lipid II (1b) that is amenable to installation of many
different peptide side chains. Precursor 3, obtained as
described,7,9 is devoid of protecting groups and can be
elaborated to make Lipid II substrates from a variety of
organisms by altering the stem peptide in the subsequent
coupling step. Synthesis of the canonical Gram-negative
pentapeptide moiety required access to an orthogonally
protected m-DAP residue. We obtained a suitably masked m-
DAP (6) by olefin cross metathesis of L-vinylglycine 4 and D-
allylglycine 5 utilizing the Grubbs second generation catalyst
and subsequent hydrogenation.10 Pentapeptide 7 was formed
from m-DAP by sequential condensation with NH2-D-Ala-D-
Ala-OMe followed by Boc-L-Ala-γ-D-Glu(OH)-OMe. The
precursor to Lipid II, Lipid I (8), was obtained through a
DMTMM-promoted condensation7a of MurNAc heptaprenyl
pyrophosphate (3) and pentapeptide 7 followed by protecting
group hydrolysis under basic conditions. Lipid I was then
converted to the desired m-DAP-containing PBP substrate,
Lipid II (1b), through MurG-catalyzed glycosylation with
UDP-GlcNAc.3a,b,11 Unlike chemical glycosylation methods,
the use of MurG provides Lipid II directly from unprotected
Lipid I, providing amounts sufficient for hundreds of enzymatic
reactions.
Figure 2. The m-DAP residue in Lipid II is essential for
transpeptidase-catalyzed cross-linking. (a) Schematic of method for
analyzing PG synthesis by PBPs.14 (b) LC/MS extracted chromato-
grams of PBP1A and L-Lys Lipid II (1a) reactions produce only A′,
representing unmodified polymer, in the presence of the inhibitor
penG (i), but both A′ and B′, representing hydrolyzed peptide side
chain, without penG (ii). PBP1A and m-DAP-Lipid II (1b) reactions
reveal fragment C in addition to fragments A and B, indicating the
formation of cross-linked PG (iv); no cross-links are formed in the
presence of penG (iii). The PGT inhibitor moenomycin prevented
formation of all fragment peaks (Figure S1). For chromatograms (i, ii):
(M+2H)/2 ions corresponding to fragments A′ and B′ were extracted:
A′: 485.2; B′: 449.7; masses corresponding to predicted cross-linked
fragments were not observed. For chromatograms (iii, vi): (M+2H)/2
ions corresponding to fragments A−D were extracted: A: 507.2; B:
471.7; C: 968.9; D: 933.4.
LC/MS analysis of 1a reactions revealed exclusively
disaccharide-pentapeptide fragment A′ in the presence of TP
inhibitor penicillin G (penG, Figure 2b, trace i) and a small
amount of disaccharide-tetrapeptide B′ due to TP-catalyzed
hydrolysis in the absence of inhibitor (Figure 2b, trace ii).
Reactions with 1b produced only A in the presence of penG
(Figure 2b, trace iii); however, fragments A, B, and an
additional fragment, C, arising from TP-catalyzed cross-linking,
Existing methods to analyze the reactions catalyzed by high
molecular weight PBPs rely on the use of authentic standards
and radiometry to identify fragments generated after post-
reaction degradation.2c,d,12 We adapted the classical HPLC
method for analysis of cell wall fragments,13 which relies on
nonvolatile salts as the mobile phase additive, so that the
separation conditions are compatible with LC/MS. The
4633
dx.doi.org/10.1021/ja312510m | J. Am. Chem. Soc. 2013, 135, 4632−4635

Journal of the American Chemical Society
Communication
were observed without added TP inhibitor (Figure 2b, trace iv).
Hence, although E. coli PBP1A polymerizes both L-Lys Lipid II
(1a) and m-DAP Lipid II (1b), it can only cross-link glycan
strands containing m-DAP. The fact that only m-DAP (Figure
1a, R = COOH) and not L-Lys (R = H) can act as a nucleophile
in the cross-linking reaction explains in vivo data showing that,
while L-Lys can be incorporated into PG in E. coli, the ε-amine
of Lys was never detected in cross-links.15 This result also
clarifies why cross-links could not be detected previously when
L-Lys Lipid II was incubated with E. coli PBPs in vitro,3d,7a as the
TP domain is selective for its native substrate. Similar to
PBP1A, E. coli PBP1B polymerizes both 1a and 1b but only
cross-links the m-DAP glycan strands (see Figure S2). These
results verify that the assay detects both glycosyltransfer and
transpeptidation activities of bifunctional PBPs and that the m-
DAP residue in E. coli PG is necessary in the peptide side chain
for cross-linking to occur in vitro.16
isolated sacculi (Figure S3). Hence, using only a few purified
components and the native E. coli substrate, it is possible to
reconstitute PG that is similar in composition to in vivo
samples.
In summary, we have synthesized the E. coli cell wall
precursor m-DAP Lipid II and used it in an LC/MS assay
designed to analyze cell wall composition. E. coli PBP1A and
PBP1B are able to polymerize both L-Lys substrate 1a and m-
DAP substrate 1b but cross-link only PG containing the
appropriate substituent in the third position of the peptide side
chain (m-DAP). The third residue in the peptide side chain is
important in the TP reaction because it contains the reactive
amine that forms cross-links. Since cell walls from different
organisms are built from precursors with varying peptide side
chains5 and PBPs are sensitive to the identity of the reactive
residues, it is important to have an efficient, flexible route to
Lipid II that is amenable to changes in the composition of the
peptide. Hence, the synthesis we described can be applied to
the study of cell wall construction in other organisms, and this
in vitro system can be used to investigate the proteins involved
in the synthesis of different bacterial cell wall structures.
Reconstructing how enzymes build and break PG will provide a
better understanding of the maintenance of a complex cellular
structure and may provide new insight into how to target the
essential bacterial cytoskeleton.
Using the LC/MS assay, we next compared the composition
of synthetic PG to isolated bacterial cell wall. Purified E. coli cell
wall lacks pentapeptide-containing PG fragments, and it was
proposed that this is due partially to the activity of the
carboxypeptidase PBP5,17a,b which removes the terminal D-Ala
residue from peptide side chains (Figure 3a).17 We compared
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, synthesis, and analysis of substrates.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
suzanne_walker@hms.harvard.edu; kahne@chemistry.harvard.
edu
■
Present Address
§Graduate School of Pharmaceutical Sciences and Pharmaceut-
ical Chemistry, Tohoku University, Sendai, Miyagi, Japan
Notes
The authors declare no competing financial interest.
Figure 3. Composition of PG produced in vitro resembles PG isolated
from E. coli cells. (a) Schematic of experimental procedure for PG
analysis shows that fragments A and C would result from degradation
of PG synthesized in vitro, while B and D would result from prior
hydrolysis of PG by E. coli PBP5,17d which liberates terminal D-Ala
residues. (b) Treatment of PBP1A and 1b reactions with PBP5
produces hydrolysis fragments B and D (i) in proportions similar to
those found in treated in vivo PG sample (ii). In vitro samples were
prepared as before,14 except quenched PBP1A reactions were treated
with PBP5 (0.8 μM) for 2 h prior to analysis. See SI for details on cell
wall isolation. (M+2H)/2 ions corresponding to fragments A−D were
extracted from each chromatogram: A: 507.2; B: 471.7; C: 968.9; D:
933.4.
ACKNOWLEDGMENTS
■
Research was supported by the NIH (R01 GM066174 to D.K.;
R01 GM76710 to D.K. and S.W.; F32 GM103056 to M.D.L.),
NERCE (AI057159), and NSF (DGE-1144152 to J.M.M). Use
of Agilent 6520 Q-TOF spectrometer was supported by the
Talpin Funds for Discovery Program (P.I.: S.W.). We thank
Sunia Trauger (Small Molecule Mass Spectrometry Facility,
Harvard) for HRMS assistance and the Bernhardt Lab
(Harvard Medical School) for overexpression plasmids (see SI).
REFERENCES
■
(1) (a) Tomasz, A. Annu. Rev. Microbiol. 1979, 33, 113. (b) Walsh, C.
Antibiotics: actions, origins, resistance. ASM Press: Washington, D.C.,
2003. (c) Lovering, A. L.; Safadi, S. S.; Strynadka, N. C. Annu. Rev.
Biochem. 2012, 81, 451.
PBP1A-generated cell wall treated with PBP5 (Figure 3b, trace
i) to cell wall isolated from E. coli cultures using standard
protocols13,18 (Figure 3b, trace ii). In both traces, we observed
cell wall fragments B and D, representing the disaccharide
tetrapeptide (tetra) and the hydrolyzed cross-linked product
(tetra-tetra), respectively.
Unlike reactions that contained only PBP1A (Figure 2b), the
in vitro products treated with PBP5 were tetrapeptide-
containing fragments like the major products generated from
(2) Lipid II containing various peptide side chains can be accessed by
treating isolated cell wall precursors with membrane preparations or
MraY/lipid phosphate, see: (a) Anderson, J. S.; Matsuhashi, M.;
Haskin, M. A.; Strominger, J. L. Proc. Natl. Acad. Sci. U.S.A. 1965, 53,
881. (b) Breukink, E.; van Heusden, H. E.; Vollmerhaus, P. J.;
Swiezewska, E.; Brunner, L.; Walker, S.; Heck, A. J. R.; De Kruijff, B. J.
4634
dx.doi.org/10.1021/ja312510m | J. Am. Chem. Soc. 2013, 135, 4632−4635

Journal of the American Chemical Society
Communication
Biol. Chem. 2003, 278, 19898. (c) Bertsche, U.; Breukink, E.; Kast, T.;
Vollmer, W. J. Biol. Chem. 2005, 280, 38096. (d) Born, P.; Breukink,
E.; Vollmer, W. J. Biol. Chem. 2006, 281, 26985. (e) El Ghachi, M.;
(13) Glauner, B.; Holtje, J. V.; Schwarz, U. J. Biol. Chem. 1988, 263,
10088.
̈
(14) Lipid II (1a or 1b, 40 μM) was incubated with E. coli PBP1A or
PBP1B (400 nM) in 50 mM HEPES (4-(2-hydroxyethyl)-1-
piperazine-ethanesulfonic acid), 10 mM CaCl2, 20% DMSO, pH =
7.5 (10 μL total volume) for 20 min at 25 °C in the presence or
absence of penicillin G (1 kU/mL). Reaction was quenched at 95 °C
for 5 min and then treated with mutanolysin (1U for 1.5 h and then
1U for an additional 1.5 h at 37 °C) and sodium borohydride (10 μL,
10 mg/mL, 30 min). Phosphoric acid (20% v/v, 1.2 μL) was added to
adjust pH to ∼4. The mixture was lyophilized, redissolved in 10 μL
water, and subjected to LC/MS analysis (ESI-MS, positive mode,
Waters SymmetryShield RP18 column, 5 μm, 3.9×150 mm).
Fragments were separated with water (0.1% formic acid) for 5 min
followed by a gradient of 0−20% acetonirile (0.1% formic acid)/water
(0.1% formic acid) over 40 min at 0.5 mL/min. Molecular ions
corresponding to expected disaccharide fragments were extracted from
the chromatogram. See SI for more details.
(15) (a) Mengin-Lecreulx, D.; Falla, T.; Blanot, D.; van Heijenoort,
J.; Adams, D. J.; Chopra, I. J. Bacteriol. 1999, 181, 5909. (b) Mengin-
Lecreulx, D.; Michaud, C.; Richaud, C.; Blanot, D.; van Heijenoort, J. J.
Bacteriol. 1988, 170, 2031.
(16) Previous studies have shown that m-DAP Lipid II forms cross-
linked PG, see refs 2c, d, and 11.
(17) (a) Kraus, W.; Holtje, J. V. J. Bacteriol. 1987, 169, 3099. (b) Li,
S. Y.; Holtje, J. V.; Young, K. D. Anal. Biochem. 2004, 326, 1.
(c) Nicholas, R. A.; Strominger, J. L. J. Biol. Chem. 1988, 263, 2034.
(d) Zhang, W.; Shi, Q.; Meroueh, S. O.; Vakulenko, S. B.; Mobashery,
S. Biochemistry 2007, 46, 10113.
́
Bouhss, A.; Barreteau, H.; Touze, T.; Auger, G.; Blanot, D.; Mengin-
Lecreulx, D. J. Biol. Chem. 2006, 281, 22761. (f) Lloyd, A. J.; Gilbey, A.
M.; Blewett, A. M.; De Pascale, G.; El Zoeiby, A.; Levesque, R. C.;
Catherwood, A. C.; Tomasz, A.; Bugg, T. D. H.; Roper, D. I.; Dowson,
C. G. J. Biol. Chem. 2008, 283, 6402. (g) Patin, D.; Barreteau, H.;
́
Auger, G.; Magnet, S.; Crouvoisier, M.; Bouhss, A.; Touze, T.; Arthur,
M.; Mengin-Lecreulx, D.; Blanot, D. Biochimie 2012, 94, 985.
(3) L-Lys-containing Lipid II can be accessed by synthetic/
semisynthetic methods, see: (a) Men, H.; Park, P.; Ge, M.; Walker,
S. J. Am. Chem. Soc. 1998, 120, 2484. (b) Ha, S.; Chang, E.; Lo, M.;
Men, H.; Park, P.; Ge, M.; Walker, S. J. Am. Chem. Soc. 1999, 121,
8415. (c) Ye, X.; Lo, M.; Brunner, L.; Walker, D.; Kahne, D.; Walker,
S. J. Am. Chem. Soc. 2001, 123, 3155. (d) Schwartz, B.; Markwalder, J.
A.; Wang, Y. J. Am. Chem. Soc. 2001, 123, 11638. (e) VanNieuwenhze,
M.; Mauldin, S.; Zia-Ebrahimi, M.; Winger, B.; Hornback, W.; Saha, S.;
Aikins, J.; Blaszczak, L. J. Am. Chem. Soc. 2002, 124, 3656. (f) Shih, H.-
W.; Chang, Y.-F.; Li, W.-J.; Meng, F.-C.; Huang, C.-Y.; Ma, C.; Cheng,
T.-J. R.; Wong, C.-H.; Cheng, W.-C. Angew. Chem. Int. Ed. Engl. 2012,
51, 10123.
(4) Liu, C.-Y.; Guo, C.-W.; Chang, Y.-F.; Wang, J.-T.; Shih, H.-W.;
Hsu, Y.-F.; Chen, C.-W.; Chen, S.-K.; Wang, Y.-C.; Cheng, T.-J. R.;
Ma, C.; Wong, C.-H.; Fang, J.-M.; Cheng, W.-C. Org. Lett. 2010, 12,
1608.
(5) Bouhss, A.; Trunkfield, A. E.; Bugg, T. D. H.; Mengin-Lecreulx,
D. FEMS Microbiol. Rev. 2008, 32, 208.
(6) (a) Chen, L.; Walker, D.; Sun, B.; Hu, Y.; Walker, S.; Kahne, D.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5658. (b) Chen, L.; Yuan, Y.;
Helm, J. S.; Hu, Y.; Rew, Y.; Shin, D.; Boger, D. L.; Walker, S. J. Am.
Chem. Soc. 2004, 126, 7462. (c) Barrett, D. S.; Chen, L.; Litterman, N.
K.; Walker, S. Biochemistry 2004, 43, 12375. (d) Barrett, D.;
Leimkuhler, C.; Chen, L.; Walker, D.; Kahne, D.; Walker, S. J.
Bacteriol. 2005, 187, 2215. (e) Barrett, D.; Wang, T.-S.; Yuan, Y.;
Zhang, Y.; Kahne, D.; Walker, S. J. Biol. Chem. 2007, 282, 31964.
(f) Yuan, Y.; Barrett, D.; Zhang, Y.; Kahne, D.; Sliz, P.; Walker, S. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 5348. (g) Fang, X.; Tiyanont, K.;
Zhang, Y.; Wanner, J.; Boger, D.; Walker, S. Mol. BioSyst. 2006, 2, 69.
(h) Perlstein, D. L.; Zhang, Y.; Wang, T.-S.; Kahne, D. E.; Walker, S. J.
Am. Chem. Soc. 2007, 129, 12674. (i) Wang, T.-S.; Manning, S.;
Walker, S.; Kahne, D. J. Am. Chem. Soc. 2008, 130, 14068. (j) Perlstein,
D.; Wang, T.-S.; Doud, E.; Kahne, D.; Walker, S. J. Am. Chem. Soc.
2010, 132, 48. (k) Paradis-Bleau, C.; Markovski, M.; Uehara, T.;
Lupoli, T. J.; Walker, S.; Kahne, D. E.; Bernhardt, T. G. Cell 2010, 143,
1110.
(18) Uehara, T.; Dinh, T.; Bernhardt, T. G. J. Bact. 2009, 191, 5094.
(7) (a) Lupoli, T. J.; Tsukamoto, H.; Doud, E. H.; Wang, T.-S.;
Walker, S.; Kahne, D. J. Am. Chem. Soc. 2011, 133, 10748.
(b) Tsukamoto, H.; Kahne, D. Bioorg. Med. Chem. Lett. 2011, 21,
5050. (c) Weerapana, E.; Glover, K. J.; Chen, M. M.; Imperiali, B. J.
Am. Chem. Soc. 2005, 127, 13766.
(8) A heptaprenyl lipid chain was used rather than the most
commonly found natural lipid (undecaprenyl) because the former
does not aggregate as extensively and thus is a better substrate for
biochemical studies (see ref 3c).
(9) (a) Gampe, C. M.; Tsukamoto, H.; Wang, T.-S.; Walker, S.;
Kahne, D. Tetrahedron 2011, 67, 9771. (b) Hesek, D.; Lee, M.;
̌
Zajícek, J.; Fisher, J. F.; Mobashery, S. J. Am. Chem. Soc. 2012, 134,
13881.
(10) Chowdhury, A. R.; Boons, G.-J. Tetrahedron Lett. 2005, 46,
1675.
(11) Ha, S.; Walker, D.; Shi, Y.; Walker, S. Protein Sci. 2000, 9, 1045.
(12) (a) Muller, P.; Ewers, C.; Bertsche, U.; Anstett, M.; Kallis, T.;
Breukink, E.; Fraipont, C.; Terrak, M.; Nguyen-Disteche, M.; Vollmer,
W. J. Biol. Chem. 2007, 282, 36394. (b) Typas, A.; Banzhaf, M.; van
den Berg van Saparoea, B.; Verheul, J.; Biboy, J.; Nichols, R. J.; Zietek,
M.; Beilharz, K.; Kannenberg, K.; von Rechenberg, M.; Breukink, E.;
den Blaauwen, T.; Gross, C. A.; Vollmer, W. Cell 2010, 143, 1097.
4635
dx.doi.org/10.1021/ja312510m | J. Am. Chem. Soc. 2013, 135, 4632−4635